Note: Descriptions are shown in the official language in which they were submitted.
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DIRECT MOLECULAR CLONING OF
A MODIFIED EUKARYOTIC CYTOPLASMIC DNA VIRUS GENOME
Background of the Invention
The present invention relates to modified genomes of
eukaryotic DNA viruses which replicate in the cytoplasm
of a_ host cell,, such as poxviruses and iridoviruses.
More specifically, the invention relates to direct
molecular cloning of a modified cytoplasmic DNA virus
genome that is produced by modifying under extracellular
conditions a purified DNA molecule comprising a
cytoplasmic DNA virus genome. The modified DNA molecule
is then packaged into infectious virions in a cell
infected with a helper cytoplasmic DNA virus. In a
preferred embodiment of the present invention, a foreign
DNA fragment comprising a desired gene is inserted
directly into a genomic poxvirus DNA at a~restriction
endonuclease cleavage site that is unique in the viral
genome, and the modified viral DNA is packaged into
virions by transfection into cells infected with a helper
poxvirus .
Cytoplasmic DNA viruses of eukaryotes include diverse
poxviruses and iridoviruses found in vertebrates and
insects . Poxviruses having recombinant genomes have been
used for expression of a variety of inserted genes. Such
poxviruses can be used to produce biologically active
polypeptides in cell cultures, for instance, and to
deliver vaccine antigens directly to an animal or a human
immune system. Construction of recombinant iridovirus
genomes for expression of foreign genes appears not to be
documented in the literature pertaining to genetic
engineering.
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Conventional techniques for construction of
recombinant poxvirus genomes comprised of foreign genes
rely in part on in vivo (intracellular) recombi~iation.
The use of intracellular recombination wx~ first
described as a process of "marker rescue" with ,~subgenomic
fragments of viral DNA by Sam & Dumbell, Ann. Virol.
(Institut Pasteur) 132E: 135 (1981). 'Tihese authors
demonstrated that a temperature-sensitive vaccinia virus
mutant could be "rescued" by intracellular recombination
with a subgenomic DNA fragment of a rabbit poxvirus. The
methods they used for intracellular.recombination are
still used today.
Construction of recombinant vaccinia viruses
comprised of non-poxvirus ("foreygn°) genes was later
described by Panicali & Paoletti, Proc. Nat'I Acad. Sci.
U.S.A. 79: 4927-4931 (1982); Mackett, et al., Proc. Nat'I
Acad. Sci. U.S.A. 79: 7415-7419 (1982); and U.S. patent
No. 4,769,330. More specifica~.ly, the extant technology
for producing recombinant poxcviruses involves two steps.
First, a DNA fragment is prepared that has regions of
homology to the poxvirus ~yenome surrounding a foreign
gene. Alternatively, an "insertion" plasmid is
constructed by in vitro Gextracellular) recombination of
a foreign gene with a Hlasmid. This plasmid comprises
short viral DNA seque~~ces that are homologous to the
region of the poxvirus genome where gene insertion is
ultimately desired. The foreign gene is inserted into
the plasmid at a sits flanked by the viral DNA sequences
and, typically, downstream of a poxvirus promoter that
will control trans.c:ription of the inserted gene. In the
second step, the insertion plasmid is introduced into
host cells infected with the target poxvirus. The gene
is then indirectly inserted into the poxvirus genome by
intracellular recombination between homologous viral
sequences in the poxvirus genome and the portion of the
plasmid including the foreign gene. The resulting
recombinant genome then replicates, producing infectious
poxvirus.
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Thus, insertion of each particular gene into a
poxvirus genome has heretofore required a distinct
plasmid comprised of the gene flanked viral sequences
selected for a desired insertion location. ~ difficulty
with this approach is that a new insertion plasmid is
required for each recombinant poxvirus_ Each plasmid
must be constructed by extracellular recombinant DNA
methods, amplified in a bacterial cell, and then
laboriously isolated and rigorously purified before
addition to a poxvirus-infected host cell.
Another problem with extant methodology in this
regard is a low yield of recombinant genomes, which can
necessitate screening hundreds ~f individual viruses to
find a single desired recombinant. The poor yield is a
function of the low frequency of individual intracellular
recombination events, compoY~nded by the requirement for
multiple events of this smrt to achieve integration of
the insertion plasmid intm a viral genome. As a result,
the majority of viral gy ones produced by intracellular
recombination methods are parental genomes that lack a
foreign gene. It is~' often necessary, therefore, to
introduce a selective marker gene into a poxvirus genome,
along with any other desired sequence, to permit ready
detection of the required rare recombinants without the
need of characterizing isolated DNAs from numerous
individual virus .clones.
Purified DNAs of eukaryotic cytoplasmic DNA viruses
are incapable of replicating when introduced into
susceptible host cells using methods that initiate
infections with viral DNAs that replicate in the nucleus .
This lack of infectivity of DNAs of cytoplasmic DNA
viruses results from the fact that viral transcription
must be initiated in infected cells by a virus-specific
RNA polymerase which is normally provided inside
infecting virions.
"Reactivation" of poxvirus DNA, in which genomic DNA
inside an inactivated, noninfectious poxvirus particle
was packaged into infectious virions by coinfection with
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a viable helper poxvirus, has been known for decades.
See, for instance, Fenner & Woodroofe, Virology il: 185-
201 (1960). In 1981 Sam and Dumbell demonstrated that
isolated, noninfectious genomic DNA of a fist poxvirus
could be packaged into infectious poxvi~us virions in
cells infected with a second, genetically distinct
poxvirus. Sam & Dumbell, Ann. Virol. ~Institut Pasteur)
132E: 135 (1981). This packaging of naked poxvirus DNA
was first demonstrated by transfection of unmodified DNA
comprising a first wildtype c~rthopoxvirus genome,
isolated from virions or infected cells, into cells
infected with a second naturally occurring orthopoxvirus
genome. However, heterologous packaging, packaging of
DNA from one poxvirus genus (orthopox, for example) by
viable virions of another genus (e.g., avipox), has not
been demonstrated yet.
The use of intracellular recombination for
constructing a recombinant poxvirus genome expressing
non-poxvirus genes was reported shortly ,after Sam &
Dumbell first reported intracellular packaging of naked
poxvirus DNA into poxyirus virions and marker rescue with
DNA fragments by intracellular recombination. See
Panicali & Paoletti, 1982; Mackett, et al., 1982. The
relevant literature of the succeeding decade, however,
appears not to document the direct molecular cloning,
i.e., construct,ion solely by extracellular genetic
engineering, of a modified genome of any eukaryotic
cytoplasmic DNA virus, particularly a poxvirus. The
literature does not even evidence widespread recognition
of any advantage possibly realized from such a direct
cloning approach. To the contrary, an authoritative
treatise has stated that direct molecular cloning is not
practical in the context of genetic engineering of
poxviruse~ because poxvirus DNA is not infectious. F.
FENNER, R. WITTEK & K.R. DUMBELL, THE POXVIRUSES
(Academic Press, 1989). Others working in the area have
likewise discounted endonucleolytic cleavage and
relegation of poxvirus DNAs, even while recognizing a
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potential for rescue by infectious virus of isolated
DNA comprising a recombinant poxvirus genome. See,
for example, Mackett & Smith J. Gen Virol. 67: 2067-
2082 (1986). Moreover, recent reviews propound the
thesis that the only way feasible to construct a
recombinant poxvirus genome is by methods requiring
intracellular recombination. See Miner & Hruby,
TIBTECH 8: 20-25 (1990), and Moss & Flexner, Ann.
Rev. Immunol. 5: 305-324 (1987).
Summary of the Invention
According to one aspect of the present invention,
there is provided a method for producing a modified
chordopoxvirus, wherein said method comprises the
steps of: (I) modifying under extracellular
conditions a first chordopoxvirus genome to produce a
modified ck~ordopoxvirus genome; (II) infecting a
cultured host cell with a second chordopoxvirus that
is heterologous to said first chordopoxvirus, said
second chordopoxvirus comprising a second viral
genome which is expressed in said cultured host cell
to package said modified chordopoxvirus genome into
an infectious virion; (III) introducing said modified
chordopoxvirus genome into said cultured host cell,
wherein said modified chordopoxvirus genome is
packaged into an infectious virion; and
(IV)recovering from said cultured host cell said
infectious virion that includes said modified
chcrdopoxvirus genome.
According to another aspect of the present
invention, there is provided a modified
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chordopoxvirus produced by the method described
herein.
According to still another aspect of the present
invention, there is provided a modified chordopoxvirus that
includes a modified chordopoxvirus genome, wherein said
modified chordopoxvirus genome comprises: (I) a first
chordopoxvirus genome, wherein said first genome
includes a cleavage site for a sequence-specif is
endonuclease and said cleavage site is a unique site
in said first genome; and (II) a first DNA sequence
inserted into said unique site in said first genome,
wherein said first DNA sequence is not naturally
occurring in a chordopoxvirus genome.
According to yet another aspect of the present
invention, there is provided a rrlodified chordopoxvirus that
includes a modified chordopoxvirus genome, wherein said
chordopoxvirus genome comprises: (I) a first
chordopoxvirus genome, wherein said genome includes
a first cleavage site for a first sequence-specific
endonuclease and a second cleavage site for a second
sequence-specific endonuclease and each of said-
cleavage sites is a unique site in said genome; (II)
and a first DNA sequence inserted into said first
genome between said first unique site and said second
unique site.
According to a further aspect of the present
invention, there is provided a modified chordopoxvirus that
includes a modified chordopoxvirus genome, wherein said
chordopoxvirus genome comprises: (I) a first chordopoxvirus
genome, wherein said first genome includes a first
DNA sequence and said first DNA sequence includes a
cleavage site for a sequence-specific endonuclease
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that is a unique site in said modified chordopoxvirus
genome; and (II) a promoter located such that a DNA
sequence inserted into said unique site is under
transcriptional control of said promoter, wherein
said first DNA sequence lacks a translation start
codon between said promoter and said unique site.
According to yet another aspect of the present
invention, there is provided a kit for producing a
chordopoxvirus genome comprising:(I) purified DNA
vector arms, produced by cleavage of a chordopoxvirus
genome at a first unique restriction site, wherein
said arms are used for the insertion of a DNA segment
into said chordopoxvirus genome, wherein said DNA
segment possesses cohesive ends compatible for
ligation with each of said vector arms, and wherein
said arms are capable of forming a viable, modified
chordopoxvirus genome upon ligation; (II) a DNA
ligase; and (III) buffer solutions and reagents
suitable for ligation of DNA segments together to
produce said modified chordopoxvirus genome.
According to still a further aspect of the
present invention, there is provided a plasmid for
use in the method described herein, wherein said
plasmid comprises a DNA segment having a cleavage
site for the bacterial restriction endonuclease NotI
at each end, wherein said DNA segment comprises a
sequence-specific endonuclease cleavage site that is
unique in said plasmid.
According to another aspect of the present
invention, there is provided a plasmid comprising a
DNA segment having a cleavage site for the bacterial
restriction endonuclease NotI at each end, wherein
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said DNA segment comprises a sequence-specific
endonuclease cleavage site that is unique in said
plasmid, and wherein said plasmid is as shown in
Figure 1.3, designated pN2 and comprising t=he
sequence of SEQ. ID. NO. 1.
According to yet another aspect of the present
invention, there is provided a plasmid comprising a
first master cloning site that includes unique sit es
of a second master cloning site of the vaccinia virus
vector designated vdTK, selected from the following
group of plasmids shown in Figure 4.3: pA0 comprising
the sequence of SEQ. ID. NO. 6, pAl comprising t he
sequence of SEQ. ID. NO. 7, and pA2 comprising t he
sequence of SEQ. ID. NO. 8.
According to one aspect of the present invention,
there is provided a plasmid comprising a modified
EcoRI K fragment of vaccinia virus DNA from which t he
K1L host range gene is deleted.
According to another aspect of the present
invention, there is provided a plasmid comprising a
segment of a chordopoxvirus genome that comprises a
thymidine kinase gene of said chordopoxvirus, wherein
said thymidine kinase gene has been modified to
prevent expression of active thymidine kinase,
wherein said plasmid is as shown in Figure 4.2 and is
selected from the group of plasmids: pHindJ-2
comprising the sequence of SEQ. ID. NO. 4, and
pHindJ-3 comprising the sequence of SEQ. ID. NO. 5.
According to still another aspect of the present
invention, there is provided a modified vaccinia
virus comprising a modification under extracellular
conditions of a wildtype vaccinia virus genome having
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a single cleavage site for the bacterial restriction
endonuclease NotI, wherein said modification
eliminates said single cleavage site, and wherein
said modification does not regenerate said wildtype
vaccinia virus genome.
According to still another aspect of the present
invention, there is provided a modified vaccinia
virus comprising a modification under extracellu 1 ar
conditions of a wildtype vaccinia virus genome having
a single cleavage site for the bacterial restrict i on
endonuclease SmaI, wherein said modificat ion
eliminates said single cleavage site, and wherein
said modification does not regenerate said wildtype
vaccinia virus genome.
According to yet another aspect of the present
invention, there is provided a modified vaccinia
virus comprising modifications under extracellular
conditions of a wildtype vaccinia virus genome having
a single cleavage site for the bacterial restriction
endonuclease NotI, a single cleavage site for the
bacterial restriction endonuclease SmaI, and a
thymidine kinase gene, wherein said modifications
comprise: (I) a modification that eliminates said
site for the bacterial restriction endonuclease NotI;
(II) a modification that eliminates said site for the
bacterial restriction endonuclease SmaI; and (III) a
modification comprising an insertion in said
thymidine kinase gene which comprises a single
cleavage site for the bacterial restriction
endonuclease NotI and a single cleavage site for the
bacterial restriction endonuclease SmaI.
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According to a further aspect of the present
invention, there is provided a modified vacci nia
virus designated vdhr comprising a modified genome
that lacks both a functional K1L host range gene and
a functional C7L host range gene, wherein s aid
modified genome comprises the genome of a C 7L-
negative strain of vaccinia virus (WR-6/2) having the
sequence of SEQ. ID. NO. 22 inserted at the K1L to cus
of wildtype vaccinia virus in place of a K1L gene.
According to a further aspect of the present
invention, there is provided a modif zed
chordopoxvirus produced by the method described
herein, wherein said first DNA sequence is expressed
in a cultured host cell resulting in production of a
protein.
According to yet a further aspect of the present
invention, there is provided a method for producing a
protein employing a modified vaccinia viral
expression system comprising the following steps: (a)
providing a modified vaccinia virus containing a
heterologous insert encoding a protein, wherein said
insert was cloned into the viral g.enome into a unique
restriction endonuclease cleavage site; (b) infecting
a vertebrate cell culture with the modified vaccinia
virus from step (a) ; (c) culturing the infected cells
under conditions resulting in express ion of the
protein; and, (d) isolating the protein produced by
the infected cells.
According to yet a further aspect of the present
invention, there is provided a method for producing a
protein employing a modified fowlpox vira 1 expression
system comprising the following steps: (a) providing
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a modified fowlpox virus containing a heterologous
insert encoding a protein, wherein said insert was
cloned into the viral genome into a unique
restriction endonuclease cleavage site recognized by
a restriction endonuclease selected from the gz-oup
consisting of Notl, Smal, ApaI, and RsrII; (b)
infecting a vertebrate cell culture with the modified
fowlpox virus from step (a); (c) culturing the
infected cells under conditions resulting in
expression of the protein; and (d) isolating the
protein produced by the infected cells.
According to another aspect of the pre sent
invention, there is provided a method for producing a
protein employing a modified chordopoxvirus expression
system comprising the following steps: (a) providing a
modified chordopoxvirus containing a heterologous
insert encoding a protein, wherein said insert was
cloned into the viral genome into a unique
restriction endonuclease cleavage site recognized by
a restriction endonuclease selected from the group
consisting of NotI, SmaI, ApaI, and RsrII; (b)
infecting a vertebrate cell culture with the modified
chordopoxvirus from step (a); (c) culturing the
infected cells under conditions resulting in
expression of the protein; and (d) isolating the
protein produced by the infected cells.
According to still a further aspect of the
present invention, there is provided a method for
producing a protein employing a modified vaccinia
viral expression system comprising the following
steps:(a) infecting cells with modified vaccinia
virus containing a heterologous insert encoding a
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virus containing a heterologous insert encoding a
protein, wherein said insert was cloned into the
viral genome into a unique restriction endonuclease
cleavage site; (b) culturing the infected cells from
step (a) under conditions resulting in expression of
the protein.
According to still a further aspect of the
present invention, there is provided a method for
producing a protein employing a modified fow lpox
viral expression system comprising the following
steps: (a) infecting cells with a modified fowlpox
virus containing a heterologous insert encoding a
protein, wherein said insert was cloned into the
viral genome into a unique restriction endonuclease
cleavage site recognized by a restriction
endonuclease selected from the group consisting of
NotI, SmaI, ApaI, and RsrII; and,(b) culturing the
infected cells from step (a) under conditions
resulting in expression of the protein.
According to yet another aspect of the present
invention, there is provided a method for producing a
protein employing a modified chordopoxvirus
expression system comprising the following steps:(a)
infecting cells with modified chordopoxvirus
containing a heterologous insert encoding a protein,
wherein said insert was cloned into the viral genome
into a unique restriction endonuclease cleavage site;
(b) culturing the infected cells from step (a) under
conditions resulting in expression of the protein.
According to another aspect of the present
invention, there is provided use of a protein
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According to yet another aspect of the present
invention, there is provided use of a prot=.ein
produced by the method described herein for the
priming of an immune response in a vertebrate.
According to one aspect of the present
invention, there is provided use of a protein
produced by the method described herein for the
preparation of a medicament for the generation of an
immune response in a vertebrate.
According to another aspect of the present
invention, there is provided use of a protein
produced by the method described herein for the
preparation of a medicament for the priming of an
immune response in a vertebrate.
It is therefore an object of the present
invention to provide a method for construct ing
modified genomes of eukaryotic cytoplasmic viruses,
particularly of poxviruses, which overcomes the
aforementioned limitations associated with
conventional techniques based on intracellular
recombination.
It is another object of the present invention
to provide cytoplasmic virus genome-construction
techniques that produce substantially higher yields
of recombinants than existing methodology.
It is a further object of this invention to
provide methods for modifying a genome of a
cytoplasmic DNA virus by direct modification of
genomic viral DNA and intracellular packaging of the
modified viral DNA into virions with the aid of
helper virus functions.
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It is another object of this invention to
provide methods for construction of a genome of a
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cytoplasmic DNA virus that produce in one
recombination reaction step modified genomes having
a foreign DNA segment inserted in each of the two
possible orientations and modified genomes having
multiple insertions of a foreign DNA segment.
It is still another object of this invent ion
to provide modified eukaryotic cytoplasmic virus
genomes suitable for direct molecular cloning of
foreign genes in a modified cytoplasmic DNA virus
genome, comprising two portions of a genomic viral
DNA produced by cleavage with a sequence specific
endonuclease at a site that is unique in the viral
genome.
It is yet a further object of this invention
to provide a cytoplasmic virus, particularly a
poxvirus, having a modified genome comprised of a
foreign DNA inserted into a unique cleavage site
for a sequence-specific endonuclease.
It is another object of this invention to
provide plasmids which facilitate construction and
transfer of gene cassettes into a cytoplasmic
virus, particularly a poxvirus, using direct
molecular cloning.
In accomplishing these and other objects,
there has been provided, in accordance with one
aspect of the present invention, a method for
producing a modified eukaryotic cytoplasmic virus
by direct molecular cloning of a modified
eukaryotic cytoplasmic virus genome, wherein the
method comprises the steps of: (I) modifying
under extracellular conditions a first eukaryotic
cytoplasmic virus genome to produce a modified
eukaryotic cytoplasmic virus genome;
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(II) infecting a cultured host cell with a second
eukaryotic cytoplasmic virus that is heterologous
to the first eukaryotic cytoplasmic virus, the
second eukaryotic cytoplasmic virus comprising a
second viral genome which is expressed in the
cultured host cell to package said modified
eukaryotic cytoplasmic virus genome into an
infectious virion; (III) introducing the modified
eukaryotic cytoplasmic virus genome into the
cultured host cell, wherein the modified eukaryotic
cytoplasmic virus genome is packaged into an
infectious virion; and (IV) recovering from the
cultured host cell the infectious virion comprised
of the modified eukaryotic cytoplasmic virus
genome.
According to one embodiment of this method,
the step of modifying the first eukaryotic
cytoplasmic virus genome under extracellular
conditions comprises a step of cleaving the first
genome with a sequence-specific endonuclease.
According to another embodiment, the step of
modifying the first eukaryotic cytoplasmic virus
genome comprises a step of inserting a first DNA
sequence into the first genome. Advantageously,
this first DNA sequence is inserted into the first
genome at a cleavage site for sequence-specific
endonuclease. It should be noted that where a
particular sequence-specific endonuclease, such as
a bacterial restriction enzyme, is described herein
by name, that name also signifies any isoschizomer
of the named nuclease.
Optionally, the step of modifying the first
eukaryotic cytoplasmic virus genome according to
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this method also comprises a step of using a
phosphatase to remove a phosphate moiety from an
end of a DNA segment that is produced by cleav Zng
the first genome with a sequence-speci tic
endonuclease.
In some embodiments of this method, the first
genome i s a vaccinia virus genome and the unique
site is a cleavage site for the bacterial
restriction endonuclease NotI or for the bacterial
restriction endonuclease SmaI. The first genome
also may comprise a second DNA sequence not
naturally occurring in a eukaryotic cytoplas rnic
virus genome where that second DNA sequence is
comprised of the unique cleavage site. For
instance, the first genome may be a fowlpox virus
genome comprising a sequence of an Escherichia coli
i~-galactosidase gene and the unique site is a
cleavage site for the bacterial restriction
endonuclease NotI that is located in that gene.
In other forms of this method, the first DNA
sequence is inserted into the first genome between
a first cleavage site for a first sequence-
specific endonuclease and a second cleavage site
for a second sequence-specific endonuclease.
Optionally, each of the first and second cleavage
sites is unique in the first genome.
According to other embodiments of the method
of this invention, at least a portion of the first
DNA sequence which is inserted into the first
genome is under transcriptional control of a
promoter. This promoter may be located in the
first DNA sequence that is inserted into the first
genome. Alternatively, the promoter is located in
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the modified eukaryotic cytoplasmic virus genome
upstream of the first DNA sequence that is inserted
into the first genome. In some cases, the promoter
is utilized by an RNA polymerase encoded by the
modified eukaryotic cytoplasmic virus genome. This
promoter may also be suitable for initiation
transcription by an RNA polymerase of the
eukaryotic cytoplasmic virus genome to be modified.
In certain methods, the promoter comprises a
modification of a naturally occurring promoter of
the eukaryotic cytoplasmic virus.
The step of modifying the first eukaryotic
cytoplasmic virus genome according to the method of
this invention may comprise a step of deleting a
DNA sequence from the first genome. Alternatively,
this step comprises a step of substituting a DNA
sequence of the first genome.
Advantageously, the step of introducing the
modified eukaryotic cytoplasmic virus genome into
the cultured host cell is carried out about one
hour after the step of infecting the first cultured
host cell with the second eukaryotic cytoplasmic
virus genome.
In one variation of this method, the first
cultured host cell is selected such that expression
of the second viral genome in the first cultured
host cell does not produce infectious virions
comprised of the second viral genome. For
instance, where the modified eukaryotic cytoplasmic
virus genome is a modified vaccinia virus genome
and the second viral genome is a fowlpox virus
genome, the selected host cultured cell is a
mammalian cell.
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In some forms of the method of modifying a
viral genome, the step of recovering infecti~us
virions comprised of the modified eukaryo t is
cytoplasmic virus genome comprises a step of
infecting a second cultured host cell w i th
infectious virions produced by the first cultu red
host cell. This is done under conditions such t hat
expression of the second viral genome in the sec and
cultured host cell does not produce infecti ous
virions comprised of the second viral genome. For
instance, when the modified eukaryotic cytoplas rnic
virus genome is a modified vaccinia virus genorne,
the second viral genome may be a fowlpox v i rus
genome, and the second cultured host cell is a
mammalian cell. Alternatively, the modif ied
eukaryotic cytoplasmic virus genome comprise s a
functional host range gene required to produce
infectious virions in the second cultured host c ell
and the second viral genome lacks that functional
host range gene. This is illustrated by the case
where the modified eukaryotic cytoplasmic virus
genome is a modified vaccinia virus genome
comprising a functional host range gene required to
produce infectious virions in a human cell and the
second cultured host cell is a human cell.
In other forms of this method, the modified
eukaryotic cytoplasmic virus genome comprises a
selective marker gene, the second viral genflme
lacks that selective marker gene, and the step of
infecting the second cultured host cell is carried
out under conditions that select for a genome
expressing that selective marker gene.
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Advantageously, expression of the selective marker
gene in the second cultured host cell confers on
the second cultured host cell resistance at a 1 evel
sufficient to select for a genome expressing the
selective marker gene.
According to another aspect of the pre sent
invention, there is provided a modified eukaryotic
cytoplasmic virus produced by direct molecular
cloning of a modified eukaryotic cytoplasmic virus
genome according to methods summarized hereinabove .
Yet another aspect of the present invention
relates to a modified eukaryotic cytoplasmic virus
comprised of a modified eukaryotic cytoplasmic
genome, wherein the modified eukaryotic cytoplasmic
genome comprises (I) a first eukaryotic
cytoplasmic virus genome, wherein the genome is
comprised of a cleavage site for a sequence-
specific endonuclease and the cleavage site is a
unique site in the genome; and
(II) a first DNA sequence inserted into the unique
site in the genome.
According to a major embodiment of this aspect
of the invention, the first DNA sequence is not
naturally occurring in a eukaryotic cytoplasmic
virus genome. In some preferred cases, the genome
is a vaccinia virus genome and the unique site is a
cleavage site for a bacterial restriction
endonuclease selected from the group consisting of
NotI and SmaI.
The genome may comprise a second DNA sequence
not naturally occurring in a eukaryotic cytoplasmic
virus genome and that second DNA sequence is
comprised of the unique cleavage site. In one
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example, the genome is a fowlpox virus genome
comprising a second DNA sequence of an Escherichia
coli f3-galactosidase gene and the unique site in
that gene is a cleavage site for the bacte r ial
restriction endonuclease NotI.
In some modified viruses of this invention, at
least a portion of said first DNA sequence that is
inserted into the unique site is under
transcriptional control of a promoter. This
promoter is located in the first DNA sequence t hat
is inserted into the genome. In some cases the
genome is a poxvirus genome and the promoter
comprises a poxvirus promoter or a modification
thereof .
Yet another aspect of the present invention
relates to a modified eukaryotic cytoplasmic virus
comprised of a modified eukaryotic cytoplasmic
virus genome, wherein the modified eukaryotic
cytoplasmic virus genome comprises (I) a first
eukaryotic cytoplasmic virus genome, wherein the
genome is comprised of a first cleavage site for a
first sequence-specific endonuclease and a second
cleavage site for a second sequence-specific
endonuclease and each of the cleavage sites is a
unique site in the genome; and (II) a first DNA
sequence inserted into the genome between the first
unique site and the second unique site.
In some forms of this modified virus the first
DNA sequence is not naturally occurring in a
eukaryotic cytoplasmic virus genome. In some cases
the genome comprises a second DNA sequence not
naturally occurring in a eukaryotic cytoplasmic
virus genome and that second DNA sequence is
CA 02515166 1992-08-25
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comprised of the first DNA sequence inserted
between the first unique site and the second unique
site. For an example, this modified virus may
comprise a genome that is a vaccinia virus genome
and each of the first unique site and the second
unique site is a cleavage site for a bacterial
restriction endonuclease selected from the group
consisting of NotI, SmaI, ApaI and RsrII.
Yet another modified eukaryotic cytoplasmic
virus of the present invention is comprised of a
modified eukaryotic cytoplasmic virus comprised of
a modified eukaryotic cytoplasmic virus genome,
wherein the modified eukaryotic cytoplasmic virus
genome comprises (I) a first eukaryotic
cytoplasmic virus genome, wherein the genome is
comprised of a first DNA sequence and the first DNA
sequence is comprised of a cleavage site for a
sequence-specific endonuclease that is a unique
site in the modified eukaryotic cytoplasmic virus
genome; and (II) a promoter located such that a
DNA sequence inserted into the unique site is under
transcriptional control of the promoter, wherein
the first DNA sequence lacks a translation start
codon between the promoter and the unique site.
This first DNA sequence may be one that is not
naturally occurring in a eukaryotic cytoplasmic
virus genome. This modified virus is exemplified
by one in which the genome is a vaccinia virus
genome and the first DNA sequence is comprised of a
multiple cloning site comprising cleavage sites for
the bacterial restriction endonuclease NotI, SmaI,
ApaI and RsrII.
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Still another aspect of this invention relates
to a kit for direct molecular cloning of a modified
eukaryotic cytoplasmic virus genome, comprising
(I) purified DNA vector arms, produced by cleavage
of a modified eukaryotic cytoplasmic virus ge riome
at a first unique restriction site, the arms
forming a viable, modified viral genome upon
ligation; (II) a DNA ligase; and (III) buffer
solutions and reagents suitable for ligation o~ DNA
segments together to produce the modified viral
genome.
In one form, this kit further comprise s a
plasmid comprised of a gene expression cassette
flanked by sites for cleavage with a segue nce-
specific endonuclease that are compatible for
insertion of that cassette into a unique cleavage
site of the modified viral genome encoded by the
DNA vector arms. The kit may further compri se a
first host cell and a second virus suitable for
packaging of the modified viral genome into
infectious virions.
According to a further aspect, this invention
relates to a plasmid comprising a DNA segment
having a cleavage site for the bacterial
restriction endonuclease NotI at each end. In this
plasmid, this DNA segment comprises a sequence-
specific endonuclease cleavage site that is unique
CA 02515166 1992-08-25
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in the plasmid. An example of this plasmid is
designated pN2 and comprises the sequence of ~EQ.
ID. NO.l. In this plasmid the DNA segment may
further comprise a selective marker gene udder
transcriptional control of a poxvirus promoter.
For example, such plasmids include plastnids
designated pN2-gpta comprising the sequence of SEQ.
ID. NO. 2, and pN2-gptb comprising the sequenc a of
SEQ. ID. NO. 3.
Another plasmid of the invention contains a
DNA segment that further comprises a poxv irus
promoter operatively linked to a DNA sequence
comprising a restriction endonuclease cleavage
site. Thus, a DNA segment inserted into this
cleavage site is under transcriptional contro 1 of
this promoter. Examples are plasmids designated
pAl-S2 comprising the sequence of SEQ. ID. NO. 11,
and pA2-S2 comprising the sequence of SEQ. ID. NO.
12. An example of such a plasmid which further
comprises a selective marker gene under contro 1 of
a separate poxvirus promoter is plasmid pN2gp t-S4,
comprising the sequence of SEQ. ID. NO. 14.
Still another plasmid comprises a segment of a
poxvirus genome that comprises a thymidine kinase
gene of that poxvirus. This thymidine kinase gene
has been modified to prevent expression of active
thymidine kinase, as in plasmids designated pHindJ-
2 comprising the sequence of SEQ. ID. NO. 4, and
pHindJ-3 comprising the sequence of SEQ. ID. NO. 5.
Another plasmid comprises a poxvirus promoter
operatively linked to a translational start colon.
This start colon is immediately followed by a
second restriction endonuclease cleavage site
suitably arranged to permit translation of an open
reading frame inserted into that second restriction
CA 02515166 1992-08-25
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endonuclease cleavage site. Examples of this
plasmid include plasmids designated pA1-S1
comprising the sequence of SEQ. ID. NO. 9 and pA2-
S1 comprising the sequence of SEQ. ID. NO. 10, and
plasmid pN2gpt-S3A comprising the sequence of SEQ.
ID. NO. 13.
One particular plasmid of this type further
comprises a DNA sequence encoding human
prothrombin, where that DNA sequence is operatively
linked to the poxvirus promoter and a start c odon
by a plasmid designated plasmid pAlS1-PT, and
comprising the sequence SEQ. ID. NO. 15.
Another plasmid further comprises a DNA
sequence encoding human plasminogen and including a
translation start codon, where that DNA sequence is
operatively linked to the poxvirus promoter. This
is exemplified by plasmids derived from pN2gpt-S4,
such as pN2gpt-GPg, encoding human glu-plasminogen
and comprising the sequence of SEQ. ID. N0. 17, and
pN2gpt-LPg encoding lys-plasminogen and comprising
a sequence of SEQ. ID. NO. 18.
Yet another plasmid of this invention, as
above, further comprises a DNA sequence encoding
human immunodeficiency virus (HIV) gp160, including
a translation start codon, operatively linked to
the poxvirus promoter by plasmid pN2gpt-gp160
comprising the sequence SEQ. ID. NO. 19. Finally,
another plasmid comprises a DNA sequence encoding
human von Willebrand factor, an example being
designated plasmid pvWF, comprising the sequence of
SEQ. ID. NO. 20.
Some plasmids of this invention comprise a
sequence-specific endonuclease cleavage site that
is unique in the genome of the poxvirus. Examples
CA 02515166 1992-08-25
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include pA0 comprising the sequence of SEQ. ID. NO.
6, pAl comprising the sequence of SEQ. ID. NO _
and pA2 comprising the sequence of SEQ. ID. NO-_ g, ,
Another plasmid comprises a modified EcoRI K
fragment of vaccinia virus DNA from which the K1L
host range gene is deleted. Two examples are
pEcoK-dhr comprising the sequence of SEQ. ID. NO.
21, and pdhr-gpt comprising the sequence of SEQ.
ID. NO. 22.
The invention also provides for a pla smid
comprising a segment of a poxvirus genome that
comprises a thymidine kinase gene of the poxvi rus,
wherein the thymidine kinase gene has been modified
to prevent expression of active thymidine ki nase
and to a plasmid selected from the group of
plasmids: pHindJ-2 comprising the sequence of SEQ.
ID. NO. 4, and pHingJ-3 comprising the sequenc a of
SEQ. ID. NO. 5.
In another aspect, the invention provides a
modified vaccinia virus comprising a modification
under extracellular conditions of a wildtype
vaccinia virus genome having a single cleavage site
for the bacterial restriction endonuclease NotI,
wherein the modification eliminates the single
cleavage site and in one example is designated vdN.
The invention further comprises a modification
under extracellular conditions of a wildtype
vaccinia virus genome having a single cleavage site
for the bacterial restriction endonuclease SmaI,
wherein the modification eliminates the single
cleavage site and in one example is designated
vdSN.
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In another aspect, the invention relates t o a
modified vaccinia virus comprising modifications
under extracellular conditions of a wildtype
vaccinia virus genome having a single cleavage site
for the bacterial restriction endonuclease Not=, a
single cleavage site for the bacterial restriction
endonuclease SmaI, and a thymidine kinase gene,
wherein the modifications comprise:(I) a
modification that. eliminates said site for the
bacterial restriction endonuclease NotI; (II) a
modification that eliminates the site for the
bacterial restriction endonuclease SmaI; and
(III) a modification comprising an insertion in
the thymidine kinase gene which comprises a single
cleavage site for the bacterial restriction
endonuclease NotI and a single cleavage site for
the bacterial restriction endonuclease SmaI and in
one example, the virus is designated vdTK.
In other embodiments, the invention relates to
a modified vaccinia virus, designated vPTl, further
comprising a DNA sequence encoding human
prothrombin, wherein the sequence comprises that
portion of plasmid pAlS1-PT comprising the sequence
of SEQ. ID. NO. 15 which lies between cleavage
sites for endonucleases NotI and RsrII, wherein
further the sequence is inserted into said vdTK
between cleavage sites for endonucleases NotI and
RsrII, to a modified vaccinia virus designated
vGPgl, further comprising a DNA sequence comprising
that portion of plasmid pN2gpt-GPg encoding human
gluu-plasminogen and comprising the sequence of
SEQ. ID. NO. 17 which lies between two cleavage
sites for endonucleases NotI, wherein further the
sequence is inserted into said vdTK in a cleavage
CA 02515166 1992-08-25
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site for endonuclease NotI, to a modified vacc inia
virus designated vLPgl, further comprising that
portion of plasmid pN2gpt-LPg encoding lys-
plasminogen and comprising a sequence of SEQ. ID.
NO. 18 which lies between two cleavage sites for
endonucleases NotI, wherein further the sequence is
inserted into said vdTK in a cleavage site for
endonuclease NotI, to a modified vaccinia virus
further comprising a DNA sequence encoding human
immunodeficiency virus (HIV) gp160, wherein said
sequence comprises that portion of plasmid pN2 gpt-
gp160 comprising the sequence of SEQ. ID. NO. 19
which lies between cleavage sites for endonucleases
NotI which lies between cleavage sites for
endonucleases Notl, wherein further the sequence is
inserted into said vdTK in a cleavage site for
endonuclease NotI, to a modified vaccinia virus
designated vS4, further comprising a DNA sequence
encoding a synthetic poxvirus promoter S4, wherein
said the sequence comprises the sequence of
oligonucleotide P-artP(11) of SEQ. ID. N0. 38,
wherein further said sequence is inserted into said
vdTK in a cleavage site for endonuclease NotI and
to a modified vaccinia virus designated vvWF,
further comprising a DNA sequence encoding human
von Willebrand factor, wherein the DNA sequence
comprises that portion of plasmid pvWF comprising
the sequence of SEQ. ID. NO. 20 which lies between
cleavage site for endonuclease NotI, wherein
further said sequence is inserted into said vS4 at
a cleavage site for endonuclease NotI.
The invention also relates to a modified ,
vaccinia virus designated vdhr comprising a
modified genome that lacks both a functional K1L
CA 02515166 1992-08-25
14f
host range gene and a functional C7L host range
gene, wherein the modified genome comprises the
genome of a C7L-negative strain of vaccinia virus
(WR-6/2) having the sequence of SEQ. ID. N0. 22
inserted at the K1L locus of wildtype vaccinia
virus in place of a K1L gene and to a modified
eukaryotic cytoplasmic virus produced by direct
molecular cloning of a modified eukaryotic
cytoplasmic virus genome according to the method of
the invention wherein the first DNA sequence is
expressed in a cultured host cell resulting in
production of a protein.
In another aspect, the invention relates to a
modified eukaryotic cytoplasmic virus produced by
direct molecular cloning of a modified eukaryotic
cytoplasmic virus genome according to the invention
wherein the modified eukaryotic cytoplasmic virus
genome is a modified poxvirus genome.
In a particular embodiment, the invention
relates to a modified vaccinia virus further
comprising a DNA sequence encoding human
immunodeficiency virus (HIV) gp160, wherein the
sequence comprises that portion of plasmid pN2gpt
gp160 comprising the sequence of SEQ. ID. No. 69
which lies between cleavage sites for the
endonuclease SmaI, wherein further the sequence is
inserted into the vdTK in a cleavage site for the
endonuclease Smal.
In another aspect, the invention relates to a
method for producing a protein employing a modified
eukaryotic cytoplasmic viral expression system
comprising Lne roiiowlng seeps:
CA 02515166 1992-08-25
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(a) providing a modified eukaryotic cytoplasmd.c
virus containing a heterologous insert encoding a
protein, wherein said insert was molecularly clop ed
directly into the viral genome into a unique
restriction endonuclease cleavage site;
(b) infecting a vertebrate cell culture with t he
modified eukaryotic cytoplasmic virus from step (a);
(c) culturing the infected cells under conditions
resulting in expression of the protein; and,
(d) isolating the protein produced by the infect ed
cells.
In another aspect, the invention relates to a
method for producing a protein employing a modified
eukaryotic cytoplasmic viral expression system
comprising the following steps:
(a) infecting cells with modified eukaryotic
cytoplasmic virus containing a heterologous ins ert
encoding a protein, wherein said insert was
molecularly cloned directly into the viral genome
into a unique restriction endonuclease cleavage
site;
(b) culturing the infected cells from step (a)
under conditions resulting in expression of the
protein.
In another aspect, the invention relates to a
method for generating an immune response in a
vertebrate against a heterologous protein comprising
the following steps:
(a) providing a modified eukaryotic cytoplasmic
virus containing a heterologous insert encoding an
immunogenic protein, wherein said insert was
molecularly clonea airecLiy -~nLC ire viral geuc<iie
CA 02515166 1992-08-25
14h
into a unique restriction endonuclease cleavage
site;
(b) administering the modified eukaryot_.ic
cytoplasmic virus to the vertebrate in an amount
sufficient to generate the immune response.
In another aspect, the invention relates to a
method for priming an immune response in a
vertebrate comprising the following steps:
(a) providing a modified eukaryotic cytoplas rnic
virus containing a heterologous insert encoding an
immunogenic protein, wherein said insert was
molecularly cloned directly into the viral genome
into a unique restriction endonuclease cleavage
site;
(b) administering the modified eukaryotic
cytoplasmic virus to the vertebrate in an amount
sufficient to prime the immune response.
Embodiments of the above-mentioned methods may
utilize as the eukaryotic cytoplasmic virus, for
example, vaccinia or fowlpox virus as well as
others.
Other objects, features and advantages of the
present invention will become apparent from the
following detailed description. It should be
understood, however,
CA 02515166 1992-08-25
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that the detailed description and the specif is examp les,
while indicating preferred embodiments of the invent ion,
are given by way of illustration only, since various
changes and modifications within the spirit and scope of
the invention will become apparent to those skills d in
the art from this detailed description.
Brief Description of the Drawings
Figure 1.1 illustrates expression of marker genes by
modified genomes of poxviruses produced by reactivation
of naked poxvirus DNA. A silver-stained polyacryl amide
gel of proteins produced in culture supernatants of cells
infected with packaged viruses (vpPg#1-vpPg#8) and with
wildtype (WT) virus controls is shown. The upper arrow
points to plasminogen marker band, the lower arrow, to
the band of major secreted 35 K vaccinia marker protein.
Lanes 1 and 9, marker proteins; lanes 2 and 10, human
plasminogen standard (10 ng); lane 3, vac cinia
recombinant vPgD (source of packaged DNA); lanes 4-7 and
il-14, vpPg#1-8; lanes 8 and 15, wildtype vaccinia (WR
WT) .
Figure 1.2 is a schematic diagram illustrating direct
molecular cloning of poxvirus genomes comprised of a gene
cassette for expression of a marker gene (the E. coli gpt
gene) under control of a vaccinia virus promoter.
Figure 1.3 is a schematic illustration of
construction of plasmids (pN2-gpta and pN2-gptb) which
are precursors for construction of gene expression
cassettes by insertion of a promoter and an open reading
frame. Such cassettes are designed for direct molecular
transfer into vaccinia virus vectors using a unique
insertion site and a selectable marker gene (gpt) driven
by a vaccinia virus promoter. MCS - multiple cloning
site. P7.5 - promoter for vaccinia 7.5K polypeptide
gene; P11 = promoter for vaccinia 11K polypeptide gene.
CA 02515166 1992-08-25
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Arrows indicate the directions of transcription frog the
promoters.
Figure 1.4 demonstrates that poxvirus genomes
produced by direct molecular cloning contain the gpt
marker gene cassette inserted at a unique (NotI) cleavage
site, as shown by Southern blot analyses of plaque-
purified viral DNAs digested with the HindIII
endonuclease using a gpt-gene probe. Lane 1, marker DNAs
(HindIII digested phage ~ DNA); lanes 2 and 3, wildtype
vaccinia virus (WR) DNA cut with HindIII (500 and 10 O ng,
respectively); lanes 4-9, DNAs of cells infected with
plaques designated 2.1.1 through 7.1.1; lanes 10-12, DNAs
of cells infected with plaques 10.1.1-12.1.1. Arrows
indicate sizes of the restriction fragments of the marker
in kilobasepairs.
Figure 1.5. further illustrates structure s of
modified poxvirus DNAs using Southern blots of NotI-
digested DNAs of cells infected with various isolates and
hybridized with a gpt-gene probe. Lane 1, marker DNAs
(HindIII digested phage ~ DNA) ; lane 2, vaccinia wildtype
(WT) DNA cut with NotI (50 ng); lanes 3-8, DNAs of cells
infected with recombinant plaques designated 2.1.1
through 7.1.1; lanes 9-11, DNAs of celis infected with
plaques 10.1.1-12.1.1.
Figure 1.6 shows a comparison of DNAs from wildtype
(WT) vaccinia and a modified clone (vp7) using ethidium
bromide staining of DNA fragments cleaved with indicated
restriction endonucleases and separated on an agarose
gel. Lanes 1 and 2, NotI digests of WT and vp7; lanes 3
and 4, HindIII digests of WT and vp7; lanes 5 and 6,
HindIII and NotI combined digests of WT and vp7; lanes 7
and 9, PstI digests of WT and vp7; lanes 9 and 10, PstI
and NotI combined digests of WT and vp7 ; lanes 11 and
12, SalI digests of WT and vp7; lane 13, marker DNAs
(ligated and HindIII digested phage ~ DNA; and phage ~X
cut with HaeIII). Arrows on the left indicate sizes of
fragments (in kilobasepairs) of NotI digest of vaccinia
CA 02515166 1992-08-25
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WT; arrows on right, markers. Note that lanes 1 and 2
contain about tenfold less DNA than the other lanes .
Figure 1.7 illustrates a Southern blot of the gel
shown in Figure 1.6 using a gpt-gene probe. Az-rows
indicate marker sizes.
Figure 1.8 presents Southern blot analyse 8 of
vaccinia virus DNAs from infected cells digested with
NotI and hybridized to a vaccinia virus probe. Lanes 1-
4, DNAs of cells infected with plaques designated A1-A4;
lanes 5-8, plaques C1-C4; lanes 9-12, plaques E1-E4; lane
13, vaccinia WT DNA; lane 14, DNA of uninfected CV-1 host
cells; lane 15, marker DNAs (HindIII digested pha~ge
DNA; and phage ~X cut with HaeIII).
Figure 1.9 shows a Southern blot of the same samples
as in the gel shown in Figure 1.8 using a gpt-gene probe.
Lanes 1-12 as in Figure 1.8; lane 13, DNA of uninfected
CV-1 host cells; lane 14, vaccinia WT DNA; lane 15,
marker DNAs (HindIII digested phage ~ DNA; and phage ~X
cut with HaeIII).
Figure 1.10 shows a Southern blot of the same viral
DNAs as in the gel in Figure 1.8, restricted with PstI,
using a gpt-gene probe. Lanes 1-12 as in Figure 1.8;
lane 13, DNA of uninfected CV-1 host cells; lane 14,
vaccinia WT DNA; lane 15, marker DNAs (HindIII digested
phage ~ DNA; and phage ~X cut with HaeIII).
Figure 1.11 outlines a schematic of the predicted
structure of the modified PstI "C" fragments of vaccinia
virus DNAs with single or double insertions of the gpt-
gene cassette. P=PstI and N=NotI cleavage sites. The
numbers indicates sizes of respective PstI fragments;
bold type numbers indicate fragments expected to
hybridize with a gpt-gene probe. Arrows indicate
direction of transcription of the gpt-gene (800 bp) by
the vaccinia virus promoter (300 bp).
Figure 2.1 presents analyses of recombinant avipox
(fowlpox, FP) genomes by digestion with the restriction
endonuclease NotI and separation by FIGE on a 1% agarose
gel. Lane 5, marker (phage ~ HindIII fragments, uncut
CA 02515166 1992-08-25
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phage ~ and vaccinia WR); lanes 1 and 2, fowlpox virus
HP1.441 DNA, uncut and cut with NotI; lanes 3 arid 4,
recombinant fowlpox virus f-TK2a DNA, uncut and cut with
NotI.
Figure 2.2 illustrates construction of fowlpox
viruses expressing foreign genes by direct mole cular
cloning. A gene expression cassette, consisting o f the
E. coli gpt gene controlled by a poxvirus promoter ( p) is
ligated with the right and left DNA arms (ra and la,
respectively) of fowlpox virus (f-TK2a) obtained by
cleavage with NotI. Packaging is performed by fowlpox
helper virus (strain HP2) in chicken embryo fibroblasts.
Figure 3.1 illustrates a process for construction of
modified poxviruses by extracellular genome engine Bring
and intracellular packaging. A gene cassette cons i sting
of the gpt gene controlled by a vaccinia virus promoter,
is ligated with the "right arm" (ra) and the "lef t arrn"
(la) of vaccinia virus DNA cleaved at a unique sit a with
the endonuclease SmaI. Packaging is done by the fowlpox
helper virus (strain HP1..441) in chicken embryo
fibroblasts. P1 - promoter of the vaccinia virus gene
coding for the 7.5 kDA polypeptide.
Figure 3.2 demonstrates that engineered vaccinia
virus genomes packaged by fowlpox helper virus contain
the expected insert at a unique SmaI cleavage site, as
determined by Southern blot analyses . Total DNA isolated
from infected cells was digested with HindIII, and the
blot was hybridized with a gpt-gene probe. Lanes 1-8,
DNAs from cells infected with plaques designated F12.2-
F12.9; lanes 9-13, plaques F13.1-F13.5; lanes 14 and 15,
HindIII-digested DNA isolated from uninfected cells and
cells infected with vaccinia (WR wildtype) virus,
respectively; lane 16, markers (HindIII- digested phage
~ DNA). The DNA in lane 8 does not hybridize because the
virus isolate #F12.9 did not replicate.
Figure 3.3 presents a schematic outline of the
expected structures of modified vaccinia virus genomes
having a gene cassette inserted into a unique SmaI site,
CA 02515166 1992-08-25
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particularly the modified HindIII "A" fragment s of
viruses with single and double insertions. H = Hi ndIII
and S - SmaI restriction endonuclease cleavage s ites.
Numbers indicate sizes of the HindIII fragments, with
those in bold type indicating fragments expect ed to
hybridize with a gpt-gene probe. The gpt gene cas sette
consists of a vaccinia virus promoter (about 300 by in
size) separated by an internal HindIII site from the gpt
sequences (about 800 bp). Arrows indicate the dire ~tion
of transcription of the gpt-gene.
Figure 4.1 shows a schematic plan for the
construction of vaccinia virus vector vdTK having a
modified thymidine kinase (tk) gene. WR-WT = wil dtype
(WT) western Reserve (WR) strain of vaccinia virus (W).
Panel A shows a method using only direct mol a cular
modification of the vaccinia virus genome, including
deletion of undesired NotI and SmaI sites. Panel B
outlines an alternative approach for deletion of a NotI
site using marker rescue techniques with vaccinia virus
and a modified plasmid. Panel C shows an alternative
method for deleting the SmaI site by marker rescue.
Figure 4.2 illustrates construction of the vaccinia
virus vector (vdTK) having the thymidine kinase (tk) gene
replaced with a multiple cloning site. The arrow
indicates the initiation and direction of transcription
of the vaccinia virus tk gene (W-tk) in the HindIII J
fragment cloned in plasmid pHindJ-1. The tk gene was
replaced, as shown, and the final plasmid pHindJ-3 was
used to insert the modified HindIII J fragment into
vaccinia virus.
Figure 4.3 outlines construction of plasmids (pAl and
pA2) which are precursors for construction of gene
expression cassettes by insertion of a promoter and an
open reading frame. Such cassettes are suitable for
direct molecular transfer into vaccinia virus vector vdTK
using directional (forced) cloning.
Figure 4.4 illustrates construction of plasmids (pAl-
S1 and pA2-S1) comprised of gene expression cassettes
CA 02515166 1992-08-25
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suitable for association of open reading frames with a
synthetic poxvirus promoter (S1) and a translation start
codon. The cassettes are designed for direct mole cular
transfer into vaccinia virus vector vdTK by forced
cloning. The S1 promoter is present in different
orientations in the two plasmids, as indicated by the
arrows showing the directions of transcription.
Figure 4.5 outlines the construction of plasmids
(pAl-S2 and pA2-S2) comprised of gene expression
cassettes suitable for association of open reading frames
already having a translation start codon with a synthetic
poxvirus promoter (S2), prior to direct molecular
transfer into vaccinia virus vector vdTK by forced
cloning. The S2 promoter is present in different
orientations in the two plasmids, as indicated by the
arrows showing the directions of transcription.
Figure 4.6 shows the construction of plasmids pN2-
gpta and pN2-gptb.
Figure 4.7 shows construction of plasmids (pN2gpt-S3A
and pN2gpt-S4) comprised of gene expression cassettes
suitable for association of an open reading frame, either
lacking (S3A) or having (S4) a translation start codon,
with a synthetic promoter (S3A or S4, respectively),
prior to direct molecular transfer into a unique site in
vaccinia virus vector vdTK. Abbreviations as in Figure
1.3.
Figure 5.1 illustrates construction of a gene
expression cassette plasmid (pAlS1-PT) for expression of
human prothrombin in vaccinia virus vector vdTK.
Abbreviations as in Figure 1.3. Arrows indicate the
direction of transcription.
Figure 5.2 presents construction of a gene expression
cassette plasmid (pN2gpt-GPg) for expression of human
glu-plasminogen in vaccinia virus vector vdTK. S4 -
synthetic poxvirus promoter; other abbreviations as in
Figure 1.3.
Figure 5.3 shows construction of a gene expression
cassette plasmid (pN2gpt-LPg) for expression of human
CA 02515166 1992-08-25
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lys-plasminogen in vaccinia virus vector vdTK.
Abbreviations as in Figure 1.3.
Figure 5.4 outlines construction of a gene expression
cassette plasmid (pN2gpt-gp160) for expression of a human
virus antigen (HIV gp160) in vaccinia virus vector vdTK.
Abbreviations as in Figure 1.3.
Figure 5.5 illustrates an approach for screen ing of
modified vaccinia viruses made by direct molecular
cloning based on concurrent insertion of a marke r, gene
(the E. coli lacZ gene) which confers a vi aually
distinctive phenotype ("blue" plaque compared to normal
"white" plaques of viruses lacking a lacZ gene).
Figure 5.6 illustrates the construction of plasmida
pTZS4-lacZa and pTZS4-lacZb.
Figure 6.1 illustrates construction of a vaccinia
virus vector (vS4) with a directional master cloning site
under control of a strong late vaccinia virus promoter
(S4) .
Figure 6.2 presents construction of a modified
vaccinia virus (wwF) for expression of von-Willebrand
factor by direct molecular insertion of an open reading
frame into vaccinia virus vector vS4. vWF ~ von
Willebrand factor cDNA. The arrow indicates the
direction of transcription from the S4 promoter.
Figure 7.1 illustrates the effect of amount of added
DNA on packaging of vaccinia virus DNA by fowlpox helper
virus in mammalian (CV-1) cells in which fowlpox virus
does not completely replicate. Five cultures were
infected with fowlpox virus and subsequently transfected
with the indicated amounts of vaccinia virus DNA. The
first column indicates a culture with no added DNA and no
fowlpox virus, and the fifth column, no added DNA but
infected with fowlpox virus.
Figure 8.1 outlines construction of a vaccinia virus
(vdhr) suitable for use as a helper virus having host
range mutations which prevent replication in some human
cell lines. hr-gene - host range gene located in the
CA 02515166 1992-08-25
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EcoRI K fragment of vaccinia virus; other abbreviations
as in Figure 1.3.
Figure 9.1 shows the construction of the pl asmids
pS2gpt-P2 and pP2gp160NIN. Arrows within plasmids show
the direction of transcription of the respective genes.
Figure 9.2 shows the schematic outline of the
construction of the viruses vP2-gp160NIN-A and vP2-
gp160MN-H.
Figure 9.3 shows maps of the PstI-E-fragment of the
wild-type vaccinia virus and of the PstI-fragments of the
chimeric viruses comprising gp160 genes. Arrows indicate
the direction of transcription of the gp160 gene.
Numbers indicate sizes of fragments in kilo-base pairs.
Figure 9.4 shows construction of the plasmid pselP
gpt-L2. Arrows indicate the direction of transcription
of the respective genes.
Figure 9.5 A) shows construction of the plasmid
pselP-gp160MN and Figure 9.5 H) shows sequences around
translational start codons of wild-type (SEQ ID N0:73)
and modified gp160-genes (SEQ ID N0:75).
Figure 9.6 is a schematic outline of construction of
the chimeric vaccinia viruses vselP-gp160MNA and vP2-
gp160MNB. Arrows indicate the direction of transcription
of the gp160-gene.
Figure 9.7 is a map of the SaII-F-fragment of the
wild-type vaccinia virus and of SaII-fragments of
chimeric vaccinia viruses vselP-gp160NINA and vP2-
gp160NINH. Arrows indicate the direction of transcription
of the gp160-gene. Numbers indicate sizes of the
fragments in kilo-base pairs.
Figure 10.1 A) shows the structure of plasmid pN2-
gptaProtS. The double gene cassette consisting of the
gpt gene controlled by the vaccinia P7.5 promoter (P7.5)
and the human Protein S gene (huProtS) controlled by a
synthetic poxvirus promoter (self) is flanked by NotI
restriction sites. Figure 10.1 H) shows sequences around
translational start codons of wild-type Protein S gene
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(SEQ ID N0:77) and the Protein S gene in the chimeras
(SEQ ID N0:79).
Figure 10.2 shows Southern blot analysis of chimeric
vaccinia viruses carrying the Protein S gene. A) Total
cellular DNAs digested with SacI and hybridized with
vaccinia wild-type SacI fragment. H) The same material
digested with NotI and probed with human protein S
sequences. C) schematic outline of the wild-type SacI-I
fragment and the chimeric SacI-fragment after ligation of
the insert.
Figure 10.3 shows western blot analysis of plasma-
derived Protein S (pdProtS; lanes 1 and 2) and of
recombinant Protein S (rProtS). Cell culture
supernatants (10 ~,1) of SK Hepl cells were assayed after
incubation periods of 24-72 h.
Figure 11.1 shows the structure of plasmid pN2gpta-
FIX. The double gene cassette consisting of the gpt gene
controlled by the vaccinia P7.5 promoter (P7.5) and the
human factor IX gene controlled by a synthetic poxvirus
promoter (self) is flanked by NotI restriction sites.
Figure 11.2 shows Southern blot analysis of the
chimeric viruses carrying a gene for human factor IX.
Panel A) Total cellular DNAs digested with SfuI and
hybridized with the human factor IX gene probe (plasmid
pHluescript-FIX). In all eight isolates (#1-6, 9 and 10)
the insert had the 'a'-orientation; m = marker; W-WT/WR
- vaccinia wild-type, WR-strain. Panel B) Predicted
genomic structures of the chimeric viruses.
Figure 11.3 shows Western blot analysis of plasma
derived factor IX (pdFIX; lanes 1 and 2) and of
recombinant factor IX expressed by chimeric vaccinia
virus in Vero cells. Cell culture supernatants (10 ~1)
were assayed after incubation for 72 h. #1-6, 9 and 10
- numbers of plaque isolates; pd FIX - plasma-derived
factor IX.
Figure 12.1 illustrates construction of the chimeric
fowlpox virus f-envIIIB by direct molecular cloning of
and HIVIIIB env gene.
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Figure 12.2 A) shows Southern blots of SspI-fragments
of chimeric fowlpox virus isolates showing orientations
of env gene inserts. Lanes 1-12, viral isolates f- LFa-1;
lane 13 and 14, HP1,441 and f-TK2a (negative controls);
lane 15, SspI digest (10 ng) of pN2gpt-gp160 (positive
control). Panel H) shows restriction maps of SspI
fragments of inserts in the two possible orientat ions in
the chimeric fowlpox virus. Numbers indicate s i zes of
SspI-fragments in kilo-base pairs. Arrows indicate
orientations of the insert which coincide with direction
of transcription of the gp160 transcription unit.
Figure 12.3 shows expression of HIV envelope
glycoproteins in chicken embryo fibroblasts (Western
blots). Lanes 1-8, viral isolates f-LF2a-h; lane s 9 and
15, gp160 standard (provided by A. Mitterer, Immuno Ag,
Orth/Donau, Austria); lanes 10-13, viral isolates f-lF2i-
1; lane 14, marker proteins; lanes 16 and 17, fowlpox
viruses HP1.441 and f-TK2a (negative controls).
Figure 12.4 shows detection of HIV gp41 produced by
chimeric vaccinia viruses in infected chicken embryo
fibroblasts (Western blots). Lanes 1-8, viral isolates
f-LF2a-h; lanes 9 and 15, gp160 standard; lanes 10-13,
viral isolates f-LF2i-1; lane 14, marker proteins; lanes
16 and 17, fowlpox viruses HP1.441 and f-TK2a (negative
controls).
Detailed Description of Preferred Embodiments
The present invention represents the first
construction of a modified genome of a eukaryotic
cytoplasmic DNA virus, as exemplified by a poxvirus,
completely outside the confines of a living cell. This
construction was accomplished using an isolated viral
genomic DNA that was cleaved by a sequence-specific
endonuclease and then religated with foreign DNA. The
resulting modified DNA was then packaged into infectious
poxvirus virions by transfection into a host cell
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infected with another poxvirus that served as a helper
virus.
The present invention enables diverse strategies for
vector development from eukaryotic cytoplasmi c DNA
viruses which have been applied previously to other DNA
viruses to solve various genetic engineering problems.
For instance, this direct cloning approach offers the
possibility of cloning genes directly in cytoplasmic DNA
viruses, such as poxviruses, that cannot be cloned in
bacterial systems, either because they are too large for
bacterial vectors or are toxic to bacteria or are
unstable in bacteria. Direct molecular cloning allows
greater precision over construction of engineered viral
genomes and under optimum conditions can increase the
speed of cloning as well as produce a variety of
constructs in a single ligation reaction, having multiple
inserts in various orientations, which permits rapid
screening for arrangements affording optimal expression
of a foreign gene.
As used in the present context, "eukaryotic
cytoplasmic DNA virus" includes iridoviruses and
poxviruses. "Iridovirus" includes any virus that is
classified as a member of the family Iridoviridae, as
exemplified by the African swine fever virus as well as
certain amphibian and insect viruses. "Poxvirus"
includes any member of the family Poxviridae, including
the subfamililes Chordopoxviridae (vertebrate poxviruses)
and Entomopoxviridae (insect poxviruses). See, for
example, H. Moss in B.N. FIELDS, D.M. KNIPE ET AL.
VIROLOGY 2080 (Raven Press, 1990). The chordopoxviruses
comprise, inter alia, the following genera from which
particular examples are discussed herein, as indicated in
parentheses: Orthopoxvirus (vaccinia); Avipoxvirus
( f owlpox) ; Capripoxvirus ( sheeppox) Leporipoxvirus
(rabbit (Shope) fibroma, myxoma); and Suipoxvirus
(swinepox). The entomopoxviruses comprise three genera:
A, B and C.
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According to one aspect of the present invent ion, a
method is provided for producing a modified eukaryotic
cytoplasmic DNA virus by direct molecular cloning of a
modified cytoplasmic DNA virus genome. This method
comprises a step of modifying under extracellular
conditions a purified DNA molecule comprising a first
cytoplasmic DNA virus genome to produce a modified DNA
molecule comprising a modified cytoplasmic DNA virus
genome.
A purified DNA molecule suitable for modification
according to the present method is prepared, for example,
by isolation of genomic DNA from virus particles,
according to standard methods for isolation of genomic
DNA from eukaryotic cytoplasmic DNA viruses. See, for
instance, Example 1, hereinbelow. Alternatively, some or
all of the purified DNA molecule may be prepared by
molecular cloning or chemical synthesis.
Modifying a purified DNA molecule comprising a virus
genome within the scope of the present invention includes
making any heritable change in the DNA sequence of that
genome. Such changes include, for example, inserting a
DNA sequence into that genome, deleting a DNA sequence
from that genome, or substitution of a DNA sequence in
that genome with a different DNA sequence. The DNA
sequence that is inserted, deleted or substituted is
comprised of a single DNA base pair or more than one DNA
base pair.
According to this aspect of the invention, the step
of modifying a DNA molecule comprising a first DNA virus
genome is performed with any technique that is suitable
for extracellularly modifying the sequence of a DNA
molecule. For instance, modifying a DNA molecule
according to the present invention comprehends modifying
the purified DNA molecule with a physical mutagen, such
as ultraviolet light, or with a chemical mutagen.
Numerous methods of extracellular mutagenesis of purified
DNA molecules are well known in the field of genetic
engineering.
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In another embodiment, the step of modifying the DNA
molecule comprises joining together DNA segments to form
the modified DNA molecule which comprises the modified
viral genome. According to one aspect of this
embodiment, some or all of the DNA segments joined
together to form the modified DNA molecule are produced
by cleaving the DNA molecule comprising the first virus
genome with a nuclease, preferably a sequence-specific
endonuclease. Alternatively, some or all of the DNA
segments joined together to form the modified DNA
molecule may be produced by chemical synthesis using well
known methods.
In some embodiments, the step of joining together DNA
segments to produce the modified DNA molecule comprises
an extracellular step of legating those DNA segments
together using a ligase, such as a bacterial or
bacteriophage ligase, according to widely known
recombinant DNA methods. Optionally, this DNA
modification step also comprises treating ends of DNA
segments cleaved from the DNA molecule comprising the
first virus genome with a phosphatase, for instance, calf
intestine phosphatase. This enzyme removes phosphate
moieties and thereby prevents relegation of one DNA
segment produced by cleaving the DNA molecule with
another such segment.
In an alternative approach to joining the DNA
segments, some or all of the DNA segments are joined by
extracellular annealing of cohesive ends that are
sufficiently long to enable transfection of the modified
DNA molecule into a host cell where legation of the
annealed DNA segments occurs.
In another embodiment of this method, the step of
modifying the DNA molecule comprising the first virus
genome includes a step of joining at least some DNA
segments resulting from cleaving a genomic DNA molecule
of the first virus together with an additional DNA
segment to produce the modified DNA molecule. In a
preferred embodiment of this aspect of the invention,
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this step comprises cleaving a genomic viral DNA molecule
with a sequence-specific endonuclease at a unique
cleavage site in the first 'virus genome, thereby
producing two DNA "arms" of the genomic virus DNA. The
two arms are then ligated together with a foreign DNA
comprising a sequence of interest.
A DNA sequence of interest as used herein to describe
the sequence of a foreign DNA segment that is ligated
with virus DNA arms comprises, in the first instance, a
DNA sequence that is not naturally occurring in a genome
of a eukaryotic cytoplasmic DNA virus. Alternatively, a
DNA sequence of interest comprises a sequence comprised
of a sequence that is naturally occurring in a genome of
a eukaryotic cytoplasmic DNA virus as well as a sequence
that is not naturally occurring in such a genome.
Furthermore, a sequence of interest may comprise only
sequences that are naturally occurring in a eukaryotic
cytoplasmic DNA virus, where such a sequence is inserted
into a location in the genome of that cytoplasmic DNA
virus different from the location where that sequence
naturally occurs. Moreover, insertion of a naturally
occurring viral sequence of interest from one DNA virus
into another, or from one part of a single viral genome
into another part of that genome, will necessarily create
a sequence that is "not naturally occurring in the genome
of a cytoplasmic DNA virus" according to the present
invention, at the junction of the viral genome and the
inserted viral sequence of interest.
The foreign DNA segment that is ligated to the two
arms of genomic virus DNA comprises ends that are
compatible for ligation with the ends of the viral DNA
arms. The compatible ends may be complementary cohesive
ends or blunt ends. The ligation step in this particular
method produces a modified DNA molecule comprising the
first virus genome with the DNA sequence of the foreign
DNA inserted into the first virus genome at the unique
cleavage site.
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This embodiment of a method in which a DNA sequence
is inserted into the genome of the first virus is
exemplified herein by, inter alia, a method for inserting
a gene expression cassette into a vaccinia virus genome
at a unique cleavage site for the bacterial restriction
endonuclease NotI or SmaI, as described in Examples 1 and
3, respectively. This embodiment is also exemplified by
insertion of a gene cassette into the genome of a
recombinant fowlpox virus vector, at a unique NotI, site
within the sequence of a bacterial gene within the
recombinant fowlpox virus genome, as described in Example
2.
Inserting a foreign DNA into a unique site in a
eukaryotic cytoplasmic DNA virus genome according to the
present invention is useful for the purpose of expressing
a desired protein, particularly a human protein. For
instance, Example 5 describes insertion of genes for
plasminogen, prothrombin and human immunodeficiency virus
glycoprotein 160 (HIV gp160) into a unique cleavage site
of a vaccinia virus vector and the use of the resulting
modified vaccinia viruses for production of these
proteins. The foreign proteins may be produced in cell
cultures, for preparing purified proteins, or directly in
human or animal hosts, for immunizing the host with a
vaccine comprising a modified virus according to the
present invention.
In certain embodiments, the step of modifying a virus
genome by inserting a DNA sequence comprises introducing
or eliminating a marker gene function for distinguishing
the modified virus genome from the first virus genome.
In one such embodiment, a DNA sequence inserted into the
first virus genome comprises a selective marker gene and
the step of recovering the infectious modified poxvirus
virions produced by the first host cell comprises a step
of infecting a second host cell with those infectious
virions under conditions that select for a poxvirus
genome expressing the selective marker gene. In a
preferred embodiment of this aspect of the invention,
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expression of the selective marker gene in the second
host cell confers on the second host cell resistance to
a cytotoxic drug. This drug is'present during infection
of the second host cell at a level sufficient to select
for a poxvirus genome expressing the selective marker
gene. In this case the drug selects for a modified virus
genome having the inserted selective marker gene and
selects against any genome lacking that marker gene.
Insertion of a DNA sequence comprising a selective
marker gene for distinguishing the modified virus genome
from the first virus genome is particularly useful when
a genomic DNA molecule of the first virus has been
cleaved at a unique cleavage site and, therefore, the
resulting viral DNA arms are likely to religate without
insertion of the desired DNA sequence. This approach is
exemplified by a method for inserting a gene for the
enzyme xanthine-guanine-phosphoribosyl-transferase of
Escherichia coli (hereinafter, the "gpt" gene) into,
inter alia, a vaccinia virus genome or a fowlpox virus
genome at a unique NotI site, as described in Examples 1
and 2, respectively.
A method for eliminating a marker gene function from
the first virus genome to distinguish the modified viral
genome from the first genome is exemplified in Example 2.
This method relates to insertion of a foreign DNA
sequence into a fowlpox virus genome into a NotI site
residing in an E. coli lacZ gene coding for
galactosidase. As described in Example 2 (avipox),
insertion of a DNA sequence into this site disrupts the
IacZ coding sequence and thereby prevents production of
~-galactosidase. Expression of this enzyme produces a
"blue plaque" phenotype for a virus carrying the lacZ
gene. Accordingly, a modified viral genome carrying an
insertion of a DNA sequence in this site exhibits a white
plaque phenotype that distinguishes the modified virus
from the first virus. In other embodiments of methods
according to this invention, a functioning E. coli lacZ
gene is transferred into the vector with another gene of
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interest to serve as a marker for modified viruses
containing the desired insert.
In still other embodiments of the method of this
invention, the step of modifying a DNA molecule comprises
introducing a new cleavage site for a sequence-specific
endonuclease into the first virus genome. One example of
this embodiment comprises inserting into a existing
unique site in a first poxvirus genome a foreign DNA
comprised of a synthetic DNA "linker", as described in
Example 6. This linker comprises a "multiple cloning
site" comprised of several closely adjacent cleavage
sites that are useful for insertion of foreign DNA into
the modified poxvirus genome. Advantageously, the
cleavage sites in the multiple cloning site are not
present in the first viral genome and, therefore, are
unique in the modified viral genome.
More particularly, the step of modifying a DNA
molecule comprising a first viral genome also includes
inserting a DNA sequence between a first and a second
cleavage site for a sequence-specific endonuclease. In
one such embodiment, the first viral genome comprises a
multiple cloning site comprised of cleavage sites that
are unique in the first viral genome. According to this
method, cleaving a DNA molecule comprising a first viral
genome at two such unique sites in the multiple cloning
site produces two viral DNA arms having cohesive ends
that are not compatible for ligation with each other.
The intervening DNA segment between the two unique
cleavage sites in the multiple cloning site is removed
from the cleaved viral DNA arms, for example, by ethanol
precipitation of these arms, as described for inserting
a human prothrombin gene into a modified poxvirus vector
in Example 5.
Inserting a DNA segment into a viral genome between
two unique cleavage sites is useful for "forced" cloning
of DNA inserts having cohesive ends compatible for
ligation with each of the vector arms. In other words,
this method involving cleavage of viral DNA at two sites
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is useful for increasing the yield of viral genomes
resulting from ligation of viral DNA arms compared to
arms prepared by cleavage of viral DNA at a single site,
because the arms of this method do not have ends
compatible for ligation. This forced cloning method also
directs orientation of the DNA inserted within the
modified viral genome because only one viral DNA arm is
compatible for ligation to each end of the inserted DNA.
The forced cloning method of the present invention
is demonstrated, for example, by insertion of a gene
expression cassette comprised of a human prothrombin gene
into a multiple cloning site of a vaccinia virus vector,
as described in Example 5.
In a preferred embodiment, the intervening DNA
segment between two unique cleavage sites in the first
viral genome is not essential for replication of the
first viral genome and, therefore, neither deleting this
sequence nor replacing it with another DNA segment
prevents replication of the resulting modified genome.
Alternatively, the intervening DNA segment is replaced by
a DNA segment comprising that portion of the intervening
sequence that is essential for viral replication linked
to an additional DNA sequence that is to be inserted into
the first viral genome.
In another aspect of the present method, the step of
modifying the first viral genome comprises eliminating an
undesirable cleavage site for a sequence-specific
endonuclease. Modifications of this type can be made
repeatedly, if necessary, for example, to delete
redundant cleavage sites for the same nuclease, thereby
ultimately producing a modified viral genome having a
unique cleavage site for a particular nuclease.
Methods that are particularly suitable for
eliminating a cleavage site from a viral genome are known
in the art. These include various general site-specific
mutagenesis methods. One particular method for
eliminating an endonuclease cleavage site from a viral
genome involves extracellular treatment of genomic viral
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DNA to select for mutant genomic DNA molecules that are
resistant to cleavage by the pertinent endonuclease.
Another method for eliminating a cleavage site from
a viral genome is by ligating a cleaved viral DNA
molecule with a DNA segment, for instance, a synthetic
DNA segment, comprising an end compatible for ligation
with the cleaved viral DNA but lacking a portion of the
recognition sequence for the nuclease that cleaved the
viral DNA. In this method, the cleavage site for the
sequence-specific endonuclease that cleaves the viral DNA
comprises a nuclease recognition sequence that extends
beyond the sequences encompassed in the cohesive ends
into the sequences immediately adjacent to the cohesive
ends. The synthetic insert comprises cohesive ends
compatible for ligation with the viral DNA arms cleaved
at a single site. However, the sequence immediately
adjacent to one cohesive end of the synthetic insert
differs from the recognition sequence that is required
for cleavage by the enzyme that cleaved the viral DNA.
Therefore, ligation of this end of the synthetic DNA
segment with a viral arm does not reconstitute a
functional cleavage site for the nuclease that cleaved
the viral DNA. This method for eliminating a cleavage
site from a viral genome is exemplified in Example 4 by
insertion of a synthetic DNA segment comprising a
multiple cloning site into a unique cleavage site of a
viral genome.
To prevent inactivation of a viral genome as a result
of modification, it is evident that the modification of
a viral genome according to the present method must be
made in a region of the viral genome that is not
essential for virus multiplication in cell culture under
the conditions employed for propagation of the resulting
modified virus. DNA virus genomic regions comprising
sequences that are nonessential for multiplication in
cell culture and otherwise suitable for modification
according to the present methods include sequences
between genes (i.e., intergenic regions) and sequences of
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genes that are not required for multiplication of the
modified viral genome.
A nonessential site suitable for modifying a selected
genome of a eukaryotic cytoplasmic DNA virus according to ,
the present invention may be identified by making a
desired modification and determining whether such
modification interferes with replication of that genome
under the desired infection conditions. More in
particular, restriction enzyme cleavage sites in a viral
genome, including unique sites in that genome, are
' identified, for instance, by digestion of genomic DNA and
analysis of the resulting fragments, using procedures
widely known in the art. The genome may be disrupted by
trial insertion of a short synthetic DNA segment into a
selected target cleavage site by the direct cloning
method of the present invention. Recovery of a virus
comprised of the trial insert at the selected target site
provides a direct indication that the target site is in
a nonessential region of that genome. Alternatively, if
no useful cleavage site exists at a particular genomic
target location, such a site may be introduced using
either direct molecular cloning or conventional genome
construction based on marker rescue techniques. In this
case, successful recovery of a virus comprised of the
inserted cleavage site at the target location directly
indicates that the target location is in a nonessential
region suitable for modification according to the present
invention.
Certain nonessential genomic regions suitable for
practicing the present invention with poxviruses have
been described. See, for instance, Goebel et al.,
Virology 179: 247-266 (1990), Table 1,
In further embodiments of the method, at least a
portion of the DNA sequence which is inserted into the
first viral genome is under transcriptional control of a
promoter. In certain embodiments, this promoter is
located in the DNA sequence that is inserted into the
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first viral genome and, therefore, controls transcription
of that portion of the inserted DNA sequence downstream
from the promoter. This approach is exemplified by
insertion into a poxviral genome of a gene cassette
comprising a promoter functionally linked to an open
reading frame, as described in Examples 1 through 5.
In another preferred embodiment, the promoter
controlling transcription of the DNA sequence that is
inserted into the first viral genome is located in the
modified viral genome upstream of the inserted DNA
sequence. This approach is illustrated by insertion of
a cDNA encoding the human von Willebrand factor protein
into a multiple cloning site that is functionally linked
to an upstream promoter in a vaccinia virus vector, as
described in Example 6.
In certain embodiments, the promoter controlling the
inserted DNA sequence is recognized by an RNA polymerase
encoded by the modified viral genome. Alternatively,
this promoter might be recognized only by an RNA
polymerase encoded by another genome, for example,
another viral or cellular genome. For example, this RNA
polymerase might be a bacteriophage T7 polymerase that is
encoded by another cytoplasmic DNA virus genome or by the
genome of a modified host cell. The T7 polymerase and
promoter have been used, for instance, in recombinant
poxviruaes to enhance expression of an inserted DNA
sequence. See, for example, Fuerst, T. R. et al., J.
Mol, eiol. 205: 333-348 (1989). Provision of the T7 RNA
polymerase on a separate genome is used to prevent
expression of a DNA sequence inserted into the modified
poxvirus genome except when the separate genome is
present.
In still other embodiments, the promoter controlling
the insert is suitable for initiation of transcription by
a cytoplasmic DNA virus RNA polymerase. In some
embodiments, the promoter comprises a modification of a
DNA sequence of a naturally occurring viral promoter.
One such embodiment is exemplified by use of a
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"synthetic" vaccinia virus promoter, such as the "S3A"
and "S4" promoters described, inter alia, in Examples 5
and 6.
The eukaryotic cytoplasmic DNA virus genomic
construction method of the present invention further
comprises a step of introducing the modified DNA molecule
comprising the modified viral genome into a first host
cell which packages the modified DNA molecule into
infectious modified cytoplasmic DNA virus virions.. The
modified DNA molecule is introduced into the first host
cell by a method suitable for transfection of that first
host cell with a DNA molecule, for instance, by methods
known in the art for transfection of other DNAs into
comparable host cells. For example, in a preferred
embodiment , the modified DNA is introduced into the first
host cell using the calcium phosphate precipitation
technique of Graham and van der Eb, Virology 52: 456-467
(1973).
In a preferred embodiment, this method for producing
a modified eukaryotic cytoplasmic DNA virus further
comprises a step of infecting the first host cell with a
second cytoplasmic DNA virus comprising a second
cytoplasmic DNA virus genome which is expressed to
package the modified DNA molecule into infectious
modified cytoplasmic DNA virus virions. In the method
comprising infection of the first host cell with a second
virus, introducing the recombinant DNA molecule into the
first host cell is carried out advantageously about one
hour after infecting the first host cell with the second
virus .
In another embodiment of this method, the necessary
packaging functions in the first host cell are supplied
by a genetic element other than a complete genome of a
second virus, such as a plasmid or other expression
vector suitable for transforming the first host cell and
expressing the required helper virus functions. Use of
a nonviral genetic element to provide helper functions
enables production of genetically stable helper cells
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that do not produce infectious helper virus. Use of such
a helper cell as a first host cell for packaging of a
modified DNA molecule advantageously produces only
virions comprised of that modified DNA.
In the method comprising infection of the first host
cell with a second virus, the second virus is selected so
that expression of the second viral genome in the first
host cell packages the modified DNA molecule into
infectious virions comprised of the modified viral
genome. Pursuant to the present invention, it is
feasible to effect intracellular packaging of a modified
DNA comprising a eukaryotic cytoplasmic DNA virus genome
by transfection into cells infected with a closely
related virus. For instance, DNA of a first poxvirus
genus is packaged by a host cell infected with a second
poxvirus of the same poxvirus subfamily, whether from the
same or a different genus.
In certain embodiments, expression of the second
viral genome in the first host cell produces infectious
virions comprised of the second viral genome as well as
of the modified viral genome. This situation obtains,
for instance, in the case of homolgous packaging of a
first poxvirus DNA from one genus by a second poxvirus of
the same genus. Here, although the transfected DNA
theoretically could be packaged directly, i.e., without
transcription of the transfected genome, homologous
packaging of the transfected DNA molecule probably
involves transcription and replication of both the
transfected DNA and the DNA of the helper virus. This
situation is illustrated, inter alia, with homologous
packaging of poxvirus DNA in Examples 1 and 2.
However, in other embodiments expression of the
second viral genome in the first host cell does not
produce infectious virions comprised of the second viral
genome. In cases involving heterologous packaging, for
instance, passive packaging alone cannot produce viable
virus particles from the transfected DNA. In such a case
it is advantageous to select a second (helper) virus
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which provides an RNA polymerase that recognizes
the transfected DNA as a template and thereby
serves to initiate transcription and, ultimately,
replication of the transfected DNA. This case is
exemplified by the reactivation of a modified
genome of an orthopoxvirus (vaccinia) vector by an
avipox (fowlpox) helper virus in a mammalian first
host cell in which the avipox virus is unable to
produce infectious virions comprised of the
avipoxvirus genome, as described in Examples 3.
The use of a heterologous virus to package the
modified DNA molecule, such as the use of fowlpox
or ectromelia (mouse pox) virus as a helper for
vaccinia virus constructs, advantageously
minimizes recombination events between the helper
virus genome and the transfected genome which take
place when homologous sequences of closely related
viruses are present in one cell. See Fenner &
Comben (1958) Virology 5:530-548; Fenner (1959)
Virology 8:499-507.
In certain embodiments of the method for using
a helper virus for DNA packaging, the step of
recovering the infectious virions comprised of the
modified viral genome comprises a step of infecting
a second host cell with infectious virions produced
by the first host cell. Advantageously, the second
host cell is infected under conditions such that
expression of the second viral genome in the second
host cell does not produce infectious virions
comprised of the second virus genome. In other
words, the second host cell is infected under
conditions that select for replication of the
modified virus and against the helper virus. This
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method is exemplified by a method in which the
modified genome is a modified vaccinia virus
genome, the second genome is a fowlpox virus
genome, and the second host cell is a mammalian
cell. In this method, the modified virus is plaque
purified in cultures of the mammalian host cell in
which fowlpox virus does not produce infectious
virions, as described in Example 3.
In another embodiment in which the second
host cell is infected under conditions that select
for the modified
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virus, the modified viral genome comprises a functional
host range gene required to produce infectious virions in
the second host cell. The second viral genome lacks this
functional host range gene. This embodiment is
illustrated by a method in which the modified viral
genome is a modified vaccinia virus genome comprising a
functional host range gene required to produce infectious
vaccinia virus in a human (MRC 5) cell which is used as
the second host cell, as described in Example 8.
In yet another embodiment involving selection for
modified virus in a second host cell, the modified viral
genome comprises a selective marker gene which the second
viral genome lacks, and the step of infecting the second
host cell is carried out under conditions that select for
a viral genome expressing the selective marker gene. For
example, expression of the selective marker gene in the
second host cell may confer on that cell resistance to a
cytotoxic drug. The drug is provided during infection of
the second host cell at a level sufficient to select for
a viral genome expressing the selective marker gene.
This approach is exemplified by a method for inserting a
gene for the E. coli gpt gene into a vaccinia virus
genome, as in Example 1, or a fowlpox virus genome, as in
Example 2, using in each case a homologous helper virus
lacking the selective marker gene.
In still another embodiment involving selection for
a modified virus in a second host cell, the modified
viral genome comprises a deletion of a selective marker
gene that is present in the second viral genome. Here,
the step of infecting the second host cell is carried out
under conditions that select against a viral genome
expressing that selective marker gene. For example,
expression of a poxvirus thymidine kinase (tk) gene in
the second host cell (i.e., a thymidine kinase-negative
host cell) renders the second (helper) virus sensitive to
the metabolic inhibitor, 5-bromo-deoxyuridine. Example
4 describes the use of these inhibitors during infection
of a second host cell to select for a vaccinia virus
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vector (vdTK) in which the tk gene is deleted and
replaced by a multiple cloning site.
Another aspect of the present invention relates to
a eukaryotic cytoplasmic DNA virus comprised of a
modified viral genome. A modified genome of a
cytoplasmic DNA virus within the scope of the present
invention comprises distinct component DNA sequences
which are distinguishable from each other, for example,
by routine nucleic acid hybridization or DNA sequencing
methods.
In certain embodiments, for instance, the modified
viral genome comprises a first genome of a first
eukaryotic cytoplasmic DNA virus. This first genome is
comprised of a cleavage site for a sequence-specific
endonuclease that is a unique site in the first genome.
In this embodiment, the sequences of the modified genome
that comprises the first viral genome are homologous to
a genome of a naturally occurring eukaryotic cytoplasmic
DNA virus. Further, the sequences of this first virus
are interrupted by a DNA sequence of interest as defined
hereinabove.
To determine whether this sequence is inserted into
a unique cleavage site in the first viral genome, as
required for this embodiment of a modified viral genome,
the sequences immediately flanking the insert are
compared with sequences of cleavage sites for sequence-
specific endonucleases.
In one form of this embodiment in which a DNA
sequence is inserted into a unique cleavage site in the
first viral genome, the inserted sequence in the first
viral genome is flanked by two identical intact cleavage
sites for a sequence-specific endonuclease and these two
sites are the only sites for this nuclease in the
complete modified genome. Each of these two sites is
comprised of combined portions of cleaved sites from the
first viral genome and the inserted DNA sequence.
More particularly, each strand of a double-stranded
DNA comprised of a cleavage site for a sequence-specific
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endonuclease may be considered to comprise a complete
cleavage site sequence (SLSR) consisting of a left
cleavage site sequence (SL) and a right cleavage site
sequence (SR) separated by the monophosphate linkage that
is disrupted by cleavage with the appropriate nuclease.
In certain forms of this embodiment, insertion of a DNA
sequence into a unique restriction site reproduces two
complete sites flanking the insert.
In other forms of this embodiment, however, insertion
of the DNA sequence into a unique cleavage site does not
recreate the original cleavage site at each end of the
inserted DNA sequence. See, for instance, the method for
elimination of a cleavage site described in Example 6.
Thus, the inserted DNA may be flanked at one end (e. g.,
the left end) by a complete cleavage site (SLSR) while
the right end terminates in a sequence that differs from
SL directly linked to an SR sequence in the first viral
genome. More generally, in any modified viral genome of
this invention, the DNA sequence inserted into a unique
site in a first viral genome will be flanked by two the
matching parts (SL and SR) of a cleaved site which does
not occur in the modified viral genome outside of the
inserted DNA.
In other embodiments, the modified viral genome is
comprised of a DNA sequence that is inserted between two
unique sites in the first viral genome. In this case, if
the f irat viral genome is a naturally occurring genome of
a eukaryotic cytoplasmic DNA virus, the insert will be
encompassed by viral sequences separated from the foreign
DNA sequence at least by recognizable SL and SR portions
of the two different original cleavage sites.
In additional embodiments, the modified viral genome
comprises a unique cleavage site located in a DNA
sequence that is not naturally occurring in a genome of
a eukaryotic cytoplasmic DNA virus. In this case, this
foreign DNA is not separated from the natural viral DNA
sequences by recognizable SL and SR portions of cleavage
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sites. In certain forms of this embodiment, the first
foreign DNA sequence is interrupted by a second foreign
DNA sequence inserted into a unique cleavage site in the
first sequence or between two such sites in the first
sequence. In these embodiments the second foreign DNA is
separated from the first foreign DNA sequences by
recognizable SL and SR portions of sequence-specific
endonuclease cleavage sites.
In this case, all sequences surrounding this second
foreign DNA sequence comprise the genome of the first
virus according to this invention.
Preferred embodiments of modified eukaryotic
cytoplasmic DNA viruses of this invention include a first
major embodiment in which the modified viral genome
comprises (I) a first genome of a first eukaryotic
cytoplasmic DNA virus that is comprised of a cleavage
site for a sequence-specific endonuclease. This site is
a unique site in the first viral genome. The 'modified
viral genome of this embodiment also comprises (II) a
first DNA sequence of interest. This DNA sequence is
inserted into the unique site in the first cytoplasmic
DNA virus genome.
In one variation of this first embodiment of a
modified eukaryotic cytoplasmic DNA virus, the first
viral genome comprised of the unique site is a naturally
occurring viral genome. This variation is exemplified
herein by a modified poxvirus genome comprised of a
naturally occurring vaccinia virus genome which has
unique cleavage sites for the bacterial restriction
endonucleases NotI and SmaI, as described in Examples 1
and 3. In this embodiment, the first DNA sequence of
interest, which is inserted into the unique site, is
exemplified by an E. coli gpt gene driven by a naturally
occurring vaccinia virus promoter inserted into the NotI
site (Example 1) or into the SmaI site (Example 3) of a
vaccinia virus genome.
In a second form of this first embodiment of a
modified virus, the first viral genome comprised of the
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unique site also comprises a second DNA sequence not
naturally occurring in a viral genome. Furthermore, this
second DNA sequence includes' the unique site for
insertion of the first DNA sequence. This variation is
exemplified herein by a modified fowlpox virus genome
comprising a DNA sequence encoding an Escherichia coli ~-
galactosidase gene, as described in Example 2. This
bacterial gene includes a cleavage site for the bacterial
restriction endonuclease NotI that is unique in the
modified fowlpox virus genome and, therefore, is
particularly convenient for insertion of foreign DNA
sequences.
In another variation of this first embodiment of a
modified virus, at least a portion of the first DNA
sequence that is inserted into the unique site is under
transcriptional control of a promoter. In some
instances, the promoter is located in the first DNA
sequence that is inserted into the first viral genome.
This holds, for instance, when the inserted DNA comprises
a gene cassette including a promoter and a functionally
linked gene, as described, inter alia, in Examples 1 and
2.
In a second embodiment of a modified cytoplasmic DNA
virus of this invention, the modified viral genome
comprises (I) a first viral genome comprised of a first
and a second cleavage site for a sequence-specific
endonuclease where each of these sites is unique in the
first virus genome. In one preferred variation of this
embodiment, the first viral genome comprises a multiple
cloning site comprised of several unique cleavage sites.
In this second embodiment, the modified viral genome
also comprises (II) a first DNA sequence not naturally
occurring in a genome of a eukaryotic cytoplasmic DNA
virus, and this first DNA sequence is inserted into the
first viral genome between the first and second unique
cleavage sites.
In a third embodiment of a modified cytoplasmic DNA
virus of this invention, the modified viral genome
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comprises (I) a first viral genome comprised of a first
DNA sequence not naturally occurring in a genome of a
eukaryotic cytoplasmic DNA virus. This first DNA
sequence is comprised of a cleavage site for a sequence-
s specific endonuclease that is a unique site in the
modified viral genome . The modified viral genome of this
embodiment further comprises (II) a promoter located such
that a DNA sequence inserted into the unique site is
under transcriptional control of the promoter. ,This
first DNA sequence does not have a translation start
codon between the promoter and the unique site used for
insertion of a DNA sequence. This embodiment is
exemplified by the vaccinia virus vector (vS4) described
in Example 6, which has a "synthetic" poxvirus promoter
located such that this promoter controls transcription of
a DNA sequence inserted into a multiple cloning site
designed for insertion of open reading frames.
Another aspect of the present invention relates to
a DNA molecule comprising a modified viral genome of a
modified eukaryotic cytoplasmic DNA virus of this
invention. In a preferred embodiment, this DNA molecule
is prepared by extraction of genomic DNA molecules from
virions of a modified eukaryotic cytoplasmic DNA virus of
this invention, or from cells infected with a modified
virus of this invention. Methods suitable for extracting
modified viral genomic DNAs from virions are known in the
art. In addition, suitable methods for preparing DNA of
eukaryotic cytoplasmic DNA viruses are described herein
in Example 1.
Still another aspect of the present invention relates
to genomic DNA arms of a eukaryotic cytoplasmic DNA virus
of this invention. These genomic DNA arms are useful for
direct molecular cloning of viral genomes comprising
foreign DNAs. More particularly, this aspect of the
invention relates to two DNA molecules, the left and
right genomic arms of a modified viral genome of a
eukaryotic cytoplasmic DNA virus. In the practice of the
direct cloning method of this invention, described above,
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either one or both of these arms may consist entirely of
a DNA sequence that is naturally occurring in a
cytoplasmic DNA virus . But the novel DNA molecule of the
present aspect of this invention is a modified arm of a
viral genome, in other words, a DNA molecule comprising
one end of a modified viral genome of a eukaryotic
cytoplasmic DNA virus. This end of the modified viral
genome comprises a DNA sequence of interest which
distinguishes this DNA molecule from genomic .arms
consisting of only a sequence that is naturally occurring
in a cytoplasmic DNA virus. In addition, the modified
viral genome from which the novel arm derives is
comprised of a unique cleavage site for a sequence-
specific endonuclease. Furthermore, this DNA molecule
has a terminus that is homologous to a product of
cleaving the unique site in the modified viral genome
with the sequence-specific endonuclease.
In a preferred embodiment, this DNA molecule
comprising a genomic arm is produced by cleavage of
genomic DNA of a modified virus at a unique site for a
sequence-specific endonuclease. Alternatively, this DNA
molecule may be produced by modifying another DNA
molecule to produce a terminus that is homologous to a
terminus produced by cleaving a unique site in a modified
viral genome. For instance, a DNA molecule according to
this aspect of the invention may be produced from an arm
of a naturally occurring genomic viral DNA. The required
DNA molecule may be produced from such a naturally
occurring viral arm, for example, by ligation to a
synthetic "adaptor" DNA segment comprised of a cohesive
end derived from cleavage site that is not present in the
first viral genome. In this instance the end of the
first viral genome and the ligated adaptor together
comprise one end of a modified viral genome.
Accordingly, this particular DNA molecule is not produced
by cleavage of a modified viral genomic DNA, but it does
comprise a terminus that is homologous to a terminus that
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is produced by cleaving a unique site in a modified viral
genome.
In another embodiment of a modified viral DNA arm of
the present invention, the DNA sequence not naturally
occurring in a genome of a eukaryotic cytoplasmic DNA
virus is comprised of the cleavage site for a sequence-
specific endonuclease that is unique in the modified
viral genome. This cleavage site further comprises a
left cleavage site sequence (SL) for the left genomic
arm, or the right cleavage site sequence (SR) for the
right genomic DNA artn, occurring complete cleavage site
sequence (SLSR) being unique in the modified viral
genome. This embodiment is exemplified, inter alia, by
DNA arms produced from a fowlpox virus vector by the
bacterial restriction endonuclease NotI, as described in
Example 2 , or by arms of a vaccinia virus vector (vS4 )
cleaved at any of several unique sites of an inserted
multiple cloning site, as described in Example 6.
Yet another aspect of the present invention relates
to a kit for direct molecular cloning of a modified viral
genome of a eukaryotic cytoplasmic DNA virus. This kit
comprises (I) purified DNA molecules of this invention.
These DNA molecules comprise either genomic viral DNA
arms of this invention or a complete, intact modified
viral genome of this invention, or both. The viral DNA
arms are useful for direct ligation to foreign DNA
segments to be cloned, while the intact viral DNAs are
useful for cloning after cleavage, for instance, with a
sequence-specific endonuclease at a site that is unique
in the modified viral genome.
The kit further comprises (II) a DNA ligase and (III)
solutions of a buffer and other reagents suitable for
ligation of DNA segments together to produce a modified
DNA molecule comprising said modified viral genome. A
suitable buffer and reagents for ligation are described,
for instance, in Example 1.
In one embodiment, this kit further comprises a
plasmid comprised of a gene expression cassette flanked
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by sites for cleavage with a sequence-specific
endonuclease. When cleaved by the appropriate sequence-
specific endonuclease, the sites flanking the cassette
produce ends that are compatible for insertion of this
cassette into a unique cleavage site of the modified
viral genome that is encoded by the DNA molecule.
In another embodiment, the cloning kit further
comprises a first host cell and a second (helper) virus
suitable for packaging the modified viral genome,into
infectious virions.
Yet another aspect of the present invention relates
to plasmids which are particularly suited to serve as
intermediates in the construction of modified cytoplasmic
DNA virus vectors of this invention. According to one
embodiment of this aspect, there is provided a plasmid
comprising a DNA segment having at each end the same
cleavage site for a sequence-specific endonuclease. This
site is also a unique site in a first cytoplasmic DNA
virus genome according to the present invention. This
DNA segment comprises a multiple cloning site comprised
of several closely adjacent sequence-specific
endonuclease cleavage sites that are unique in the
plasmid and, therefore, useful for insertion of foreign
DNA segments into the plasmid.
This plasmid is useful for insertion of genes into
a unique cleavage site of the DNA segment for subsequent
transfer of that segment into a unique cleavage site of
a cytoplasmic DNA virus using the direct molecular
cloning method of this invention. This plasmid is
exemplified by the plasmid pN2 (see Example 1, Figure
1.3) which has a DNA segment comprising a multiple
cloning site flanked by NotI sites and containing the
following additional bacterial restriction enzyme
cleavage sites in the stated order: XbaI, SpeI, HamHI,
SmaI, PstI, EcoRI, EcoRV, HindIII and ClaI.
Another plasmid of the present invention comprises
a DNA segment having at each end a cleavage site that is
a unique site in a cytoplasmic DNA virus. The DNA
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segment of this plasmid also comprises several
restriction enzyme cleavage sites that are unique in the
plasmid. This DNA segment further comprises a selective
marker gene (e. g., an E. coli gpt gene) under
transcriptional control of a cytoplasmic DNA virus
promoter (e. g., the vaccinia virus P7.5 promoter). This
plasmid is exemplified by two plasmids designated pN2-
gpta and pN2-gptb which contain a DNA segment flanked by
NotI sites and comprising an E. coli gpt gene under
transcriptional control of a vaccinia virus P7.5
promoter. This plasmid was created by insertion of the
promoter-gene cassette into the SmaI site of the plasmid
pN2, as described in Figure 1.3.
In a further modification of the above plasmid, the
DNA segment further comprises a second poxvirus promoter
operatively linked to a DNA sequence comprising a
restriction endonuclease cleavage site. This plasmid, as
exemplified by the plasmid pN2gpt-S3A (Figure 4.7) can be
used to insert open reading frames lacking their own
initiation codon for transfer into a vaccinia virus
vector. Similarly, the plasmid pN2gpt-S4 (Figure 4.7)
can be used to insert complete open reading frames
including an AUG translation start codon.
In another embodiment, this plasmid further comprises
a DNA sequence encoding human plasminogen, wherein the
DNA sequence is operatively linked to the poxvirus
promoter and start codon. This plasmid is exemplified by
plasmid pN2gpt-GPg, encoding human glu-plasminogen, and
by plasmid pN2gpt-LPg, encoding lys-plasminogen, in which
the coding region for amino acids 1-77 of human
plasminogen is deleted (Figures 5.2 and 5.3).
In a related form, this plasmid further comprises a
DNA sequence encoding human immunodeficiency virus (HIV)
gp160, wherein the DNA sequence is operatively linked to
the poxvirus promoter and start codon. T h i s i s
exemplified by plasmid pN2gpt-gp160, having the gp160
gene controlled by the synthetic vaccinia virus promoter
S4 (Figure 5.4).
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Another plasmid of the present invention comprises
a segment of a cytoplasmic DNA virus genome in which the
viral thymidine kinase (tk) gene is located. In this
plasmid, the coding region of the tk gene has been
modified (deleted) to prevent expression of active tk
enzyme. This plasmid is useful as an intermediate in
construction of a cytoplasmic DNA virus vector having a
defective tk gene, using conventional methods of marker
rescue, as described for the vaccinia virus tk gene,
using plasmid pHindJ-3. In a related embodiment, a
plasmid comprising a modified tk gene region of a
cytoplasmic DNA virus further comprises a multiple
cloning site comprised of several closely adjacent
sequence-specific endonuclease cleavage sites that are
unique in the plasmid. Furthermore, each of these sites
is absent in a cytoplasmic DNA virus into which the
modified tk gene region is to be inserted. Therefore,
after insertion of the modified tk gene region comprising
these unique sites into that viral genome, these sites
are useful for insertion of foreign DNA segments into the
cytoplasmic DNA virus genome carrying the modified tk
gene region, according to the direct cloning method of
the present invention.
This plasmid comprising a modified tk gene region
containing a multiple cloning site is exemplified by
plasmid pHindJ-3 in which the modified vaccinia virus tk
gene region of plasmid pHindJ-2 has inserted a multiple
cloning site with the unique sites NotI, SmaI, ApaI and
RsrII, flanked by SfiI sites (Figure 4.2). To further
facilitate forced cloning in a vaccinia virus vector,
each of the two SfiI sites is also made unique in the
vector by exploiting the variable nature of the SfiI
recognition sequence, as detailed in Example 4.
In still another embodiment, a plasmid comprises a
sequence-specific endonuclease cleavage site that is
unique in the genome of that virus. Such plasmids are
particularly suitable for construction of gene
expressions cassettes for transfer into a vector having
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the aforementioned unique site. The plasmid pA0
exemplifies the basic plasmid that contains a master
cloning site comprised of the unique sites of the master
cloning site of the vdTK vaccinia virus vector (Figure
4 . 3 ) . The related plasmids pAl and pA2 were designed f or
insertion of DNA segments, for instance, synthetic or
natural promoter fragments and were constructed by
inserting into the Xhol site of pA0 a linker comprising
a second multiple cloning site of frequently cutting
enzymes that do not cleave pAO. Hoth plasmids have the
same structure except for the orientation of the second
multiple cloning site (Figure 4.3).
In yet another embodiment, a plasmid comprises a
poxvirus promoter operatively linked to a translational
start codon, wherein this start codon is immediately
followed by a second restriction endonuclease cleavage
site suitably arranged to permit translation of an open
reading frame inserted into the second restriction
endonuclease cleavage site. This plasmid is exemplified
by plasmids pAl-S1 and pA2-S1 which provide the strong
synthetic poxvirus promoter S1, including a translational
start codon, followed by a single EcoRI site suitable for
insertion of open reading frames that do not have an
associated start codon (Figure 4.4). Plasmids pAl-S2 and
pA2-S2 are similar to pAl-S1 and pA2-S1 but have a
different poxvirus promoter, S2 (Figure 4.5).
In a related embodiment, the plasmid above further
comprises a DNA sequence encoding human prothrombin,
wherein said DNA sequence is operatively linked to said
poxvirus promoter and said start codon. This plasmid is
exemplified by the plasmid pAlS1-PT (Figure 5.1) in which
a modified prothrombin cDNA is inserted into the single
EcoRI site of the plasmid pAl-S1.
Another plasmid of the present invention comprises
a modified EcoRI K fragment of vaccinia virus DNA from
which the K1L host range gene is deleted. The helper
virus vdhr lacking both the K1L and C7L host range genes
is constructed from the C7L-negative strain WR-6/2 by
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marker rescue with a modified EcoRI K fragment from which
the K1L host range gene is deleted. See Figure 8.1.
This modified EcoRI R fragment comprises a selective
marker gene (the E. coli gpt gene) to facilitate
selection for recombinant WR-6/2 genomes comprising the
modified EcoRI K fragment using intracellular marker
rescue as described by Sam & Dumbell, 1981. The
exemplifying plasmid is designated pEcoK-dhr (Figure
8.1) .
In a further step pEcoK-dhr is linearized with NotI
and ligated with a 1.1 kb P7.5-gpt gene cassette derived
from plasmid pN2-gpta (Example 4) by NotI digestion. The
resulting plasmid pdhr-gpt (Figure 8.1) is used in marker
rescue experiments to generate the helper virus vdhr
according to the marker rescue method of Sam & Dumbell,
1981.
The present invention is further described below with
regard to the following illustrative examples.
In the examples that follow, certain constructs are
illustrated with tables detailing their characteristics.
In those tables, the following abbreviations are used:
CDS = coding sequence
rc = reverse complementary sequence
rcCDS = reverse complementary coding sequence
arabic numbers are positions of nucleotides
ATG = translational start codon
EMBL ID = Identifier in EMBL DATABANK
EXAMPLE 1. Direct molecular cloning of foreiga DNA
comprising a selective marker gene (the
gpt gene of B. coli) into a unique (NotI)
cleavage site is the genome of an
orthopoxvirus (vaecinia)
This example demonstrates direct molecular cloning
of a gene expression cassette into a poxvirus genome,
according to the present invention, by intracellular
packaging of genetically engineered poxvirus DNA. In
addition, this example illustrates use of a genetic
selection procedure for efficient recovery of modified
vaccinia viruses containing an inserted selective marker
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gene. The experimental results also reveal that
recombination frequently occurs between the DNA to be
packaged and that of the infecting helper virus during
packaging when the helper virus DNA is homologous with
the DNA to be packaged.
More particularly, a first direct molecular cloning
experiment described below shows that a marker gene (gpt-
gene) cassette can be inserted as a NotI restriction
fragment in NotI-cleaved vaccinia virus DNA. and
subsequently packaged in vaccinia virus-infected
mammalian cells. One of nine plaques examined comprised
virus having the predicted structure for a single insert
of the gpt-gene in the "a" orientation (see Figure 1.11) .
The structure of this clone (designated vp7) was stable
during large scale replication in the absence of the
selection agent.
In a second series of cloning experiments, seven of
twelve clones examined had the expected structure. In
this series, however, four small plaques (E1-E4) of
slowly replicating viruses were included, although
preferably these are not normally selected in the
practice of the present invention. Recombinants having
multiple inserts of the selective marker gene were also
obtained under selective conditions. The stability of
these multiple inserts was not examined in the absence of
the selective agent which is known to stabilize certain
otherwise unstable structures. See Falkner & Moss, J.
Virol. 64: 3108-3111 (1990).
The relatively low yield of predicted structures is
not expected given the known precision of genetic
engineering methods for site-specific cleavage and
ligation of DNA molecules. However, the particular
sequence selected for insertion in this model system, the
gpt-gene cassette, comprised vaccinia virus DNA sequences
of the P7.5 promoter which are homologous to two
endogenous promoters in the vaccinia vector which drive
two vaccinia virus 7.5-kD polypeptide genes located
within the inverted terminal repetitions of the vaccinia
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genome. See Venkatesan, B., Baroudy B. M. & Moss,
B., Cell 25: 805-813 (1981). This P7.5 promoter
has been used to construct vaccinia virus
recombinants by conventional intracellular
recombination and can be stably integrated into the
vaccinia thymidine kinase gene. Macket & Smith
(1986) J. Gen. Virol. 67:2067-2082. Occasionally,
however, submolar amounts of DNA fragments appear
during analyses of conventional recombinants, which
may result from secondary recombination events.
Where a P7.5 promoter is inserted near the
endogenous P7.5 promoters (i.e., within several
kilobases), only recombinants that have an inverted
repeat structure are stable, and this observation
has been exploited to develop a deletion procedure
based on insertion of a tandemly repeated P7.5
promoter segment. Spehner, D., Drillien, R. &
Lecocq, J.-P. J. Virol. 64: 527-533 (1990).
In the present case of insertion of the gpt-
gene cassette into the NotI site of vaccinia virus,
the distance between the P7.5 promoters of the left
inverted terminal repetition and that of the
inserted cassette is about 30 kb, probably close
enough to cause destabilizing secondary
recombination events. In fact, only the structures
of a few slowly replicating, unstable clones had an
insert in the "b" orientation which would produce a
tandem repeat arrangement of the inserted and
endogenous promoters. Thus, the rare occurrence of
this structure can be explained most likely by the
closeness of the locations of the P7.5 promoters of
the gpt-gene cassette and the endogenous P7.5
promoters and the known instability of tandemly
repeated copies of the P7.5 promoter.
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In contrast, the virus vp7 and several other
isolates (A1, A4, C1 and C2) had inserts in the "a"
orientation and were stable. The structural
analysis of one isolate, C4, was consistent with a
head-to-tail double insert.
The titers of packaged gpt-gene positive
viruses in the second series of cloning experiments
(five different samples) were approximately 1 x 105
pfu per 8 x 106 cells,
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while in the first experiment a titer of 1-2 x 102 pfu
was obtained from the same number of cells. The titer of
modified viruses will be influenced by several factors,
including ligation and packaging efficiencies, reaction
and culture conditions in the cloning procedure, and by
the amount of care taken to avoid shearing of the high
molecular vector DNA during handling. Titers of about
lOs pfu per 8 x 106 cells are generally expected under the
standard conditions described hereinbelow.
While the present example shows that the unique
intergenic NotI site of vaccinia virus can be used for
insertion of foreign DNA, it also illustrates the need to
consider whether a proposed insert may contain viral
sequences of a type and orientation that are known or
likely to cause instability of modified viruses. Inserts
lacking homology with viral sequences near the insertion
site (e.g., within 30 kb) are to be preferred for
stability. Accordingly, inserts comprising only short
synthetic promoter sequences that are recognized by the
transcription system of the vector are preferred to those
containing large segments of viral DNA including natural
promoters of the viral vector. See, for instance, the S1
promoter in Example 4, below.
The following materials and methods were used
throughout this and all subsequent examples, except where
otherwise specified.
Purification of orthopox virus and DNA: Vaccinia
virus (wildtype Western Reserve (WR) strain; American
Type Culture Collection No. VR 119) was purified by two
successive sucrose gradients according to Mackett, et al.
in D.M. GLOVER, DNA CLONING: A PRACTICAL APPROACH, 191-
211 (IRL Press, 1985). Viral DNA was prepared by the
proteinase K-SDS procedure according to Gross-Hellard et
al., Eur. J. Hiochem. 36: 32-38 (1973).
Engineering of isolated poxvirus DNA: Viral DNA
(typically 2 to 5 ~.g) was cleaved with appropriate
amounts of one or more sequence-specific endonucleases
(for example, the bacterial restriction endonuclease
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NotI), optionally treated with calf intestine
alkaline phosphatase (Boehringer, Inc.), and
purified by phenol extraction and ethanol
precipitation, according to routine recombinant DNA
methods. The resulting viral DNA arms were ligated
with a five to fifty-fold molar excess of the DNA
fragment to be inserted, having ends compatible for
ligation with the viral arms. An aliquot of the
ligation reaction was analysed by field inversion
gel electrophoresis.
More particularly, in the second series of
experiments (A-E) described below, 2 ~g of NotI-
digested vaccinia DNA that was not treated with
phosphatase were ligated with 200-600 ng of gpt-
gene cassette insert in~a volume of 30 ~l with 5-15
units of T4 iigase for 48 h at 12 C, as summarized
in Table 1.
In vivo packaging in mammalian cells: 8 x 106
African Green monkey (CV-1) cells were infected
with helper virus (either vaccinia WR wildtype or
WR6/2 virus, or other viruses as indicated) at 0.2
pfu/cell for 2 h. For the initial demonstration of
packaging with intact DNA isolated from virions, 20
~g of viral (vPgD) DNA were used. For packaging of
extracellularly engineered genomes, 1 ~g of DNA
purified from a ligation reaction were used. DNAs
were transfected into cells by the calcium
phosphate precipitation technique (Graham, F.L. &
van der Eb, 1973). The cells were incubated for 15
min at room temperature and then nine ml of medium
(DMEM, loo fetal calf serum, glutamine and
antibiotics) per one ml precipitate were added to
the cells. After four hours the medium was changed
and further incubated for two days.
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Crude virus stocks were prepared according t o
standard procedures. Mackett et al., in D.N~_
Glover, DNA Cloning: A Practical Approach, 191-21 1
(IRL Press, 1985). Plaque assays and selecti on .
conditions for the E. coli gpt gene are known i n
the art . See Falkner & Moss, J. Virol . 62 : 184 9 -
1854 (1988); and Boyle & Cougar, Gene 65: 123-1 2 8
(1988) .
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Field inversion gel electrophoresis (FIGE). Viral
DNA was separated on a 1% agarose gel in
Tris/Acetate/EDTA buffer (40 mM Tris/20 mM glacial acetic
acid/2 mM EDTA, pH 8.0) with a microcomputer controlled
power supply (Consort Model E790). To separate the whole
range of fragments, four programs were run successively,
as follows: program 1: 5 h at 7 V/cm forward pulse (F)
6 sec, reverse pulse (R) 3 sec, pause 1 sec; program 2:
5 h at 7 V/cm, F 4 sec, R 2 sec, pause 1 sec; program 3:
5 h at 7 V/cm, F 2 sec, R 1 sec, pause 1 sec; and program
4: 5-10 h at 7 V/cm, F 8 sec, R 4 sec, pause 1 sec.
Construction of plasmid pN2: The plasmid Bluescript
II SK- (Stratagene, Inc.) was digested with HindII and
ligated to NotI linkers (Pharmacia, Inc.). The resulting
plasmid, pN2, has a multiple cloning site flanked by Notl
sites.
More particularly, the multiple cloning site of pN2
consists of the following sites in the stated order:
NotI, XbaI, SpeI, HamHI, SmaI, PstI, EcoRI, EcoRV,
HindIII, ClaI and NotI. The inserted NotI linker
sequence of pN2 and twenty bases of the 5' and 3'
flanking regions of pHluescript II SK- (Stratagene, Inc.
La Jolla, USA) are shown in SEQ. ID. NO. 1. The insert
sequence starts at position 21 and ends at position 28.
(The first "T" residue at the 5'-end corresponds to
position number 2266, the last "G" residue at the 3'-end
to position number 2313 of the plasmid pN2).
Construction of plasmids pN2-gpta and pN2-gptb: The
1.1 kb HpaI-DraI fragment (containing the P7.5 promoter
gpt gene cassette) was isolated from the plasmid pTKgpt
Fla (Falkner & Moss, 1988) and inserted into the SmaI
site of the plasmid pN2 (Figure 1.3). The two resulting
plasmids are orientational isomers and were designated
pN2 -gpta and pN2 -gptb. The vaccinia virus P7 . 5 promoter-
E. coli gpt-gene cassette and twenty bases of the 5'-and
3' -flanking regions of pN2 are shown for pN2-gpta in SEQ.
ID. NO. 2. The insert starts at position 21 and ends at
position 1113. The A-residue of the translational
CA 02515166 1992-08-25
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initiation codon of the gpt-gene corresponds to position
519. The T-residue of the translational stop codon of
the gpt-gene corresponds to position number 9?5. (The.
first "C" residue at the 5'-end corresponds to the
position number 2227, the last "T" residue at the 3'-end
to position number 3359 of the plasmid pN2-gpta).
The reverse complementary form of the vaccinia virus
P7.5 promoter-E. coli gpt-gene cassette and twenty bases
of the 5'- and 3'-flanking regions of pN2 are shown for
pN2-gptb in SEQ. ID. NO. 3. The insert starts at
position 21 and ends at position 1113. The T-residue of
the (reverse complement of the) translational initiation
codon CAT corresponds to position 615. The A-residue of
the (reverse complement of the) translational stop codon
of the gpt gene corresponds to the position number 159.
Other standard techniques of recombinant DNA analysis
(Southern blot, PAGE, nick translation, for example) were
performed as described. J. SAMBROOK et al., MOLECULAR
CLONING (Cold Spring Harbor Laboratory Press, 1989).
Packaging of naked viral DNAs To establish
conditions needed for packaging of naked poxvirus DNA by
a helper virus, intact DNA isolated from virions of an
exemplary recombinant vaccinia virus (vPgD) was
transfected into monkey (CV-1) cells infected with a
helper virus (vaccinia WR wildtype). The selected
recombinant virus has several readily assayable
phenotypic markers. Thus, the vPgD genome has
incorporated into the viral thymidine kinase (tk) locus
a gene for a drug resistance marker (a gene for the
enzyme xanthine-guanine-phosphoribosyl-transferase of
Escherichia coli; i.e., the "gpt" gene) and a gene for a
conveniently detected marker protein (human plasminogen).
This virus was originally constructed from a vaccinia
virus strain [WR 6/2; Moss et al., J. Virol. 40: 387-95
(1960), which has a deletion of about 9 kb and,
consequently, does not express the viral major secreted
35K protein gene described by Kotwal et al., Nature 335:
176-178 (1988)]. The expected phenotype of the packaged
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virus, therefore, includes: tk-negative (i.e.,
replication in the presence of bromodeoxy-uridine);
gpt-positive (i.e., replication in the presence of
mycophenolic acid and xanthine); expressing the
human plasminogen gene; and not expressing the
secreted 35K protein.
Eight gpt-positive plaques from the above
packaging experiment were analysed. All were tk-
negative, and, as shown in Figure 1.1, all
expressed plasminogen. Six of these isolates
(lanes 5, 6, 7, 11, 12 and 14) did not express the
35K secreted vaccinia protein and thus showed all
the characteris- tics of the transfected genomic
DNA. Two of the plaques also expressed the 35K
protein marker (lanes 4 and 13) and therefore were
recombinants between the helper wild-type virus
(lanes 8 and 15) and the input viral genomes.
This equipment established that naked poxvirus
DNA extracted from virions is packaged when
transfec- ted into helper virus-infected cells
under the tested conditions. Therefore, these
conditions were employed for transfection of
genomic poxvirus DNA that had been modified by
direct molecular cloning, as outlined in Figure
1.2.
Packaging of extracellularly engineered
poxvirus DNA: The genome of vaccinia virus contains
a single cleavage site for the NotI sequence-
specific endonu- clease in the region known as the
HindIII F fragment. Inspection of the sequence
around this site (Geobel et al., 1990 Virology
179:247-266) revealed that it is located in an
intergenic region that is unlikely to be essential
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for viral replication. A marker gene expression
cassette was constructed in two plasmids (pN2-gpta
and pN2-gptb; Figure 1.3) by insertion of the E. ,
coli gpt gene in each of the two possible
orientations. The gpt gene was controlled by the
pro- moter of the vaccinia virus gene coding for
the 7.5 kDa protein described in Cochran et al., J.
Virol. 54: 30-37 (1985) (labelled P1 in Figure 1.2
and P7.5 in
Figure 1.3). The entire marker gene cassette
resided on a
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single 1.1 kb NotI fragment of these plasmids. This
restriction fragment from pN2 -gpta was ligated with NotI
digested WR wildtype DNA and transfected into cells that
had been infected with helper virus (WR).
In a first cloning experiment, Southern blot analyses
of the genomic structures of phenotypically gpt-positive
progeny plaques was carried out . The viral isolates were
plaque-purified three times and amplified under gpt-
selection. The HindIII-digested DNA fragments of .cells
(CV-1) infected with the different viruses were separated
on a 1% agarose gel by a combination of normal
electrophoresis and field inversion gel electrophoresis.
The gel was then blotted and hybridized with 32P-labelled
vaccinia WR DNA and a labelled probe containing gpt
sequences. The results confirmed that all phenotypically
marker-positive clones contained the 1.1 kb gpt insert.
Figure 1.4 shows blots of HindIII DNA fragments from
cells infected with the nine virus isolates (lanes 4-12) ;
plaques 2.1.1 to 7.1.1 and 10.1.1 to 12.1.1). The
expected 0.8 kb HindIII fragment that contains the gpt
sequences can be observed. In lanes 2 and 3, where
HindIII-digested wildtype virus DNA (100 and 50 ng,
respectively) were loaded, no cross-hybridization to
viral sequences was visible.
In the next experiment, total DNAs of CV-1 cell
cultures infected with the nine different plaques were
digested with NotI. The Southern blot of the separated
fragments is shown in Figure 1.5. Unexpectedly, two
bands were visible in most virus isolates, the predicted
1.1 kb insert and a second, larger fragment. Only plaque
number 7.1.1 (lane 8) showed the expected single 1.1 kb
band. While the hybridization signal of the larger
fragment is equally strong in all examined DNAs, the
intensity of the 1.1 kb band varied from DNA to DNA,
indicating that the 1.1 kb insert may be present in
different molar amounts in different genomes. The
wildtype virus control (lane 2) did not hybridize to the
gpt-gene probe .
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The same blot was also hybridized with a vaccinia
virus DNA probe. Three fragments are expected, of about
145 kb, 45 kb and 1.1 kb. The blot patterns obtained
included the expected bands but also showed an additional
band at about 5 kb. Only plaque 7.1.1 did not have the
unexpected 5 kb band.
The orientation of the DNA insert in selected
engineered vaccinia genomes was also investigated by
Southern blotting. As shown in Figure 1.2, the insert in
viral DNAs may be in either the "a" or "b" orientations
which are distinguishable by digestion of the DNAs with
appropriate restriction enzymes. Following preliminary
analyses, isolate 7.1.1 was designated clone vp7,
appeared to have the genomic structure of the expected
modified virus and therefore was expanded and purified.
The DNA of this clone was compared with that of wildtype
virus by digestion with several restriction enzymes and
separation on an agarose gel by field inversion gel
electrophoresis (Figure 1.6). In a NotI digest of vp7
stained with ethidium bromide (lane 2), only the 145 kb
and 45 kb bands contained sufficient DNA mass to be
visible, since the band for the 1.1 kb insert was
estimated to contain only about 3 ng DNA. However,
hybridization with a gpt-specific probe revealed a weak
band at 1.1 kb (Figure 1.7, lane 2). In digests with
HindIII, the expected bands at 1.4 and 0.8 kb were
observed. As predicted, the 0.8 kb band hybridized with
the gpt-gene probe ( Figures 1. 6 and 1. 7 , lanes 4 ) . In
double digests with NotI and HindIII, the expected 0.8 kb
fragment was also observed (Figures 1.6 and 1.7, lanes
6) .
In digests of vp7 DNA with PstI, a predicted 4.1 kb
fragment containing gpt sequences was observed (Figures
1.6 and 1.7, lanes 8; the 4.1 kb ethidium bromide-stained
band in Figure 1.6 is actually a doublet of 4.1 kb
fragments, one of which contains the gpt insert). Upon
cleavage with both PstI and NotI, the gpt gene cassette
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was released as a 1.1 kb fragment (Figures 1.6 and 1.7,
lanes 10 ) .
The patterns of digests obtained with these and other
restriction nucleases, including SalI (Figures 1.6 and
1.7, lanes 12), are consistent with the interpretation
that vp7 is a stable modified virus that has the gpt-gene
integrated into the NotI site of the vaccinia virus
genome in the "a" orientation (see Figure 1.11).
A second series of cloning experiments were .done
under slightly modified conditions (see Table 1 and
methods, above). Five different ligation reactions (A-E)
were set up containing constant amounts of NotI-cleaved
vaccinia vector DNA and increasing amounts of insert DNA.
Packaging was done under standard conditions in vaccinia
virus-infected CV-1 cells. The titers of gpt-positive
vaccinia viruses in all cases were about 1 x lOs pfu per
8 x 106 cells. The plaque population in all cloning
experiments was heterogeneous in size: about half had a
normal size while the other half were smaller than
normal.
Table 1. Effect of ratio of insert to vector DNA
on yield of modified viruses
Experiment A B C D E
NotI-cleaved
vector DNA (~.g) 2 2 2 2 2
gpt-gene insert (~.g) 0.2 0.2 0.4 0.4 0. 6
insert molar excess 17 17 34 34 51
T4 ligase (units) 5 15 5 15 15
gpt-positive virus (lOs) 1.12 0.88 0.96 0.96 1.16
(pfu/8 x 106 cells)
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Twelve gpt-positive plaques were isolated, four each in
three series designated series A, C and E, comprising 8
normal-sized (large) plaques (A1-4 and C1-4) and 4 small
plaques (E1-4). Each of these plaques was analyzed by ,
infecting CV-1 cells in gpt-selective medium, isolating
total cell DNAs and digesting them with restriction
nucleases, separating the fragments by FIGE and blotting
the onto a nitrocellulose membrane.
In Figure 1.8, the NotI-digested DNA samples
hybridized with the vaccinia virus DNA probe are shown
(A1-4, lanes 1-4; C1-4, lanes 5-8; E1-4, lanes 9-12).
Due to overloading of the gel, the bands smeared somewhat
but the essential features are clearly visible. The 145
kb and the 45 kb bands provided the main signal. A weak
band at about 5 kb of unknown origin can be seen in some
of the samples. The 1.1 kb band, comprising the P7.5-
promoter-gpt-gene cassette, makes up only 0.6% of the
viral genome and contains only 300 by of hybridizing
sequence (i.e., the P7.5 promoter). Therefore, this band
was not expected to give a detectable hybridization
signal under the conditions used. In a longer exposure
of the blot, when the larger bands are heavily
overexposed, the 1.1 kb bands did become visible.
As to the nature of the small plaque phenotype, small
plaques E1, E3 and E4 produced only weak hybridization
signals (Figure 1.8, lanes 9-12) indicating that the
virus in these plaques had not replicated as extensively
as those in norn~al-sized plaques (lanes 1-8), while
isolate E2 failed to produce a detectable amount of DNA
(lane 10).
The samples shown in Figure 1.8 were also hybridized
with the gpt-gene probe (Figure 1.9). The expected
single hybridization signal was obtained with plaques Al,
A4, C1, C2, C4, E3 and E4 (Figure 1.9, lanes 1, 4, 5, 6,
8, il and 12). The plaque A2 (lane 2) had the gpt-gene
integrated into the 45 kb band. (The weak signal in the
145 kb band may be due to contamination with a second
minor species or to secondary recombination events.) The
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plaque A3 (lane 3) has gpt-gene sequences integrated into
the 145 kb and 45 kb bands, while the plaque C3 (lane 7)
has an integration of those sequences into the 145 kb
band and into the NotI site. The plaques A2, A3 and C3
are probably recombinants that arose by illegitimate
intracellular recombination of homologous sequences
present in the model gene cassette insert and in the
inverted repetitions of the viral DNA.
As with the vaccinia virus DNA probe, the small
plaques E1-E4 produced only weak hybridization signals
(Figure 1.9, lanes 9-12) indicating that the virus in
these plaques had not replicated as extensively as those
in normal-sized plaques. The wildtype virus DNA and
uninfected CV-1 cell DNA did not hybridize with the gpt
gene probe (Figure 1.9, lanes 13 and 15).
The orientation and copy number of the gpt-gene
inserts were determined by digesting the samples shown in
Figure 1.9 with PstI and Southern blot analysis. The
expected sizes of new PstI fragments resulting from
insertion of the gpt-gene are shown in Figure 1.11.
Hybridization with the gpt-gene probe revealed that the
patterns of plaques A1, A4, C1 and C2 (Figure 1.10 lanes
1, 4, 5 and 6) comprised a single PstI fragment of 4.1 kb
as expected for a single insert in the "a" orientation
(Figure 1.11). For plaque E1, a weak hybridization
signal from a 21 kb band, which was observed only in long
exposures of the blot, was consistent with the "b"
orientation of the gpt-gene insert.
The structures of the viral DNAs from plaques C4 and
E3 (Figure 1.10, lanes 8 and 11) were consistent with
double tandem inserts in the "b" orientation. In this
case hybridizing fragments of 21 and 1.1 kb are expected
(Figure 1.11). The structure of the virus in plaque E4,
comprising two fragments of 4.1 and 1.1 kb, is consistent
with a tandem insertion of two gpt-genes in the "a"
orientation. The DNA from plaques A2, A3 and C3
exhibited more complex patterns indicative of insertions
at multiple sites which were not further analyzed.
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In summary, in the second cloning experiment five of
eight normal-sized plaques had genomic structures
expected for insertion of a single gpt-gene cassette into
the unique NotI site of the vaccinia virus genome. The
slower growing small-sized plaques exhibited unstable
structures which were lost during subsequent plaque
purification steps.
EXAMPLE 2. Direct molecular cloning of a selective
marker gene (S. coli gpt) into a unique
(NotI) cleavage site of a modified avipoxvirus
genome (fowlpox virus clone f-TR2a)
This example illustrates the general applicability
of direct molecular cloning of modified cytoplasmic DNA
virus genomes by illustrating an application to modified
avipoxvirus genomes that are engineered in vitro and
packaged in vivo. Avipoxviruses have the largest genomes
of the poxvirus family. The genome of fowlpox virus
(FPV) is about 300 kb in size, and heretofore FPV
recombinants expressing foreign genes have been
constructed only by marker rescue techniques [see, for
instance, Boyle and Coupar, Virus Res. 120: 343-356
(1988); Taylor et al., Vaccine 5: 497-503 (1988)].
The present example illustrates production of a
modified fowlpox virus by direct molecular cloning of a
gene expression cassette consisting of a poxvirus
promoter driving the E. coli gpt gene into a unique NotI
site in the genome of a recombinant fowlpox virus, f-
TK2a. This NotI site is located in a lacZ gene which was
previously inserted into this recombinant by
intracellular recombination. Engineered DNA is packaged
in primary chicken embryo fibroblasts infected with the
HP2 helper fowlpox virus which replicates more slowly
than the f-TK2a recombinant. Selection for gpt-positive
plaques leads to isolation of engineered fowlpox viruses .
Since the lacZ marker gene is inactivated by an insertion
at the NotI site, the progeny virus are distinguished
from vector virus lacking an insert, by a colorless
CA 02515166 1992-08-25
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phenotype in the blue plaque assay for lacZ gene
expression.
Purification of fowlpox virus and DNA: The fowlpox
virus (FPV) strain HP1 [Mayr & Malicki, Zentralblatt f.
Veterinarmedizin, Reihe B, 13: 1-12 (1966)] and the
attenuated strain HP1.441 (passage number 441 of HP1)
were obtained from A. Mayr, Munich. The fowlpox virus
strain HP2 was derived from HP1.441 by plaque
purification. Primary chicken embryo fibroblasts (CEF)
were prepared as described in European patent application
publ. # 0 338 807. The cells were grown in tissue
culture medium 199 (TCM 199; Gibco BRL) supplemented with
5% fetal calf serum, glutamine and antibiotics. Fowlpox
virus was purified by two successive sucrose gradients
according to Joklik, W. K., Virology 18: 9-18 (1962).
Viral DNA was prepared by the proteinase K/SDS procedure
according to Gross-Hellard et al., Eur. J. Biochem. 36:
32-38 (1973).
Construction of a fowlpox virus vector (f-TR2a)
having a unique (NotI) cleavage site in an inserted DNA
segment: The vaccinia virus tk-gene, together with the
E. coli IacZ gene was inserted into the intergenic region
between the tk-gene and the 3' -orf of fowlpox virus. The
plasmids pTKm-Wtka and pTKm-Wtkb were constructed by
cloning the functional vaccinia virus tk-gene into the
intermediate plasmid pTKm-sell. Upon intracellular
recombination of pTKm-Wtka and pTKm-Wtkb with wildtype
fowlpox virus DNA two novel FPV vectors, termed f-TK2a
and f-TK2b, respectively, were created. Each vector
contains two functional tk-genes, the endogenous FPV gene
and the inserted vaccinia virus tk-gene, in addition to
the inserted lacZ gene, any of which can be used as a
non-essential site for insertion of foreign DNA. In
particular, the NotI site in the lacZ gene is a unique
cleavage site in the f-TK2a and b vectors and, therefore,
is advantageous for direct molecular cloning of foreign
DNA into these vectors. Complete details of the
construction of the fowlpox virus vectors f-TK2a and f-
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TK2b are disclosed in EP 0,538,469.
In vivo packaging in avian cells: 8x106 CEF
cells are infected with 0.2 pfu/cell of helper
virus (HP2) for 2h. For packaging engineered FPV
genomes, 1 ~.g of purified ligation reaction product
is used. Cells are transfected with DNAs by the
calcium phosphate precipitation technique (Graham
and van der Eb, 1973 Virology 52:456:467) and
incubated for 15 min at room temperature. Nine ml
medium (TCM 199, 10% fetal calf serum, glutamine
and antibiotics) per one ml precipitate are added
to the cells. After four hours the medium is
changed and further incubated for two days. Crude
virus stocks are prepared according to standard
procedures (Mackett et al. in D.M. Glover, DNA
cloning: A Practical At~proach 191-211 (IRL Press,
1985). Plaque assays and gpt-selection are done as
described in EP 0,538,496.
Direct molecular cloning into a unique NotI
cleavage site of fowlpox virus genome: The
recombinant FPV strain f-TK2a (Scheiflinger et al.,
1991) is suitable as a vector for directly cloning
a gene cassette, for instance a model gpt gene
cassette as described herein, into a unique Notl
cleavage site. This Notl site of the vector is in
the coding region of a IacZ gene, which serves as a
color screening marker that is inactivated upon
gene insertion. Thus, IacZ-positive viruses form
blue plaques in the presence of the chromogenic
substrate X-Gal, while viruses with inserts in this
NotI site show a white plaque phenotype. The
genome of the f-TK2a vector also has incorporated
the vaccinia virus thymidine kinase (tk) gene that
also serves as an alternate gene insertion region.
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Both the lacZ and tk genes were inserted into the
fowlpox ,virus genome in the intergenic region
between the fowlpox thymidine kinase gene and the
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3'-open reading frame, by conventional methods
(Scheiflinger et al., 1991).
Patterns of DNA cleavage by NotI were established for
the genomic DNAs of FPV viruses HP1.441 and the vector
strain f-TK2a (Figure 2.1). HP1.441 was derived from a
virulent FPV strain through attenuation by serial passage
in chicken embryo fibroblasts. HP1.441 is the 441th
passage of HP1 and is used as a vaccine strain against
fowlpox (Mayr & Malicki, 1966) and is well adapted for
rapid replication in cell culture.
DNA from HP1.441 was analyzed as a reference for the
FPV vector strain f-TK2a which is a derivative of
HP1.441. The restriction analysis of the HP1.441 DNA
(Figure 2.1, lanes 1 and 2) showed that this strain has
no NotI sites. Cleavage of vector f-TK2a DNA with NotI
resulted in two large fragments of about 100 and 200 kb
(Figure 2.1, lane 4).
Direct molecular construction of a fowlpox virus
expressing the gpt gene: A model gene expression
cassette comprising the E. coli gpt gene was constructed
in the plasmid pN2-gpta which contains the gpt gene
driven by an early/late poxvirus promoter flanked by NotI
sites (Figure 1.3).
For cloning into the vector f-TK2a, the gpt- gene
cassette is excised from its plasmid and ligated with
NotI cleaved genomic DNA of f-TK2a as outlined in Figure
2.2. Ligated DNA is transfected into fowlpox helper
virus-infected CEF cells. Gpt- positive plaques that
remain white under an overlay containing X-Gal are
further analyzed by Southern blotting after infection of
chicken embryo fibroblaats. Total cell DNA is isolated
and the separated Notl fragments are subjected to
Southern blotting with 3zP-labelled DNAs of the helper
fowlpox (HP2) and gpt gene sequences, as described in
Example 1. Gpt-positive viruses containing the gpt gene
on the 1.1 kb NotI fragment indicating that correct
ligation has occurred in the cloning step.
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Production of modified viruses with both insert
orientations in one construction step: The present
example also illustrates how viruses having a single copy
of the inserted gene cassette in either orientation, as
well as viruses containing multiple copies of the
inserted gene, can be recovered from a single direct
molecular cloning step. The orientation of the DNA
insert in selected engineered fowlpox genomes is
determined by Southern blotting of DNAs cleaved .with
appropriate restriction enzymes. As shown in Figure 2.2,
the DNA inserted into a viral DNA may be in either the
"a" or "b" orientations. For preliminary analyses of
insert number and orientation with the present model gene
cassette, for instance, total DNA of cells infected with
selected plaques is digested with the restriction
endonuclease ClaI and NotI and separated on a 0.8%
agarose gel. The blot is hybridized with a gpt gene
probe and a fowlpox virus probe.
In the NotI-digested DNA samples of recombinant
viruses, the gpt cassette is excised as a 1.1 kb
fragment. Cleavage with ClaI of DNAs having an insert in
the a or b orientation also results in different
characteristic fragments hybridizing with a gpt gene
probe, as determined from the structures presented in
Figure 2.2.
EXAMPLE 3. Heterologous packaging of engineered
orthopox (vaccinia) virus genomic DNA by
an avipox (fowlpox) helper virus sad
subsequent selection for recombinants in
host cells of a species in which the
helper virus cannot replicate
Heterologous packaging of poxvirus DNA, for instance,
packaging of an orthopoxvirus DNA by an avipox virus, has
not been reported. However, the present example
demonstrates that in vi vo packaging of extracellularly
engineered vaccinia virus DNA can be achieved by fowlpox
virus in chicken embryo fibroblasts. The use of a vector
virus having a different host range from that of the
CA 02515166 1992-08-25
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helper virus provides a simple and efficient procedure
for purifying an engineered virus in one plaque assay
step. Thus, in the present example, the recombinant
orthopoxvirus was recovered by plaque assay on mammalian ,
(CV-1) cells which do not support full replication of the
avipox helper virus. Inclusion of a dominant selective
marker in the DNA inserted into the vector advantageously
facilitates the use of selective plaque assay conditions
for elimination of viruses comprising vector DNA lacking
the desired insert.
Another advantage of the heterologous packaging
approach is the reduced potential for recombination
between vector and helper viruses. For example, orthopox
and avipox viruses belong to different genera, have
different morphologies and replication facilities, and
share only minimal sequence homology as demonstrated by
a lack of cross-hybridization under standard
hybridization conditions. Therefore, homologous
recombination of the genomes of avipox and orthopox
viruses is exceedingly unlikely and use of these two
viruses can practically eliminate undesirable
recombination events that frequently occur between
homologous sequences of closely related viruses [Fenner
& Comben, Virology 5: 530-548 (1958); Fenner, Virology 8:
499-507 (1959)]. An alternative approach for preventing
vector-helper recombination during packaging is to use
recombination deficient virus strains or host cells.
In this example, a model expression cassette
comprising a marker gene (the E. coli gpt gene driven by
a poxvirus promoter) was inserted extracellularly into a
unique SmaI site of vaccinia virus DNA. The use of this
restriction enzyme to cleave the viral DNA produces blunt
ends which advantageously may be ligated to blunt-ended
DNA inserts prepared by any other nuclease that produces
blunt ends, or, for example by using a polymerase or
exonuclease to create blunt ends from an insert having
single-stranded ends.
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For packaging, the engineered genomic DNA was
transfected into fowlpox virus-infected host cells in
which both vaccinia and fowlpox viruses can replicate
(chicken embryo fibroblasts). Since the host range of
the fowlpox helper virus is restricted to avian cells,
vaccinia virus clones were selected by plaque-
purification of progeny from the transfected cells on
mammalian host cells (African Green Monkey Kidney CV-1
cells). Simultaneous selection for gpt gene expression
was used to isolation of only modified vaccinia viruses.
In contrast to the conventional method of producing
poxvirus recombinants where in one intracellular genetic
cross usually only one copy of a foreign gene can be
inserted in a single orientation, in the present example,
both possible orientations of a single insert, as well as
double insertions of the model gene cassette were
identified as products of a single extracellular genomic
modification reaction.
The experimental results in the present example show
that the packaging efficiency of ligated vaccinia virus
DNAs by fowlpox helper virus was low compared to
packaging of intact vaccinia virus DNA with fowlpox
virus, which produces yields in the range of 5x10' to
1x104 pfu per 6x106 chicken embryo fibroblasts after three
days of replication. In one packaging experiment
(producing plaques designated the "F12" series, infra)
the yield of packaged modified virus was 9x102 pfu, and
in a second experiment (producing the "F13" series),
5x102 pfu, per 6x106 chicken cells . One source of this
relatively low packaging frequency in these experiments
is the lack of dephosphorylation treatment of the vector
DNA arms which, therefore, were able to religate
efficiently without any insert. Such treatment was
omitted because dephosphorylation of blunt-ended DNA
fragments is usually inefficient. This problem can be
overcome by construction of host virus strains having
multiple cleavage sites with "sticky" ends that enable
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directional ("forced") cloning, thereby making the
insertion of foreign DNA fragments much more efficient.
Another factor influencing the packaging efficiency
is interference at the cellular level between the helper
and the packaged virus. Under standard packaging
conditions, within three days of incubation the helper
virus (fowlpox) usually replicates to titers of about
1x10s pfu per 6x106 chicken embryo fibroblasts. The large
excess of fowlpox virus compared to packaged vaccinia
virus creates conditions that produce negative
interference phenomena and inhibits replication of the
packaged virus.
This interference is minimized by using mammalian
cells for packaging in combination with fowlpox helper
virus as described in Example 7. In that case, the host
cells do not support full replication of the helper
fowlpox virus. Although, no testing of ligated vaccinia
virus DNA for packaging efficiency by fowlpox virus has
been made in a mammalian host cell, a packaging yield of
2x106 pfu per 8x106 mammalian (CV-1) cells was obtained
with uncleaved vaccinia virus DNA.
In each viral recombinant generated by intracellular
recombination with a given insertion plasmid an insert
has one orientation depending on the polarity of the
homologous flanking regions in that plasmid. Due to
transcriptional interference phenomena, for instance (Ink
& Pickup, 1989), expression levels for genes inserted
into a poxvirus vector depend on the orientation of the
foreign gene relative to the viral genome. Therefore, it
is desirable to obtain in one reaction step modified
viruses having either possible orientation. One of the
advantages of the procedure in this example is that both
possible orientations of the inserted DNA are obtained in
one ligation reaction, allowing immediate screening for
variants having the highest expression level. The
preferred orientation of the cassette of this example in
the selected SmaI insertion site of vaccinia virus is the
"b" orientation, as evidenced by the fact that the
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majority of modified viruses had this genomic structure.
In this cassette the P7.5 promoter controlling the
foreign gene is in the inverted repeat orientation
relative to the endogenous 7.5 kDa polypeptide gene. As
discussed in Example 1, the endogenous 7.5 kDa
polypeptide genes are located in the inverted terminal
repetitions of the vaccines genome. The distance of the
P7.5 promoter of the gpt-gene and the P7.5 promoter in
the left terminal repetition is about 20 kb. The. "a"
orientation should therefore be less stable and less
frequently obtained, in accordance with the observation
that this orientation was found only twice. However, the
viral isolates F13.4 (orientation a) and vF12.5
(orientation b) were propagated to large scale with gpt-
selection and were found to have stable predicted
structures. The stability of the various structures
comprising multiple inserts without selection remains to
be determined.
The legations contained several-fold excess of insert
over the vector, thereby favoring insertion of multiple
copies of the cassette as observed. However, it is
unclear why in this example double insertions were more
frequent than in Example 1. Due to internal
recombination events only certain configurations of
multiple inserts are expected to be stable. Further
studies to evaluate stability of viruses with multiple
inserts and the optimal ratio of vector to insert for
stability and expression level which depends on copy
number can all be conducted as necessary for each
construct, according to the teachings of this
application.
Purification of virus and DNA: The viruses and
methods of Examples 1 and 2 were used.
Engineering of viral DNA: Viral DNA purified from
virions was cleaved with SmaI and purified by one phenol
extraction and three chloroform extractions. In the
first experiment below, 2 ~.g of cleaved virus DNA were
legated with 400 ng (34 fold molar excess) of the insert
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fragment (the 1.1 kb HpaI-DraI fragment excised
from plasmid pTKgpt-Fls) in a volume of 30 ~.l for
40 h with 15 units of T4 ligase (Boehringer, Inc.).
The second ligation experiment was done under the
same conditions except that a seventeen-fold molar
excess of the 1.1 kb SmaI insert and 5 units of
ligase were used.
In vivo heterologous packaging in avian cells:
Chicken embryo f ibroblasts ( 6x106 ) infected with the
helper virus (0.5 pfu/cell of HP1.441) and
incubated for 2 h. Two ~.g of ligated DNA was
transfected into the infected cells and treated.
further as described for the homologous packaging
procedure in Example 1. The initial plaque assay
was done in CV-1 cells as described in Example 1.
Demonstration of packaging of modified
vaccinia virus DNA by fowlpox helper virus: The
design of this experiment is shown in Figure 3.1.
Vaccinia virus genomic DNA was prepared from
sucrose gradient purified virions, cut with the
restriction endonuclease SmaI, and ligated with the
blunt-ended foreign gene cassette. Ligated DNA was
transfected into fowlpox virus-infected chicken
embryo fibroblasts for packaging. Progeny virus
was identified by plaque assay on mammalian (CV-1)
cells which do not support complete replication of
fowlpox virus to produce infectious virions.
In more detail, first, the HpaI-DraI fragment
bearing the model gene cassette (containing the gpt
gene driven by the vaccinia virus P7.5 promoter)
was excised from the plasmid pTKgpt-FIs (Falkner &
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Moss, 1988 J: Viro1 62:1849-1854) and ligated
directly into the unique SmaI site of vaccinia
wildtype virus (WR strain). The gpt gene was
selected to permit positive selection of modified
viruses (Boyle & Coupar, 1988, Virus Res.
120:343:356; Falkner & Moss, 1988 J. Virol 62:1849-
1854). The single SmaI site in vaccinia virus DNA
is located in the open reading frame A51R in the
HindIII A fragment of the genome. The A51R gene
is non-essential for viral replication in cell
culture (Geobel et al., 1990 Virology 179:247-266).
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Ligated material was transfected into chicken embryo
fibroblasta infected with fowlpox helper virus. After
three days the cells were harvested and a crude virus ,
stock was prepared. Packaged vaccinia virus was
identified by plaque assay on an African Green monkey
kidney cell line (CV-1) in medium that selects for cells
infected with a virus carrying the gpt gene. This
selection scheme prevents viruses containing self-ligated
wildtype vaccinia virus DNA from forming plaques while
allowing modified viruses containing an inserted model
gpt gene cassette to do so.
The packaging frequency was low in initial
experiments. The titer of gpt-positive vaccinia virus in
the crude stock prepared from 6x106 chicken embryo
fibroblasts was in the range of 1 x 102 to 1 x 103 pfu.
Thirteen gpt-positive plaques were amplified under
gpt-selection in CV-1 cells. Total DNA of infected cells
was isolated, digested with HindIII, separated on a 0.7%
agarose gel and further processed for analysis by
Southern blotting with a gpt-gene probe. As shown in
Figure 3.2, several viruses having blot patterns
predicted for different modified genomic structures were
obtained.
In lanes 2, 4, 11 and 13 (corresponding to plaques
#F12.3, F12.5, F13.3 and F13.5) a single hybridizing
fragment of about 45 kb is visible, that is expected when
one copy of the gene cassette is inserted into the viral
genome in the "b" orientation into the viral genome (see
Figure 3.3). An expected novel fragment of 5.2 kb is
also present in all cases, and also appears when the same
DNAs are tested as in Figure 3.2 using a vaccinia virus
probe.
Two viruses having patterns consistent with the "a"
orientation were obtained in lanes 7 and 12
(corresponding to plaques #F12.8 and F13.4), where a
single gpt-hybridizing fragment of about 5.7 kb is
expected. The 5.7 kb fragment in lane 7 is more visible
in longer exposures of the autoradiograph. The pattern
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seen in lane 5 (plaque F12.6) may represent a single
insert in the "a" orientation, but the expected 5.7 kb
band is somewhat larger for unknown reasons.
The pattern of three viral isolates is consistent
with a tandem insertion in the "a" orientation (lanes 1,
6 and 10, corresponding to plaques #F12.2, F12.7 and
F13.2). In these cases two gpt-positive hybridizing
fragments, of 5.7 and 1.1 kb, are expected (see also
Figure 3.3). Fragments of 5.7 and 1.1 kb were. also
observed in equimolar amounts with the viral DNA in a
blot hybridized with a vaccinia virus probe.
The genome of the isolate in lane 3 (plaque F12.4)
probably contains a tandem duplicate insert in the "b"
orientation. In this case two fragments, of 45 kb and
1.1 kb, are expected to hybridize with the gpt-gene.
The viral DNA in lane 9 (plaque F13.1) may comprise
a head-to-head double insertion. In this case a 45 kb
and a 5.7 kb fragment hybridizing with a gpt-gene probe
are expected. However, in addition such a DNA should
contain a novel 0.6 kb fragment that hybridizes with a
vaccinia DNA probe, and, in fact, this fragment was
detected on a blot hybridized with a vaccinia probe.
Nevertheless, the expected 5.7 kb fragment was somewhat
smaller than predicted and produced a hybridization
signal that was weaker than expected. Therefore,
confirmation of the structure of this recombinant
requires more detailed analysis.
Further analysis revealed that the viruses F12.7 and
F12 . 3 , interpreted above as having double insertions with
tandem 'a' structures, and the virus F12.4, interpreted
above as having double insertions with tandem 'b'
structures, actually have multiple tandem inserts in the
'a' or 'b' orientations, respectively. The Southern blot
analysis of Figure 3.2 does not distinguish between
double tandem and multiple tandem inserts.
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EXAMPLE 4. Construction of an orthopoxvirus
(vaccinia) vector (vdTK) with a
directional master cloning site and
plasmids with compatible expression
cassettes
This example demonstrates application of the methods
of the present invention to create novel poxvirus cloning
vectors by direct molecular modification and cloning of
existing poxvirus genomes. In particular, this example
describes a vaccinia virus vector (vdTK) which allows
directional insertion (i.e., "forced cloning") of foreign
genes into a short "multiple cloning site" segment
comprised of several different endonuclease cleavage
sites each of which is unique in the vector genome.
Forced cloning eliminates the need for selection or
screening procedures to distinguish the desired
recombinants from vector virus lacking an insert because
incompatibility of DNA ends cleaved by different
nucleases prevents religation of the vector arms without
a foreign insert. Consequently, the forced cloning
approach is the most efficient way to insert a foreign
gene into a viral vector.
The directional vector vdTK is created by inserting
a multiple cloning site (comprised of unique NotI, SmaI,
ApaI and RsrII sites) in place of the thymidine kinase
(tk) gene of vaccinia virus (see Figure 4.1A). This
nonessential locus is the site most frequently used for
insertion of foreign genes into vaccinia virus, mainly
because positive selection for tk-negative viruses is
available. Thus, when ligated vdTK vector DNA is
packaged by a tk-positive helper virus, the vector virus
may be positively selected from the excess of helper
virus. Further, insertion of foreign DNA into the
vaccinia virus tk-locus by conventional methods generally
results in stable recombinants.
The multiple cloning site of the new vdTK vector is
comprised of NotI and SmaI cleavage sites which are
unique in the vector. Prior to insertion of the multiple
cloning site, NotI and SmaI Cleavage sites preexisting in
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the wildtype vaccinia virus (WR strain) are deleted by
direct molecular modifications according to the present
invention. Viruses having the desired modifications are
detected by screening techniques based on the polymeraee
chain reaction (PCR) method for amplification of specific
nucleic acid sequences. This example also describes
a set of plasmids which facilitate expression of DNAs
encoding complete or partial open reading frames in the
vdTK vaccinia vector. The present invention comprehends
insertion of open reading frames directly into a poxvirus
expression vector having all appropriate regulatory
elements suitably placed for expression of the inserted
open reading frame. However, the instant vdTK vector is
not equipped with such regulatory sequences for
expression of an inserted open reading frame that lacks
its own transcription and translation signals.
Accordingly, the plasmids of this example provide
convenient gene expression cassettes for routine linkage
of open reading frames to poxvirus promoters and,
optionally, to a translation start codon. An open
reading frame and associated regulatory sequences are
then efficiently transferred into the vdTK vector master
cloning site by forced cloning. Modified viruses having
the insert in either orientation can be obtained by using
one of two plasmids having the expression cassette in the
desired orientation within its master cloning site. The
gene expression cassettes of the plasmids exemplified
here have two nested sets of restriction enzyme cleavage
sites to facilitate cloning of open reading frames into
the vdTK vector. The cassettes have a master cloning
site comprised of the same unique sites as the master
cloning site of the vdTK vector. In addition, in the
middle of this master cloning site the cassettes contain
a variety of sites for frequently cutting enzymes that
are useful for insertion of open reading frames into the
cassettes. Thus, DNAs inserted into a cassette by means
of the frequent cutter sites are flanked on either side
by several different unique sites which are suitable for
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forced cloning of the cassette into the master cloning
site of the vdTK vector.
This example also describes gene expression cassettes
suitable for insertion into a single unique site in the
vaccinia virus vector vdTK. To overcome the reduced
cloning efficiency of using a single enzyme for cleaving
the vector DNA, the expression cassettes of these
plasmids include the E. coli gpt gene as a selective
marker.
The vdTK vaccinia vector system is preferentially
used in conjunction with the heterologous packaging
procedure described in Examples 3 and 7. The plasmids
containing the gpt marker can also be used with
homologous helper virus lacking the gpt marker. Examples
of constructs for expression of polypeptides using the
vdTK vector and related plasmid system are presented
hereinbelow in Example 5.
In addition to the above advantages, the expression
cassette plasmids of this invention also provide a means
of overcoming a general problem of incompatibility
between the ends of cleaved poxvirus vector DNAs and many
insert DNAs, as a convenient alternative to the common
use of synthetic adaptor DNA segments. Thus, isolation
of DNA fragments encoding open reading frames usually is
facilitated by use of restriction endonucleases having
recognition sequences which are short and, consequently,
randomly occur at high frequencies in all natural DNA
sequences. On the other hand, such frequently cutting
enzymes generally are not suitable for efficient direct
cloning into genomes as large as those of poxviruses, for
instance, because such enzymes cleave large DNAs into
many fragments. Religation of these fragments would
occur in random order, producing few intact viral
genomes. Therefore, insertion sites in a vaccinia vector
preferably are cleavage sites of infrequently cutting
restriction endonucleases which are unlikely to be used
for isolation of open reading frame fragments or insert
DNAs in general. The present plasmids overcome this
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general incompatibility by allowing efficient insertion
of fragments from frequent cutters into the plasmid
followed by efficient transfer into the vaccinia vector
using infrequently cutting enzymes.
Deletion of the unique NotI cleavage site from
wildtype vaecinia (wR) viruss The unique NotI site of
vaccinia virus may be eliminated by insertion into this
site of a "NotI deletion adaptor" segment having cohesive
ends compatible for ligation with NotI-cleaved DNA but
lacking sequences required for recognition by the NotI
endonuclease. Thus, the sequences formed by the ligated
cohesive ends of the NotI-cleaved viral DNA and viral DNA
and adaptor are not cleavable by NotI. This adaptor also
contains several selected restriction endonuclease
cleavage sites for directed insertion of DNA fragments.
More particularly, one ~.g of vaccinia virus WR wild
type DNA is cut with NotI and ligated with one ~g of the
double-stranded NotI-deletion adaptor. The adaptor
consists of two partially complementary strands: odNl
(SEQ. ID. NO. 16) and odN2 (SEQ. ID. N0. 23). The
central part of the adaptor contains the restriction
endonuclease cleavage sites StuI, DraI, SspI and EcoRV.
Annealed adaptor oligonucleotides are used for the
ligation reaction. The ligated material is transfected
into fowlpox virus-infected chicken embryo fibroblasts
and packaged as described in Example 3.
An alternative procedure for deleting the single NotI
site of vaccinia virus (WR strain) is outlined in Figure
4.1, panel B. In the first step, vaccinia virus DNA is
cut with SacI, the SacI "I" fragment is isolated from low
melting point agarose and cloned into the SacI site of a
suitable plasmid, such as pTZl9R (obtainable from
Pharmacia, Inc.). The resulting plasmid, pTZ-SacI, is
cut with NotI, treated with Klenow polymerase to fill in
the sticky ends and religated. The ligated material is
transfected into E. coli cells (HB101). The colonies are
isolated according to standard cloning procedures. The
resulting plasmid, pTZ-SacIdN has the NotI site deleted
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and is used in a reverse gpt-selection experiment as
described by Isaacs, S. N.. Kotwal, G. & Moss B. Virology
178: 626-630 (1990), modified as follows:
CV-1 cells (8 x 106) are infected with 0.2 pfu of the '
viral isolate vp7, a vaccinia virus that has integrated
into the single NotI site a gpt-gene cassette (see
Example 1). Subsequently, a calcium-phosphate
precipitate containing 20 ~g of DNA from the modified
SacI fragment prepared from the plasmid
pTZ-SacIdN is transfected into the cells. The cells axe
further treated as described in the packaging procedure
in Example 1. Crude virus stocks are used to infect
mouse STO cells (obtained from the American Type Culture
Collection, Rockville, MD; ATCC# CRL 1503) in the
presence of 6-thioguanine (6-TG). This is a negative
selection procedure that requires the loss of the gpt-
gene for a virus to replicate (Isaacs et al., 1990) and,
therefore, leads in the present case to integration of
the modified SacI "I° fragment and, thereby, deletion of
the gpt gene. All plaques growing in the presence of 6-
TG should lack the gpt gene and contain a modified SacI
I fragment. The estimated yield is in the range of 0.1-
0.2 % of the total plaques (i.e., the normal frequency of
recombinants in this type of marker rescue experiment).
Since the selection procedure is extremely efficient
(Isaacs et al., 1990) identification of the correct
structures is not expected to require examination of
large numbers of clones. However, whether the first
procedure above or this alternative procedure is used to
delete the single NotI of vaccinia virus, the following
screening procedure may be used to identify the desired
construct.
Identification by PCR-screening of virus (vdN) having
the NotI site deleted: Vaccinia virus clones having the
NotI site deleted may be identified by analysis of
plaques growing in a cell line (CV-1) that does not
support the growth of the fowlpox helper virus . The DNAs
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of viruses in individual plaques are analysed by a
PCR-based screening method, as follows.
The first primer for the PCR reaction is the
oligonucleotide odNl (SEQ. ID. NO. 16) and the
second primer was odN3 (SEQ. ID. NO. 24). The
sequence of second primer is located in the
vaccinia virus genome about 770 by downstream of
the first primer sequence. The template is total
DNA from I x 106 CV-1 cells infected with half the
virus of a single plaque. DNA is prepared by
standard techniques and about 50 ng is used for the
PCR reaction. The PCR reactions are carried out
according to standard techniques using commercially
available PCR kits. Positive PCR reactions produce
a DNA fragment of about 770 bp. Such a virus
having the NotI site deleted is designated "vdN".
Deletion of the unique SmaI restriction site
from vaccinia virus vdN: The WR strain of vaccinia
virus contains a single SmaI site in an open
reading frame (A51R) which is not essential for
virus replication in cell cultures (Goebel et al.,
1990 Virology 179:247-266). Although this site may
be used for foreign gene insertion, in the present
example, however, this site is deleted in favour of
creating a more versatile vaccinia virus vector by
introducing a new unique SmaI site as part of
multiple cloning site cassette.
Accordingly, vdN virus DNA (1 ~.g) is cut with
SmaI and ligated with an excess of hexamer linker
having the recognition sequence for the restriction
nuclease HindIII (odSl, 5'-AAGCTT-3'). Insertion
of this linker into the vaccinia virus SmaI
cleavage site results in destruction of the SmaI
recognition sequence and the introduction of a new
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HindIII recognition sequence. The ligated material
is packaged by transfection into cva cells that
have been infected with fowlpox virus, as described
in Example 7.
Alternatively, the single SmaI site of
vaccinia virus (WR strain) is deleted according to
the procedure outlined in Figure 4.1, panel C, by
modifying a cloned fragment of vaccinia virus DNA
instead of directly
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modifying the complete vaccinia virus DNA. In a first
step, vaccinia virus DNA is cut with SalI, the SaII F-
fragment is isolated from low melting point agarose and
cloned into the SalI site of a suitable plasmid, such as
pTZ9R (obtainable from Pharmacia, Inc.). The resulting
plasmid, pTZ-SalF, has two SmaI sites, one in a multiple
cloning site and the other in the vaccinia sequences
(Figure 4.1, panel C). pTZ-SalF is partially digested
with Smal and I-SceI linkers are added, as follows:. first
strand, I-SceI linker 1 (SEQ. ID. N0. 25) and its
complementary strand, I-SceI linker 2 (SEQ. ID. NO. 26).
The correct plasmid having the SmaI site deleted from the
vaccinia sequences is identified by cleavage with Smal
and I-SceI. The final plasmid, pTZ-SalFdS, is used to
introduce the SmaI deletion into a vaccinia virus genome
using the reverse gpt-gene selection experiment as
described for deletion of the NotI site, except that
preferred virus to be modified is the isolate F12.5, a
virus that has integrated into the single SmaI site a
gpt-gene cassette (see Example 3).
The resulting insertion of a site for endonuclease
I-SceI advantageous for direct molecular cloning because
this enzyme, isolated from yeast, recognizes an l8mer
site and, therefore, cuts random DNA sequences extremely
infrequently. For instance, I-SceI cuts the yeast genome
only once. Thierry, A., Perrin, A., Hoyer, J., Fairhead,
C., Dujon, B., Frey, B. & Schmitz, G. Nucleic Acids Res.
19: 189-190 (1991). I-SceI is commercially available
from Boehringer, Inc. Advantageously, an I-Scel site is
introduced into a vector having nv preexisting sites for
that enzyme, thereby creating a new vector with a single
site that can be used for gene insertions. Whether a
vaccinia virus DNA or other vector DNA contains a site
for I-Scel cleavage can be determined by routine
restriction analyses of the vector DNA.
Where this alternative procedure for deletion of the
SmaI site from vaccinia virus DNA is used, the order of
steps for constructing the vector vdTK is as follows:
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deletion of the SmaI site resulting in virus vdS (see
above); deletion of the NotI site by insertion of the
NotI gpt-gene cassette (see Example 1) into the single
NotI site of vdS by cloning and packaging, resulting in
the virus vdSNgpt and reverse gpt-selection as described
above, using vdSNgpt and pTZ-SacIdN as substrates for the
marker rescue experiment; and deletion of the tk-gene as
outlined in below in the present example.
An alternative procedure by which the vector.vdTK
actually was constructed is as follows . The SmaI site of
vaccinia wild-type virus was deleted, creating the
intermediate virus vdS. In a second experiment the NotI
site was deleted from vaccinia wild-type virus creating
the intermediate virus vdN. The virus vdSN was obtained
by co-infection using both viruses of Cv-1 cells and PCR
screening of the recombinant virus (that was created by
a simple genetic cross-over event). The viability of the
different intermediates was determined by titrations.
Table 4.1 A shows the results after individual
isolates from the vdN cloning experiment were plaque
purified five times (to insure that wildtype virus-free
clones were obtained) and then amplified. After
titration, crude virus stocks of the first amplification,
together with wild-type control (WR-WT), were used to
infect CV-1 cells at 0.1 pfu/cell. These cells were
harvested after 48h and used to prepare crude stocks
which were re-titered. These results are shown in Table
4.1 H. Isolates vdN/A1 #6.1111 and vdN/A1 #10.1111 were
designated as clones vdN#6 and vdN#10, respectively, and
used for large scale virus preparations.
Table 4.2 A shows the results after single isolates
of the vdS cloning experiment were plaque purified five
times and then amplified and titered. Crude stocks of
the first amplification, together with wild-type control
(WR-WT), were used to infect CV-1 cells at 0.1 pfu/cell.
The cells were harvested after 48h and the resulting
crude stocks were re-titered. These results are shown in
Figure 4.2 B. The isolates vdS# '1.11 were designated as
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clones vdS#2 and vdS#7, respectively, and used for large
scale virus preparations. In each case, the virus
isolate showing the best growth characteristics was
selected to be amplified and grown to large scale.
Table 4.1
Viability Studies of the Viral Intermediate v~lN
A) Titer after first amplification of six viral
vdN-isolates (pfu/ml crude stock):
vdN/A1# 2.1111 1.0 x 10' pfu/ml
vdN/A1# 4.1111 1.3 x lOs pfu/ml
vdN/A1# 6.1111 9.0 x 10' pfu/ml
vdN/A1# 8.1111 8.0 x 10' pfu/ml
vdN/Al# 10.1111 4.0 x 10' pfu/ml
vdN/A1# 12.1111 1.1 x l0a pfu/ml
B) Titer after second amplification:
vdN/A1# 2.1111 3.6 x l0a pfu/ml
vdN/A1# 4.1111 2.5 x lOa pfu/ml
vdN/A1# 6.1111 5.9 x lOs pfu/ml
vdN/A1# 8.1111 4.2 x 10$ pfu/ml
vdN/A1# 10.1111 4.3 x l0a pfu/ml
vdN/A1# 12.1111 2.2 x 108 pfu/ml
WR-WT 5.4 x l0E pfu/ml
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Table 4.2
Viabilitv Studies of the Viral Intermediates vdS
A) Titer after first amplification of five viral
vdS-isolates (pfu/ml crude stock)
vdS# 2.11 4.1 x 10' pfu/ml
vdS# 3.11 6.5 x 10' pfu/ml
vdS# 4.11 8.0 x 10' pfu/ml
vdS# 5 .11 2 . x 10' pfu/ml
?
vdS# 7.11 4.7 x 10' pfu/ml
B) Titer after second amplification
vdS# 2.11 1.6 x l0a pfu/ml
vdS# 3.11 1.4 x l0a pfu/ml
vdS# 4.11 8.0 x 10' pfu/ml
vdS# 5.11 1.3 x 10 pfu/ml
vdS# 7.11 1.7 x 10 pfu/ml
WR-WT 2.8 x pfu/ ml
lOs
Identification by PCR-screening of virus (vdSN)
having the SmaI site deleted: Clones of the vdSN
vaccinia virus having the SmaI site deleted are
identified by PCR screening as follows.
The first primer for the PCR reaction is the
oligonucleotide odS2 (SEQ. ID. NO. 27) and the second
primer is the oligonucleotide odS3 (SEQ. ID. NO. 28).
The sequence of oligonucleotide odS2 is located in the
vaccinia genome about 340 by upstream of the SmaI site,
while that of oligonucleotide odS3 is located about 340
by downstream of this site. The template is total DNA of
CV-1 cells infected with a virus plaque as described
above for vdN identification. The PCR-amplified band of
about 680 by is tested for susceptibility to SmaI, with
resistance to SmaI cleavage indicating insertion of the
HindIII or I-SceI linker, while wildtype control DNA is
cut into two pieces of about 340 bp. A vaccinia virus
having the desired insertion of a linker in the Smal site
is designated vdSN.
Deletion of the coding region of the thymidine kiaase
gene from vaccinia virus vdSN: From vaccinia virus vdSN,
a novel vector strain (designated vdTK) is developed by
replacing the thymidine kinase (tk) gene, which is
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located in a genetically stable region of the vaccinia
genome, with a segment comprised of several unique
restriction endonuclease cleavage sites (Figure 4.1A).
The thymidine kinase (tk) coding sequence is first
deleted f rom a plasmid (pHindJ-1 ) comprising a segment of
the vaccinia genome (the HindIII J segment) in which the
tk gene is located (see Figure 4.2). In place of the
tk-gene, a multiple cloning site with the unique sites
NotI, SmaI, ApaI and RsrII, flanked by SfiI sites is. then
inserted. Finally, the modified virus segment is
transferred into the vaccinia virus genome vdSN which was
then designated vdTK (Figure 4.1A). To further
facilitate forced cloning, each of the two SfiI sites
also may be made unique in the vector by exploiting the
variable nature of the SfiI recognition sequence
(GGCCNNNN'NGGCC). The sequences of two SfiI sites are as
follows: SfiI(1), GGCCGGCT'AGGCC (SEQ. ID. N0. 29) and
SfiI(2), GGCCATAT'AGGCC (SEQ. ID. NO. 30). This plasmid
containing the final modification of the tk gene (pHindJ-
3) is constructed from precursor plasmid pHindJ-1 by
loop-out mutagenesis, and deletion of the tk-gene is
confirmed by sequence analysis.
Construction of precursor plasmid pHindJ-1: Vaccinia
wildtype virus DNA was cut with HindIII and the resulting
fragments were separated on a 0.8% low melting point
agarose gel. The HindIII J fragment was excised under
W-light and prepared according to standard techniques.
The fragment was inserted into the single HindIII site of
the plasmid pTZl9R (Pharmacia, Inc.) resulting in pHindJ
1.
Coastructioa of plasmid pHindJ-2: Plasmid pHindJ-1
is transfected into E. coli strain NM522 and single-
stranded DNA is prepared by superinfection with the
helper phage M13K07 according to the protocol supplied by
Pharmacia. The single-stranded DNA serves as the
template for site directed mutagenesis with the primer
odTKl (SEQ. ID. NO. 31). This primer is complementary to
the promoter region and the region around the
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translational stop codon of the tk-gene. In its central
part it contains the unique restriction sites HamHI,
HpaI, NruI and EcoRI. The mutagenesia procedure is
carried out with a mutagenesis kit provided by Amersham,
Inc., according to the manual provided by the supplier.
For construction of pHindJ-2, the tk-gene sequence
has been described in Weir J.P. & Mosa B. J. Virol. 46:
530-537 (1983). The tk-gene sequence is accessible in
the EMBL Data Library under the identifier (ID) PVHZNLJ.
The sequence of the vector part (pTZl9R) of the plasmid
is available from Pharmacia, Inc. The sequence around
the deleted vaccinia virus thymidine kinase (tk)-gene in
the plasmid pHindJ-2 is shown in SEQ. ID. N0. 4. The 5'
region of the tk-gene (bases #1-19 in the present
listing; bases #4543-#4561 in ID PVHINLJ) is followed by
the unique restriction sites HamHI, HpaI, NruI and EcoRI
and the 3' region of the tk-gene (bases # 44-#67 present
listing; bases #5119-#5142 in ID PVHINLJ). Bases # 4562
to 5118 in ID PVHINLJ, which contain part of the
tk-promoter and the tk-gene coding region, are deleted in
pHindJ-2.
Construction of the plasmid pHiadJ-3: Plasmid
pHindJ-2 is digested with HamHI and EcoRI and a double-
stranded linker containing the unique restriction sites
NotI, SmaI, RsrII and ApaI, flanked by SfiI sites is
inserted. The linker consists of oligonucleotides P-J(1)
(SEQ. ID. NO. 32) and P-J(2) (SEQ. ID. NO. 33).
The modified sequence of pHindJ-3 is shown in SEQ.
ID. N0. 5. The inserted multiple cloning site
corresponds to oligonucleotide P-J(1). The inserted
sequence starts at position 21 and ends at position 99.
The flanking sequences are the same as described in
pHindJ-2, supra.
To insert the tk-deletion into vaccinia virus,
plasmid pHindJ-3 is digested with HindIII and a shortened
HindIII J fragment having a tk-gene deletion is used for
a marker rescue experiment as described by Sam and
Dumbell, 1981. Viruses having the tk-gene deleted are
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isolated by tk-negative selection (Mackett et al., 1982)
and identified by subsequent PCR screening.
More particularly, the modified HindIII fragment
present in pHindJ-3 is excised with HindIII and isolated
with a low melting point agarose gel. The marker rescue
is performed essentially as described by Sam and Dumbell
(1981) with the following modifications. 5 x 106 CV-1
cells are infected with 0.2 pfu per cell of vaccinia
virus vdSN. After one hour of incubation, one ml . of a
calcium-phosphate precipitate containing 1 ~,g of the
modified HindIII J fragment is transfected into the
infected cells. After two days growth a crude virus
stock is prepared as described in Example 1 and titrated
on human 143H tk-negative cells in the presence of
bromodeoxy-uridine (BrdU) as described by Mackett et al.,
1982. Tk-negative plaques may be further analyzed by PCR
screening.
Identification of the thymidine kinase deletion virus
(vdTR) by PCR-screening: The first primer for the PCR
reaction is oligonucleotide odTK2 (SEQ. ID. NO. 34), the
sequence of which is located about 300 by upstream of the
tk-gene. The second primer, odTK3 (SEQ. ID. NO. 35), is
located about 220 by downstream of the stop codon of the
tk-gene. The template is total DNA of CV-1 cells
infected with a virus plaque, as described for vdN
screening. The amplification product resulting from
virus having the tk-gene deletion is about 520 bp, while
the wildtype control produces a fragment of about 1.1 kb.
Construction of plasmids comprising gene expression
cassettes for transfer to the vdTR vector: The plasmid
pA0 is the basic plasmid that contains a master cloning
site comprised of the unique sites of the master cloning
site of the vdTK vaccinia virus vector. Plasmid pA0 was
constructed by replacing the multiple cloning site of a
commercially available plasmid with a segment comprised
of the unique sites of the vdTK vector and an XhoI site,
as illustrated in Figure 4.3.
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More in particular, to delete the multiple cloning
site of the pBluescript II SK- phagemid (Stratagene) , the
plasmid was digested with Sacl and Asp718. The large
vector fragment was ligated with an adaptor consisting of
the annealed oligonucleotides P-A(0.1) (SEQ. ID. N0. 36)
and P-A(0.2) (SEQ. ID. NO. 37).
The multiple cloning site of pA0 (corresponding to
the oligonucleotide P-A(0.1)) and twenty bases of the 5'-
and 3'-flanking regions of pBluescriptII SK- are shown in
SEQ. ID. NO. 6. The insert starts at position 21 and
ends at position 95. (The first "A" residue at the
5'-end corresponds to position number 2187, the last "G"
residue at the 3' -end corresponds to position number 2301
of the plasmid pA0).
Construction of the plasmids pAl and pA2: The
plasmids pAl and pA2 were designed for insertion of DNA
segments, e.g., synthetic or natural promoter fragments.
They were constructed by inserting into the Xhol site of
pA0 a linker comprising a second multiple cloning site of
frequently cutting enzymes that do not cleave pAO. Both
plasmids have the same structure except for the
orientation of the second multiple cloning site (Figure
4.3) .
The pA0 plasmid was digested with XhoI and ligated
with an adaptor consisting of the annealed
oligonucleotides P-A ( 1.1 ) and P-A ( 1. 2 ) . Plasmids of both
possible orientations of the adaptor were isolated and
designated pAl and pA2.
The multiple cloning site of pAl (corresponding to
the oligonucleotide P-A(1.1)) and twenty bases of the 5'
and 3'-flanking regions of pA0 are shown in SEQ. ID. NO.
7. The insert starts at position 21 and ends at position
83. (The first "C" residue at the 5'-end corresponds to
position number 2222, the last "C" residue at the 3'-end
corresponds to position number 2324 of the plasmid pAi).
The multiple cloning site of pA2 (corresponding to
the oligonucleotide P-A(1.2)) and twenty bases of the 5'
and 3' -ends of pA2 are shown in SEQ. ID. N0. 10. The
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insert starts at position 21 and ends at position 195.
(The first "C" residue at the 5'-end corresponds to
position number 2252, the last "G" residue at the 3'-end
corresponds to position number 2466 of the plasmid
pA2-S1) .
Construction of plaamids pAl-Sl and pA2-Sl: Plasmids
pAl-S1 and pA2-S1 provide the strong synthetic poxvirus
promoter S1, including a translational start codon,
followed by a single EcoRI site suitable for insertion of
open reading frames that do not have an associated start
codon. Promoter S1 is a modified version of a strong
poxvirus late promoter designated P2.
Plasmids pAl-S1 and pA2-S1 are obtained by inserting
a first double-stranded promoter fragment into the NdeI
and BamHI site of pAi or pA2, respectively, by forced
cloning (Figure 4.4, panel A) In particular, vector pAl
is digested with NdeI and BamHI and ligated with an
adaptor consisting of the annealed oligonucleotides P
P2m1.1 and P-P2m1.2. The resulting plasmid is designated
pAl-S1.
The synthetic promoter sequence of pAl-S1
(corresponding to the oligonucleotide P-P2m1.1) and
twenty bases of the 5'- and 3'-flanking regions of pAl
are shown in SEQ. ID. NO. 9. The insert starts at
position 21 and ends at position 193. (The first "C"
residue at the 5 'end corresponds to position number 2228,
the last "G" residue at the 3 'end corresponds to position
number 2440 of the plasmid pAl-S1).
The vector pA2 was digested with NdeI and BamHI and
ligated with an adaptor consisting of annealed
oligonucleotides P-P2m1.1 and P-P2m1.2, as for pAl-S1,
above. The resulting plasmid is designated pA2-S1.
The synthetic promoter sequence of pA2-S1
(corresponding to the oligonucleotide P-P2m1.2) and
twenty bases of the 5'- and 3'-flanking regions of pA2
are shown in SEQ. ID. N0. 10. The insert starts at
position 21 and ends at position 195. (The first "C"
residue at the 5 'end corresponds to position number 2252,
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the last "G" residue at the 3 'end corresponds to position
number 2466 of the plasmid pA2-S1).
Construction of plasmids pAl-S2 and pA2-S2:
The plasmids pAl-S2 and pA2-S2 contain the strong
synthetic promoter S2, a modified version of a strong
late synthetic poxvirus promoter described by Davison &
Moss, J. Mol. Hiol. 210: 771-784 (1989). These plasmids
do not provide a translational start codon with the
promoter and, therefore, are suited for insertion of
complete open reading frames that include a start codon.
The promoters have different orientations with respect to
the vdTK master cloning site in these two plasmids.
Plasmids pAi-S2 and pA2-S2 are obtained by forced
cloning of a second double-stranded promoter fragment
into the HpaI and EcoRI sites of pAl and pA2,
respectively (Figure 4.5, panel A). More particularly,
plasmid pAl is digested with the enzymes HpaI and EcoRI,
and ligated with a synthetic linker sequence consisting
of annealed oligonucleotides P-artP(5) and P-artP(6).
The resulting plaamid is designated pAl-S2.
The synthetic promoter sequence of pAl-S2
(corresponding to the oligonucleotide P-artP(5) and
twenty bases of the 5'- and 3'-flanking regions of pAl
are shown in SEQ. ID. N0. 11. The insert sequence starts
at position 21 and ends at position 68. (The first "T"
residue at the 5'-end corresponds to position number
2240, the last "A" residue at the 3'-end corresponds to
position number 2327 of the plasmid pAl-S2).
Similarly, the plasmid pA2 is digested with the
enzymes HpaI and EcoRI, and ligated with the annealed
oligonucleotides P-artP(5) and P-artP(6) as for pAi-S2.
The resulting plasmid is designated pA2-S2. The
synthetic promoter sequence of pA2-S2 (corresponding to
the oligonucleotide P-artP (6) and twenty bases of the 5'
and 3'-flanking regions of pA2 are shown in SEQ. ID. NO.
12. The insert starts at position 21 and ends at
position 72. (The first "T" residue at the 5'-end
corresponds to position number 2263, the last "A" residue
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at the 3'-end corresponds to position number 2354 of the
plasmid pA2-S2).
After insertion of an open reading frame into any of
the plasmids pAl-S1, pA2-S1, pAl-S2 or pA2-S2, the entire
expression cassette can be excised and inserted by forced
cloning into corresponding sites in the virus vector
vdTK. The cassette can be inserted into the virus genome
in either orientation depending on the cloning plasmid
used.
Construction of plasmids comprising expression
cassettes with a selective marker (pN2gpt-S3A and pN2gpt-
S4): Resides plasmids designed for forced cloning,
described hereinabove, two additional plasmids were
constructed for transferring genes into one unique (Notl)
site in a poxvirus vector with the help of the E. coli
gpt selectable marker gene. They also provide two
additional poxvirus promoters besides the S1 and S2
promoters described hereinabove.
The plasmid pN2gpt-S3A (Figure 4.7) can be used to
insert open reading frames lacking their own initiation
codon. The genes to be transferred into vaccinia virus
(the gpt marker and the open reading frame) can be
excised either with Notl alone or with two enzymes, for
example, NotI and SmaI (or RsrII or ApaI). The excised
fragment is then inserted into the corresponding sites)
of the virus vector vdTK.
The plasmid pN2gpt-S4 ( Figure 4 . 7 ) can be used to
insert complete open reading frames including an AUG
translation start codon. The cassettes consisting of the
gpt-marker gene and the open reading frame can be excised
as described for pN2gpt-S3A. The promoters S3A and S4
are modified versions of strong poxvirus late promoters.
These plasmids were constructed by first making
plasmids pN2 -gpta and pN2 -gptb ( Figure 4 . 6 ) which contain
an E. coli gpt gene driven by the vaccinia virus P7.5
promoter, flanked by several unique restriction sites
including NotI (Figure 1.3). Insertion of the S3A or S4
promoter-fragment into the unique PstI and ClaI sites in
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pN2-gptb resulted in the plasmids pN2gpt-S3A and pN2gpt-
S4.
Construction of plasmids pN2-gpta and pN2-gptb: See
Example 1 and Figure 4.6
Construction of plasmid pN2gpt-S3A: The parental
plasmid pN2-gptb was digested with PstI and ClaI and
ligated with a synthetic linker sequence consisting of
the oligonucleotides P-artP(7) and P-artP(8) (SEQ. ID.
NO. 40). The resulting plasmid was designated pN2gpt
S3A.
The synthetic promoter sequence of pN2gpt-S3A
(corresponding to the oligonucleotide P-artP(7)) and
twenty bases of the 5'- and 3'-flanking regions of
pN2-gptb are shown for pN2gpt-S3A in SEQ. ID. NO. 13.
The inserted DNA sequence starts at position 21 and ends
at position 107. (The first T-residue at the 5'-end
corresponds to position number 3328, the last A-residue
at the 3'-end to position number 3454 of the plasmid
pN2gpt-S3A).
Construction of plasmid pN2gpt-S4: The plasmid pN2-
gptb was digested with PstI and ClaI and ligated with an
adaptor sequence consisting of the oligonucleotides P-
artP(9) and P-artP(10) (SEQ. ID. NO. 41). The resulting
plasmid was designated pN2gpt-S4.
The synthetic promoter sequence of pN2gpt-S4
(corresponding to the oligonucleotide P-artP(9)) and
twenty bases of the 5'- and 3'-flanking regions of
pN2-gptb are shown for pN2gpt-S4 in SEQ. ID. NO. 14. The
inserted DNA sequence starts at position 21 and ends at
position 114. (The first "T" residue at the 5'-end
corresponds to base #3328, the last "A" residue at the
3' -end to position base #3461 of the plasmid pN2gpt-S4) .
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EXAMPLE 5. Expression of polypeptides is a vaccinia
virus vector (vdTK) by direct molecular
insertion of gene,expressioa cassettes
This example demonstrates the facility with whiclZ
cloned genes can be inserted into a vaccinia virus vector
(vdTR) of the present invention for rapid creation of
poxvirus expression constructs using direct molecular
insertion of gene expression cassettes described in
Example 4. Here, use of the vdTR vector-cassette system
to make constructs for expressing several particular
model polypeptides is described, including human blood
proteins (prothrombin and variants of plasminogen) and a
human virus antigen (HIV gp160).
Construction of a modified vaccinia virus (vPT1)
expressing human prothrombia: Human prothrombin (PT)
serves as a model for foreign protein expression in a
vaccinia virus vector of the present invention. A cDNA
encoding prothrombin has been shown previously to be
expressible by a conventionally constructed recombinant
vaccinia virus, as disclosed in Patent Application
w091/11519 by Falkner et al. ("the Falkner
application").
A modified prothrombin cDNA is excised as a 2.0 kb
EcoRI fragment from the plasmid pTRgpt-PTHHb, and
inserted into the single EcoRI site of the plasmid pAl-S1
(Example 4 , Figure 4 . 4 ) resulting in the plasmid pAlS1- PT
(F3gure 5.1). In the expression cassette of this
plasmid, the prothrombin cDNA is driven by the synthetic
poxvirua promoter S1 which also provides a translation
initiation codon.
The sequence of human prothrombin has been published:
Degen S. J. F., MacGillivray R. T. A. & Davie, E.
H~ochemistry 22: 2087-2097 (1983). This sequence is
accessible in the EMHO Data Library under the Identifier
(ID) HSTHR1. The sequence in ID HSTHR1 is not complete;
it lacks the first 19 by of the prothrombin coding
region. The present inventors have sequenced the missing
CA 02515166 1992-08-25
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part of the cDNA in ID HSTHR1 and present this
hereinbelow.
Due to the many modifications and base changes, the
full sequence of the present human prothrombin cDNA clone
including the S1 promoter and 20 bases of plasmid
flanking sequences is shown in SEQ. ID. N0. 15.
Hy the engineering steps outlined in the Falkner
application (PCT/EP-91/00139), the cDNA was modified as
follows: two additional codons (bases #22-27) were
introduced resulting in the incorporation of two new
amino acids; the 3'-untranslated sequence was removed by
introduction of an EcoRI site: bases #1963-1965
(#1920-1922 ID HSTHR1) were changed from TGG to GAA by
site directed mutagenesis.
One base pair change was found in the present
PT-cDNA, that results in a novel NcoI site: base #525
(#482 in ID HSTHR1) is changed from C to A. This is a
silent mutation because the CCC codon (Pro) is changed to
CCA (Pro) which results in a new NcoI site. (The first
base of SEQ. ID. NO. 15 from pAlS1-PT corresponds to base
#2394 and the last base to #4381 of the full sequence of
plasmid pAlS1-PT).
For transfer into the vaccinia virus vector vdTR, the
cassette is excised from the plasmid pAlS1-PT with NotI
and RsrII endonucleases and isolated after separation on
a low melting point agarose gel. The virus vector vdTK
DNA is cleaved with NotI and RsrII, extracted with phenol
and precipitated with ethanol. The small NotI-RsrII
connecting fragment of the multiple cloning site of the
vector DNA is lost during the ethanol precipitation step.
The vaccinia vector arms are ligated with a twenty-fold
molar excess of cassette. Packaging of ligated vaccinia
virus DNA with fowlpox helper virus in chicken cells is
described in Example 3. Packaged viruses from plaques
produced by infection of in CV-1 cells are plaque
purified again and small crude stocks are prepared. The
virus isolates may be further analyzed by Southern
blotting and expression analysis as described in the
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Falkner application. A viral isolate having the correct
genomic structure for insertion of the prothrombin cDNA
is designated vPTl. A similar recombinant vaccinia virus
produced by marker rescue induced prothrombin expression '
in Vero cells at levels of activity of about 50-60 mU/ml
of cell culture supernatant. See the Falkner
application.
Construction of a vaecinia virus (vGPgl) expressing
human glu-plasminogen: The native form of plasminogen
(Pg) has an amino terminus starting with the amino acid
glutamic acid (glu) and is therefore called glu-
plasminogen (glu-Pg). A partially processed form of
plasminogen that lacks the first 77 amino terminal amino
acids (the activation peptide) is called lys-plasminogen
(lys-Pg). The affinity of lys-Pg for its substrate
fibrin is much higher than that of glu-Pg. In addition,
recombinant lys-Pg is considerably more stable than glu-
Pg in supernatants of cell cultures infected with a
(conventional) vaccinia recombinant carrying the glu-Pg
gene.
The complete human plasminogen cDNA (including its
translational start and stop codons) was excised from a
plasmid (phPlas-6) as a Hall-SmaI fragment. The sequence
of human plasminogen has been published by Forsgren M,
Raden H, Israelsson M, Larsson K & Heden L-0. FENS
Letters 213: 254-260 (1987) and is accessible in the EMBO
Data Library (GenBank) under the Identifier (ID) HSPMGR.
Therefore sequences of this plasmid have not been
included in the instant Sequence Listing because this
plasmid is not a unique source of the plasminogen DNA
sequence. However, the coding region of the present
plasminogen sequence differs from the published sequence
in at least one nucleotide: the "A" residue at position
#112 (ID HSPMGR) is a "G" residue in the instant DNA,
resulting in an amino acid substitution (Lys~Glu).
The plasminogen cDNA was inserted into the HpaI site
of the plasmid pN2gpt-S4 (Example 4, Figure 4.7) , which
was selected for constructing a gene expression cassette
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with a selectable marker because the plasminogen cDNA
contains two ApaI sites and one RsrII site and therefore
does not allow the use of the expression cassettes
designed for forced cloning. The resulting plasmid was
designated pN2gpt-GPg (Figure 5.2).
The joining region of the S4 promoter including the
initiation codon of plasminogen (base #32 this listing;
base #55 in ID HSPMGR) is shown for pN2gpt-GPg in SEQ.
ID. NO. 17. The coding region of glu-plasminogen was
omitted in the sequence listing. The sequence continues
with the stop codon (base #35 this listing; base #2485 in
ID HSPMGR) and 25 bases of the 3'-untranslated
plasminogen sequence. This sequence is followed by 29
bases of the multiple cloning site of phPlas6 and by 20
bases of the multiple cloning site of plasmid pN2gpt-S4.
To transfer the glu-plasminogen gene cassette into
a vaccinia virus genome, the NotI fragment of pN2gpt-GPg
containing the two genes and their promoters (the P7.5
promoter controlling the gpt-selection marker, and the
S4-promoter controlling the glu-plasminogen gene) is
isolated from a low melting point agarose gel and
purified. This cassette is ligated with arms of vaccinia
virus vdTK DNA cut with NotI. Packaging and plaque
purification are described in Example 3. A virus having
the correct structure for the inserted plasminogen-gene
cassette is designated vN2gpt-GPg. This virus is used
for expression of plasminogen in CV-1 cells as described
for an analogous vaccinia virus constructed by marker
rescue techniques. Secreted glu-Pg in cell culture
supernatants was detected at a level of about 1.5 ~g/106
cells after 24 hours of infection with a conventionally
constructed vaccinia virus under standard conditions for
cultivation of vaccinia virus vectors for expression of
foreign proteins in cell culture. The glu-plasminogen in
the cell culture supernatant was detectable only in the
presence of a protease inhibitor (50 ~g/ml of aprotinin) .
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Construction of a vaccinia virus (vLPgl) expressing
human lys-plasminogen: A sequence encoding lys-
plasminogen was prepared by deletion of the 231 by coding
region for the first 77 amino acids (Glut to Lys77) of
plasminogen from the complete plasminogen cDNA as shown
in Figure 5.3. This sequence was inserted into the gene
expression cassette of a plasmid (pN2gpt-S4) having a
selectable marker gene (E. coli gpt) , resulting in the
plasmid designated pN2gpt-LPg (Figure 5.3).
In this plasmid, the pre-sequence (coding for the
signal peptide that mediates secretion) is directly fused
with the first nucleotide of lysine residue 78 in
plasminogen. The novel signal peptide cleavage site
created by the fusion is similar to many known signal
cleavage sites. See, for instance, von Heinje, Eur. J.
Eiochem. 133: 17-21 (1983).
In addition, an NcoI site was introduced at the site
of the initiation codon of the Pg cDNA to facilitate
cloning into the single NcoI site of the plasmid pN2gpt-
S4 and to achieve the optimal context of the promoter and
the Pg-coding region. To facilitate excision of Pg cDNA
with NcoI, one of two internal Ncol sites (NcoI(2);
Figure 5.3) was deleted from the Pg cDNA, as follows.
The plasmid phPlas6 was transferred into E. coli
strain NM522 and single-stranded DNA was prepared by
superinfection with the helper phage M13K07. The first
round of mutagenesis was done with two oligonucleotides,
oNcoi and oNco2, using the single-stranded phPlas6 DNA as
a template with a commercially available mutagenesis kit
(Amersham, Inc.). The oligonucleotide Ncol converts two
A-residues upstream of the plasminogen start codon into
two C-residues, resulting in an NcoI site around the
start codon without changing the coding region of the
plasminogen pre-sequence. The oligonucleotide oNco2
converts a T into a C residue within the internal NcoI
site (NcoI(2)) of the Pg cDNA, producing a silent
mutation that inactivates this NcoI site.
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The coding region for amino acids 1-77 of plasminogen
was deleted by second loop-out mutagenesis step using 42-
base oligonucleotide oNco3. All'mutations were confirmed
by sequencing and restriction analysis.
The plasmid having the three mutations, phLplas, was
linearized with SmaI and partially digested with NcoI.
The 2.2 kb NcoI-SmaI fragment was isolated and inserted
into plasmid pN2gpt-S4 that had been cut with NcoI and
Smal. The resulting plasmid was designated pN2gpt-LPg.
Due to the many modifications of the plasminogen cDNA
in pN2gpt-LPg, the full sequence of the NcoI-SmaI
fragment of pLplas including 20 bases of the S4 promoter
and 20 bases of the downstream plasmid region of
pN2gpt-S4 is shown in SEQ. ID. N0. 18. The plasminogen
cDNA sequence was modified as follows: the former two
A-residues at positions #19 and #20 (bases #53 and 54 in
ID HSPMGR) were changed into two C-residues, resulting in
an NcoI site; base #21 this listing (#55 in ID HSPMGR) is
the A-residue of the plasminogen start codon; base #2220
(base #2485 in ID HSPMGR) is the T-residue of the stop
codon; base #111 in ID HSPMGR (base #77 this listing) was
joined with base #343 in ID HSPMGR (base #78 this
listing) resulting in the deletion of the sequence coding
for the "activation peptide"; the T-residue #926 (base
#1191 in ID HSPMGR) was changed into a C residue
(conservative exchange) resulting in the disappearance of
an internal NcoI site.
To transfer the lys-plasminogen gene cassette into
a vaccinia virus genome, the NotI fragment of pN2gpt-LPg
containing the gene expression cassette comprised of two
promoter-gene combinations (the P7.5 promoter-gpt gene
and the S4 promoter-lys-plasminogen gene) is ligated with
NotI cleaved vaccinia virus vdTK vector DNA and packaged
as described in Example 7. An isolate having the proper
structure for the inserted gene cassette, designated
vN2gpt-LPg, is used for expression of lys-plasminogen in
CV-1 cells under conditions used previously for a
conventionally constructed recombinant under standard
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conditions for cultivation of vaccinia virus expression
vectors for production of proteins in cell culture.
Secreted lys-Pg in cell culture supernatants was detected
at a level of about 1.0-2.0 ~Cg/106 cells after 24 hours
of infection with the conventional recombinant. The lys-
plasminogen in the cell culture supernatant was stable
without addition of a protease inhibitor.
Construction of a vaccinia virus (vgp160-1) for
expressing human immunodeficiency virus glycoprotein.160
(8IV gp160): The complete open reading frame of HIV
gp160 is obtained on a 2.5 kb EcoRV fragment containing
excised from replicative form (RF) DNA of an M13 phage
[mpPEenv; Fuerst et al., Mol. Cell. 9iol. 7: 2538-2544
(1987)]. This fragment is inserted into the plasmid
pN2gpt-S4 as outlined in Figure 5.4. In the resulting
plasmid, pN2gpt-gp160, the gp160 gene is controlled by
the synthetic vaccinia virus promoter S4.
The sequence of HIV gp160 has been published by
Ratner, L. et al. Nature 313: 277-284 (1985). The
sequence of clone BH8 is accessible in the EMHO Data
Library (GenBank) under the Identifier (ID) HIVH3HH8.
Therefore, the gp160 sequence is not included in SEQ. ID.
NO. 19, but the joining region of the S4 promoter and an
EcoRV HIV-gp160 fragment including the initiation codon
of gp160 gene (base #28 this listing; base 226 in ID
HIVH3BH8) is shown. The EcoRV HIV-gp160 fragment stems
from the M13 phage (replicative form) mpPEenv described
in Fuerst, T.R., Earl, P. & Moss, B. Mol. Cell. Hiol. 7:
2538-2544 (1987). The sequence continues with the stop
codon (base #31 this listing; base #2779 in ID HIVH3HH8)
and one half of the downstream EcoRV site. This sequence
is followed by 20 bases of the multiple cloning site of
plasmid pN2gpt-S4. The first base (T) of this listing
corresponds to base #3368, the last base (G), to #5973 in
the sequence of pN2gpt-gp160.
To transfer the HIV gp160 gene-expression cassette
into a vaccinia virus genome, the NotI fragment
containing both gene-promoter combinations (the P7.5
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promoter-gpt selection marker and the S4 promoter-gp160
gene) is ligated with NotI-cleaved DNA of the vaccinia
virus vector vdTK and packaged as described in Example 7.
An isolate having the correct structure of insertion of
the cassette, designated vN2gpt-gp160, is used for
expression of gp160 in African green monkey (Vero) cells
under conditions used previously for a conventionally
constructed recombinant. Barrett et al., AIDS Research
and Human Retroviruses 6: 159-171 (1989).
Construction of a vacciaia virus vector providing for
screening for modified viruses carrying insertions by
coinaertion of a lacZ gene: To demonstrate the screening
for insertion by coinsertion of an E. coli lacZ gene in
combination with the direct cloning approach, the plasmid
pTZgpt-S3AlacZ provides a useful model construct (Figure
5.5). The plasmid pTZl9R (Pharmacia, Inc.) was cut with
Pvull, and the large 2.5 kb vector fragment was prepared
and ligated with NotI linkers (Boehringer, Inc.). The
resulting plasmid, pTZ-N, has a deletion of the multiple
cloning site that is located within the sequences of the
alpha complementation peptide in the pT219R plasmid.
Therefore, possible recombination events between the lacZ
gene to be inserted into pTZ-N and the sequences of the
alpha complementation peptide are excluded.
To construct a gene expression cassette for direct
molecular cloning, the 1.2 kb NotI fragment, containing
the gpt-gene cassette and the S3A promoter, is excised
from pN2gpt-S3A (Example 4) and inserted into pTZ-N
resulting in the plasmid pTZgpt-S3A. The 3.0 kb EcoRI
lacZ fragment (excised from plasmid pTKgpt-Fls/3; Falkner
& Moss, 1988) is inserted into the single EcoRI site of
pTZgpt-S3A. The resulting plasmid designated pTZgpt-
S3AlacZ.
The 4.4 kb NotI fragment of this plasmid, consisting
of the two marker genes (E. coli gpt and lacZ), is
ligated with NotI cleaved DNA of the virus vdTK (Example
4). The ligation and packaging conditions are described
CA 02515166 1992-08-25
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in Example 3. The estimated yield of modified viruses in
the case of gpt-selection is described in Example 3.
An additional vaccinia virus~vector was constructed
as follows. The plasmids pTZS4-lacZa and pTZS4-lacZb
provided useful model constructs (Figure 5.6). Plasmid
pTZ-N was constructed as above. The gene expression
cassette, the 1.2 kb NotI fragment containing the gpt-
gene cassette and the S4 promoter was excised from
pN2gpt-S4 (Example 4) and inserted into pTZ-N resulting
in the plasmid pTZgpt-S4. A 3.3 kb SmaI-StuI IacZ
fragment was excised from plasmid placZN*, which was
constructed by digesting the plasmid pFP-Zsart (European
Patent Application No. 91 114 300. -6, Recombinant Fowlpox
Virus) with NotI and ligating pFP-Zsart with the
oligonucleotide P-NotI- (5'-GGCCAT-3'). This 3.3 kb
SmaI-StuI lacZ fragment was inserted into the single SmaI
site of pTZgpt-S4. The resulting plasmids were
designated pTZS4-lacZa and pTZS4-lacZb.
The 4.5 kb NotI fragment of this plasmid was ligated
with the NotI cleaved DNA of the virus vdTK and packaged
as described above.
The combination of lacZ and gpt-selection in a single
cloning step offers no advantage because all gpt-positive
plaques will contain the lacZ gene. However, for the
construction of viruses having insertions in different
sites, a second screening procedure is desirable. The
marker of first choice is the gpt marker, but IacZ
screening offers an alternative method for detection of
inserts, for instance, when the target viral genome
already contains a copy of a selectable marker such as
the E. coli gpt gene.
For such screening, two ml of 1/10, 1/100 and 1/1000
dilutions of crude virus stocks prepared after packaging
(see Example 3) is plated on 30 large (diameter of 8.5
cm) petri dishes (10 petri dishes per dilution). The
blue plaque assay is done according to standard
procedures. Chakrabarti, S., Brechling, K. & MOSS, H.
Mol. Cell. Biol. 5: 3403-3409 (1985).
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EXAMPLE 6. Construction of a vaccinia virus vector
(vS4) with a directional master cloning
site under transcriptional control of a
strong late vaccinia virus promoter
The present example describes a vaccinia virus
cloning vector (vS4) that is designed for direct
molecular insertion of a complete open reading frame into
a master cloning site that is functionally linked to a
vaccinia virus promoter. Accordingly, use of this vector
according to methods of the present invention enables
insertion of genes directly into a poxvirus vector
without separate construction of an insertion plasmid, as
required in conventional construction of recombinant
poxviruses by intracellular recombination. This vector
also obviates the need for separate construction of a
gene expression cassette for transfer into a vaccinia
virus vector by direct molecular insertion, as described
hereinabove.
The master cloning site of vector S4 is located in
the genetically stable central region of the vaccinia
virus genome and is comprised of several cleavage sites
that are unique in the vector, thus pernlitting
directional insertion. The S4 promoter immediately
upstream of the master cloning site is a strong synthetic
variant of a late vaccinia virus promoter. This
expression vector is suitable for direct cloning and
expression of large open reading frames which include a
translation start codon, as illustrated here by a cDNA
encoding a human blood protein, the von Willebrand factor
(vWF) .
Construction of the vaccinia virus vector vS4: An
adaptor containing the synthetic vaccinia virus promoter
S4 is inserted into the vaccinia virus vector vdTK
(Example 4, Figure 4.1) at the unique NotI site (Figure
6.1). Insertion of the selected adaptor oligonucleotides
inactivates the upstream NotI site while the downstream
NotI site remains functional as a unique cloning site.
More particularly, DNA (1 ~.g) of the vector vdTK
(Example 4, Figure 4.1) is cleaved with NotI and ligated
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with (0.5 ~,g) annealed oligonucleotides P-artP(11) (SEQ.
ID. NO. 38) and P-artP(12) (SEQ. ID. NO. 39). The
ligation mix is packaged and plaques are identified as
described in Example 3. Plaques are subjected to PCR
screening as described (Example 4, Identification of the
virus vdTK by PCR screening). An isolate having the
insert in the correct orientation is designated vS4.
Insertion of the von Willebrand factor cDNA into vS4:
Plasmid pvWF contains the complete von Willebrand factor
cDNA flanked by NotI sites. The sequence of human vWF
has been published: Honthron, D. et al . , Nucl. Acids Res.
14: 7125-7128 1986). The sequence is accessible in the
EMHO Data Library under the Identifier (ID) HSVWFR1.
SEQ. ID. NO. 20 shows the junction in the virus genome of
wWF of the viral S4 promoter and the 5'-untranslated
region of the present vWF cDNA in the plasmid pvWF up to
the translational start codon (base #249 in this listing;
base #100 in ID HSVWFR1). The coding region of vWF was
omitted in the instant sequence listing. The sequence
continues with the stop codon (base #252; base #8539 in
ID HSVWFR1) and the 3'-untranslated sequence up to the
NotI site (base #304) and twenty bases of overlap with
the 3'-region of the viral genome of wWF.
The vWF cDNA fragment is released with NotI, isolated
and ligated with vS4 vector DNA that has been cleaved
with NotI and treated with phosphatase, as illustrated in
Figure 6.2.
One ~g of ligated DNA is packaged as described in
Example 7. Plaques are picked and analyzed by PCR
screening. The first primer for the PCR reaction is
oligonucleotide odTK2 which is located about 300 by
upstream of the tk-gene; the reverse primer ovWFl is
located in the vWF gene about 50 by downstream of the
initiation codon. PCR amplification occurs only when the
vWF insert is in the correct orientation relative to the
S4 promoter in the vector. PCR-positive plaques are
identified and analyzed further. Alternatively, if the
yield of desired modified virus is low, on the order of
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0.1 to 0.01%, then they may be identified by in situ
plaque hybridization methods adapted from those known in
the art. See, for instance, Villareal, L. P. & Herg, P.
Science 196:183-185 (1977).
A virus clone having the cDNA insert by PCR or
hybridization and further showing the expected
restriction pattern with PvuII is designated vvWF. Such
vectors may be tested for expression of von Willebrand
factor ae described for other human proteins in Example
5, modified as appropriate according to genetic
engineering principles well known by one skilled in this
art.
EXAMPLE 7. Heterologous packaging of orthopox
(vaccinia) virus genomic DNA by an avipox
(fowlpox) helper virus and simultaneous
selection for modified virus in host cells
of a species in which the helper virus
cannot replicate
Example 3 describes packaging of modified vaccinia
virus DNA with fowlpox helper virus in avian cells and
subsequent isolation of progeny virus plaques in
mammalian (CV-1) cells in which the avipox helper virus
cannot replicate. The present example illustrates
packaging of vaccinia virus DNA by fowlpox directly in
CV-1 cells, thereby permitting simultaneous packaging and
host range selection for packaged virus. Resides
eliminating helper virus from the initial stock of
progeny, this procedure circumvents the tedious
requirement for producing primary cultures of chicken
embryo fibroblasts for each packaging experiment.
Instead, continuous mammalian cell lines that are
commonly used for vaccinia virus replication also can be
used for packaging vaccinia virus with fowlpox helper
virus.
It is known that fowlpox virus (FPV) replicates
completely only in avian cells; no viable progeny virus
is obtained from infected mammalian cells. The precise
point in the life cycle of FPV at which replication is
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aborted in mammalian cells is not known. However, FPV is
known to produce viral proteins in mammalian cells and
even to induce protective immunity in mammals when used
as a live vaccine. Taylor et al., Vaccine 6: 497-503
(1988). Nevertheless, FPV has not been shown previously
to have a capacity for packaging heterologous poxvirus
genomic DNA, particularly directly engineered vaccinia
virus DNA.
In an initial experiment, CV-l cells (5 x 106) were
infected with one pfu/cell of fowlpox virus (strain
HP1.441) and incubated for one hour. Subsequently, a
calcium-phosphate precipitate (one ml containing one ~.g
of vaccinia virus wildtype DNA) was transfected into the
infected cells. After 15 min at room temperature, 10 ml
of medium (DMEM, 10% fetal calf serum) were added. The
cells were incubated for four hours, and the medium was
changed. The cells were then incubated for six days, and
a crude virus stock was prepared. The progeny virus were
titered on CV-1 cells. Typical vaccinia plaques were
visible after two days.
The dependence of packaging efficiency on the amount
of genomic viral DNA was determined over a range of DNA
amounts from 0.1 to 10 ~,g per 5 x 106 CV-1 cells. See
Figure 7.1. Amounts of DNA in excess of 1 ~g (e.g., 10
~,g) produced a coarse calcium-phosphate precipitate that
reduced the efficiency of transfection in terms of pfu/~.g
of input DNA. Figure 7.1.
The dependence of the packaged vaccinia virus yield
on the incubation time for packaging was analyzed using
a constant amount of vaccinia virus wildtype DNA (1 ~.g)
and a constant amount of FPV helper virus (1 pfu/cell)
under the conditions described above for the initial
experiment in this example except that the medium added
15 minutes after transfection was changed after four
hours, and the cells were then incubated for an
additional 1 to 5 days before preparing a crude virus
stock (total volume of 2 ml). Virus stock from control
cells infected with FPV only and incubated for 5 days
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produced no visible' plaques. This experiment was
repeated three times and a typical outcome is shown in
Table 7.1, below.
Table 7.1. Effect of incubation time on yield of
vaccinia virus from DNA packaging by fowlpox helper virus
in mammalian (CV-1) cells.
Incubation Time Titer
(hours) (pfu/ml)
24 1. 0 x lOZ
48 4.6 x 104
72 5 . 0 x lOs
96 5.6 x 106
120 2.1 x 10~
The titer of packaged vaccinia virus, detected by
plaque assay on mammalian (CV-1) cells, rose continually
from about 10z pfu/ml at 24 hours to about 2 x 10' after
120 hours. Incubation times in the range of 48 to 72
hours produced convenient levels of packaged vaccinia
virus (between 104 and 106 pfu/ml) and, therefore, are
suitable for routine packaging of vaccinia virus DNA by
fowlpox virus in mammalian cells.
Vaccinia DNA can be packaged in maam~alian cells
abortively infected with fowlpox virus. It was shown
previously that fowlpox virus can also infect mammalian
cells, but the viral life cycle is not completed in these
non-typic host cells. Depending on the cell type, viral
growth stops either in the early or in the late stage and
viable fowlpox virus is not formed (Taylor et al., 1988) .
These findings prompted an investigation into packaging
vaccinia DNA in a continuous mammalian cell line.
Confluent monolayers of CV-1 cells were infected with
0.05 pfu per cell of the FPV strain HP1.441 and then
transfected with a ligation mixture consisting of NotI
cleaved vaccinia virus DNA and a gpt gene cassette having
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NotI flanking sites. More particularly, vaccinia DNA (1
fig) was digested with NotI and ligated with indicated
amounts of insert DNA (P7.5 gpt gene cassette). The
unique NotI site in vaccinia virus is located in an
intergenic region in the HindIII F fragment. Goebel, et
al., Virology, 179:247 (1990). After incubation for
three days the cells were harvested and the crude virus
stock was titered on CV-1 cells in the presence (+MPA)
and in the absence (-MPA) of gpt-selective medium.. The
outcome is summarized in Table 7.2.
Table 7.2
Titers
after
abortive
packaging
a t. insert (nq) titers chimeras
(pfu xl0~z/6x106
cells)
- MPA* + MPA
1. 200 17.2 1.6 9.3
2. 200 42:5 5.1 12.3
3. 400 64.0 3.8 5.9
4. 400 26.8 3.8 14.2
5. 210.0
*MPA, mycophenolic acid.
The most important result was that fowlpox virus
could package the modified vaccinia DNA in a cell type
that prevents its own growth. Moreover, the yield of
chimeric plaques was in the range of 5-10%. This
compares favorably with the classical in vivo
recombination technique, in which usually about 0.1% of
the total plaques are recombinants. Ligation of the
vector arms alone (Table 7.2, experiment #5) resulted in
a higher titer compared to ligation experiments 1-4 with
insert, probably due to lack of contaminants present in
the agarose-purified insert molecules.
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Some of the isolated viruses were plaque-purified and
further characterized. They showed the typical HindIII
restriction patterns of vaccinia virus and, in addition,
foreign gene bands characteristic for the two possible
orientations of the single insert. With insertion into
the NotI site, no viruses with multiple inserts were
observed.
Heterologous packaged chimeric vaccinia viruses do
not cross hybridize with fowlpox virus. In order to
study the effects of heterologous packing by FPV on the
structure of chimeric vaccinia viruses, DNAs of isolates
F13.4, F12.5, F13.2, F13.2 and F12.4, together with those
of four purified isolates from the NotI cloning
experiment and the fowlpox virus controls, were digested
with HindIII, and the resulting fragments were separated
by electrophoresis and analyzed by Southern hybridization
with a fowlpox virus probe prepared from sucrose
gradient-purified virions. No cross hybridization of the
vaccinia viruses with FPV DNA was observed.
EXAMPLE 8. Homologous packaging of engineered
vaccinia virus genomic DNA by a vaccinia
virus host range mutant (vdhr) that is
unable to replicate in a human cell line
The present example illustrates construction and
utilization of a helper poxvirus comprised of deletions
that limit its host range, particularly the ability to
replicate in certain human cell lines. Therefore,
modified vaccinia virus free of helper virus can be
prepared by packaging of vector DNA with this mutant
helper virus and isolating clones of the engineered virus
by infecting appropriate human cells.
This mutant helper virus is derived from host range
mutants of vaccinia virus which are unable to replicate
in a variety of human cells and which display altered
cytopathic effects on many other cells that are
permissive for infection by wildtype vaccinia virus.
See, for example, Drillien et al., Virology 111: 488-499
(1981). In particular, the genome of this helper virus
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comprises mutations of two host range genes which
together prevent it from replicating in human (MRC
5) cells in which only vaccinia virus genomes
having at least one intact host range gene can
replicate.
Construction of the host range mutant vaccinia
virus vdhr: The genomic location and DNA sequence
of one vaccinia virus gene required for replication
in human cells has been described by Gillard et
al., Proc. Natl. Acad. Sci. USA 83: 5573-5577
(1986). Recently, this gene has been designated K1L
(Goebel et al., 1990 Virology 179:247-266). A
second vaccinia virus host range gene has been
mapped {Perkus et al., J. Virology 63: 3829-2836
(1989)}. This second gene (designated C7L
according to Geobel et al., 1990 Virology 179:247-
266) lies in a region encompassing parts of the
HindIII C and HindIII N fragments. This region is
deleted in the vaccinia virus WR6/2 strain (Moss et
al., J. Virol. 40: 387-395 (1981)). Strain WR-6/2
therefore lacks the C7L host range gene.
The helper virus vdhr lacking both the K1L and
C7L host range genes is constructed from the C7L
negative strain WR-6/2 by marker rescue with a
modified EcoRI K fragment from which the K1L host
range gene is deleted. See Figure 8.1. This
modified EcoRI K fragment comprises a selective
marker gene (the E. coli gpt gene) to facilitate
selection for modified WR-6/2 genomes comprising
the modified EcoRI K fragment using intracellular
marker rescue as described by Sam & Dumbell, 1981
Ann. Virol. (Institut Pasteur) 132E:135. A
conditional lethal mutant which lacks the ability
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to grow on human cell lines has also been described
by Perkus et al., 1989 J. Virol 63:3829-3836.
More particularly, the 5.2kb EcoRI K fragment
of vaccinia virus wildtype DNA is subcloned into
the plasmid pFP-tkl8i. The resulting plasmid is
designated pFP-EcoKl. The vaccinia virus host
range gene K1L (Gillard et al. , 1986) is selected
and simultaneously a unique NotI site is introduced
by loopout mutagenesis using the oligonucleotide P-
hr(3) (SEQ. ID. NO. 42). The resulting plasmid is
designated pEcoK-dhr.
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The plasmid pFP-tkl8i was constructed by modification
of the plasmid pFP-tk-10.4 (see Falkner et al., European
patent application number 89303887.7, publication EPA 0
338, 807, Examples 3 and 8, the entire disclosure of which
is hereby incorporated herein by reference). Plasmid
pFP-tk10.4 was digested with NcoI and ligated with an
adaptor consisting of annealed nucleotides P-NcoI(1) and
P-NcoI(2), resulting in the introduction of a multiple
cloning site into the single NcoI site of the FPV tk-.gene
with the restriction endonuclease cleavage sites EcoRI,
NotI and HindIII.
The sequence of vaccinia virus has been published by
Goebel, S. J. et al., Virology 179: 247-266 (1990). It
is accessible in the EMBO Data Library (GenHank) under
the Accession Number M35027. The sequence of the vaccinia
virus host range gene K1L has been published by Gillard.
S. et al., Proc. Natl. Acad. Sci. USA 83: 5573-5577
(1986) and is accessible in the EMBO Data Library
(GenHank) under the Identifier (ID) PXVACMHC. Therefore,
the coding sequence of the K1L gene is not included in
SEQ. ID. NO. 21. In pEcoK-dhr the K1L gene is deleted
and replaced by a NotI site. The joining region between
the PXVACMHC sequence and the NotI site insert is shown
(bases #1-20 of this listing correspond to bases #72 -91
in ID PXVACMHC). The coding region of K1L was deleted
and replaced by a NotI site followed by two G residues
(bases #21-30 in the sequence listing). The sequence
continues with 20bp flanking region (bases #31-50 this
listing; bases #944-963 in ID PXVACMHC).
In a further step pEcoK-dhr is linearized with Notl
and ligated with a 1.1 kb P7.5-gpt gene cassette derived
from plasmid pN2-gpta (Example 4) by NotI digestion. The
resulting plasmid pdhr-gpt is used generate the helper
virus vdhr.
The NotI cassette (comprising the P7.5 promoter-gpt-
gene cassette) inserted into pEcoK-dhr and twenty bases
of the 5' and 3' flanking regions are shown for pdhr-gpt
in SEQ. ID. NO. 22. The flanking region (bases #1-20
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this listing) correspond to bases #72-91 in ID PXVACMHC
(see SEQ. ID. NO. 21 for pEcoK-dhr). The inserted DNA
sequence starts at position 21 (the first "G" of a NotI
site) and ends at position 1189 (the last "C" residue of
a NotI site). The A-residue of the translational
initiation codon of the gpt-gene corresponds to position
#548. The T-residue of the translational stop codon of
the gpt gene corresponds to position number #1004. The
sequence continues with 20 bases of flanking region
(bases #1192-1209 this listing; bases #944-961 in ID
PXVACMHC). The two "G" residues #1190 and 1191 in this
listing, correspond to position 29 and 30 of pEcoK-dhr.
To transfer the Eco K fragment into vaccinia virus,
the plasmid is transfected into primary chicken embryo
fibroblasts cells infected with the vaccinia virus
deletion mutant WR-6/2. Modified viruses are selected as
gpt-positive (using mycophenolic acid). A gpt-positive
is plaque-purified three times in CEF cells and
designated vdhr.
Characterization of the vdhr helper virus:
The structure of gpt-positive vaccinia virus vdhr is
analyzed by Southern blotting and host range tests. The
vdhr virus is capable of forming plaques on chicken
embryo fibroblasts and two monkey cell lines (BSC40 and
Vero) but is defective for replication in the human cell
line MRC-5.
Packaging of engineered vaccinia virus DNA using the
host range mutant vdhr as a helper virus: A construct for
expression of a cDNA encoding human prothrombin
demonstrates the utility of this approach. The product
from a ligation mixture described in Example 5, Figure
5.1, is transfected into chicken embryo fibroblasts
infected with vdhr as a helper virus. After 2 days the
cells are harvested and a crude virus stock is prepared.
Packaged virus is assayed for plaque formation on human
(MRC 5) cells in which the desired vaccinia virus
replicates but the mutant vdhr helper virus does not.
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After three days the cells are stained with neutral
red and plaques are selected for further analysis by
Southern blotting. Modified vaccinia virus clones having
the desired structure are identified. Viruses which have
undergone recombination with the highly homologous helper
virus are also expected.
Example 9- Construction of novel chimeric vaecinia
viruses encoding HIV gp160 (vP2-gp160",ntA,
vP2-gp160~,,B aad vselP-gp160",ni) aad
expression of recombiaaat gp160",Q,t is Vero
cells.
The present example illustrates construction by
direct molecular cloning of a vaccinia virus recombinant
for large scale production of gp160 of the HIV-i,,,a,,
isolate. Production of the gp160 of the HIVE isolate
described by Ratner et al., Nature 313: 277-284 (1985),
using a conventionally constructed vaccinia virus
expression vector, has been described by Harrett et al.,
AIDS Research and Human Retroviruses 5: 159-171 (1989).
The HIVE isolate, however, is a rare HIV variant.
Efforts at developing vaccines based on HIV envelope
proteins should include more representative HIV-1
isolates such as the MN-isolate. Gurgo et al., Virology
164: 531-536 (1988); Carrow et al., Aids Research and
Human Retroviruses 7: 831-839 (1991). Accordingly, the
present vaccinia virus vectors were constructed via
direct molecular cloning to express the gp160 protein of
the HIV,,Q,, isolate .
Construction of the plasmid pP2-gp160~ and of the
chimeric viruses vP2-gpt160rINA and vP2-gp160"nrH: The
strategy of inserting the gp160-gene into vaccinia virus
involved: (i) modifying the gp160-gene by removing the
large 5'-untranslated region (5'-UTR) and introducing a
suitable cloning site upstream of the start codon; (ii)
cloning the modified gp160-gene downstream of the strong
late fowlpox virus P2-promoter (European Patent
CA 02515166 1992-08-25
-114-
Application No. 91 114 300.-6, August 26, 1991), and
(iii) inserting a blunt-ended fragment consisting of the
P2-gp160 and P7.5-gpt-gene cassettes into the single
restriction endonuclease cleavage sites of appropriate
viral host strains, e.g. into the SmaI site of the host
vaccinia strain vdTIC (Example 4), the SmaI or the NotI
sites of the vaccinia strain WR. 6/2 (Moss et al., J.
Virol., 40: 387 (1981)) or the vaccinia wild-type strain
WR.
For these purposes, a new SmaI site was introduced
into the plasmid pN2gpt-S4 (Example 4), resulting in the
plasmid pS2gpt-S4 (Fig. 9.1, SEQ ID N0:62) . Subsequently
the S4-promoter was exchanged by the P2-promoter
resulting in the plasmid pS2gpt-P2 (SEQ ID N0:63). This
plasmid allows the cloning of complete open reading frame
(orfs) but can also be used to clone incomplete orfs
lacking their own start codon; the start codon is
provided, for instance, when cloning into the single NcoI
site (CCATGG) of this plasmid. Construction of the
plasmids and viruses is described in further detail
below. For the modification of the gp160-gene, a PCR-
generated proximal fragment was exchanged leading to a
gp160-gene cassette with a minimal 5'-UTR. This cassette
is present in the final construct, the plasmid pP2-
gp1601,,Q,, (Figure 9 .1, SEQ ID NO: 69 ) . Additional
characteristics of the plasmid are shown in the following
table.
pP2~160mn (6926bo) (SEO ID N0:69)
Location Description
3 0 1 pS2gpt-P2 sequences
- 3529
2396 - 2851 rcCDS of E. coli gpt gene
2851 T of rc initiation codon TAC of the gpt
gene
2395 A of the rc stop codon of TTA
3081 - 3323 rc of vaccinia P7.5 promoter
3 5 3358 3526 P2 promoter sequence according to EP application
-
Avipox "intergenic region".
3534 - 6001 CDS of the HIV-1 strain Na1 gp160 sequence
(EN~L ID
RfiHIVMNC)
3534 A of the initiation codon ATG of the gp160NaT
40 6102 T of the stop codon TAA of the gp160I4r1
6173 - 6926 pS2gpt-P2 sequences
CA 02515166 1992-08-25
-115-
The plasmids were constructed as follows.
pS2gpt-S4: The plasmid pN2gpt-S4 (Example 4) was
digested with XbaI and ligated with a SmaI-adaptor (SEQ ID
N0:43: 5'-CTAGCCCGGG-3') inactivating the XbaI and
creating a Smal site. The resulting plasmid was
designated pS2gpt-S4 (SEQ ID N0:62). Additional
characteristics of this plasmid are shown in the following
table.
pS2c~Dt-S4 (4145bp) (SBO ID N0:62)
Location Descrit~tion
1 - 2226 pN2gpt-S4 sequences of SFsQ ID N0:14. Position
1
corresponds to the first nucleotide G 'S-TGGCACTTT
TCGGGGAAAT-3'.
2227 - 2236 SmaI-adaptor 5'-CTAGCCCGGG-3'.
2396 - 2851 rcCDS of fi. coli gpt gene
2851 T of rc initiation codon TAC of the gpt gene
2395 A of the rc stop codon of TTA
3081 - 3323 rc of vaccinia P7.5 promoter
3358 - 3451 S4-promoter of SEQ. ID#14 (oligonucleotide
P-
artP(9) see p. 120)
2237 - 4145 pN2gpt-S4 sequences of SEQ. ID No. 14
pS2gpt-P2: The S4-promoter segment of plasmid
pS2gpt-S4 was removed by cleavage with PstI and HpaI and
replaced with a 172 by PstI-F~paI P2-promoter segment.
This promoter segment was generated by PCR with the
plasmid pTZgpt-P2a (Falkner et al., European Patent
Application No. 91 114 300.-6, August 26, 1991) as the
template and the oligonucleotides P-P2 5'(1) and P-P2
3'(1) as the primers. The PCR-product was cut with PstI
and HpaI and ligated the PstI and HpaI-cut large fragment
of pS2gpt-S4. The sequence of P-P2 5'(1) (SEQ ID N0:44)
is: 5'-GTACGTACGG CTGCAGTTGT TAGAGCTTGG TATAGCGGAC
AACTAAG-3'; the sequence of P-P2 3'(1) (SEQ ID N0:45) is:
5'-TCTGACTGAC GTTAACGATT TATAGGCTAT AAAAAATAGT ATTTTCTACT-
3'. The correct sequence of the PCR fragment was
confirmed by sequencing of the final plasmid, designated
pS2gpt-P2 (SEQ ID N0:63). The sequence primers used were
P-SM(2) (SEQ ID N0:46), 5'-GTC TTG AGT ATT GGT ATT AC-3'
and P - SM ( 3 ) ( S EQ ID NO : 4 7 ) , 5 ' - CGA AAC TAT CAA AAC GCT TTA
TG-3'. Additional characteristics of the plasmid pS2gpt-
P2 are shown in the following table.
CA 02515166 1992-08-25
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g~2crnt-P2 (4277bn) (Sfi0 ID N0:63)
Location Description
1 - 3357 pS2gpt-S4 sequences
2396 - 2851 rcCDS of fi. coli gpt gene
2851 T of rc initiation codon TAC of the gpt gene
2395 A of the rc atop codon of TTA
3081 - 3323 rc of vaccinia P7.5 promoter
3358 - 3526 P2 promoter sequence according to fiP application
Avipox "intergenic region".
3527 - 4277 pS2gpt-S4 sequences
pD~leva2: The plasmid pMNenvl was provided by R.
Gallo (National Cancer Institute, Bethesda, Maryland). It
contains the gp160-gene of the HIV MN-strain cloned as a
3.1 kb EcoRI-PvuII fragment in the vector pSP72 (Promega,
Inc.). The 0.6kb EcoRI-Asp718 fragment of pNlNenvl was
replaced with a 0.13kb EcoRI-Asp718 fragment, removing
large parts of the 5' untranslated region of the gp160-
gene. This 0.13kb fragment was generated by PCR using the
plasmid pMNenvl as the template and the oligonucleotides
P-MN(1) and P-MN(2) as the primers. The forward primer P-
MN(1) introduced, in addition, a StuI site 1 by upstream
of the start codon. The sequence of P-MN(1) (SEQ ID
N0:48) is 5'-AGCTAGCTGA ATTCAGGCCT CATGAGAGTG AAGGGGATCA
GGAGGAATTA TCA-3'; the sequence of P-MN(2) (SEQ ID N0:49)
is 5'-CATCTGATGC ACAAAATAGA GTGGTGGTTG-3'. The resulting
plasmid was designated pNINenv2. To exclude mutations the
PCR generated fragment in this plasmid was sequenced with
the primers P-Seq (2) (SEQ ID N0:50) 5'-CTG TGG GTA CAC
AGG CTT GTG TGG CCC-3' and P-Seq(3) (SEQ ID N0:51) 5'-CAA
TTT TTC TGT AGC ACT ACA GAT C-3'.
pP2-gp160D~T: The 2.7kb StuI-PvuII fragment,
containing the MN gp160-gene, isolated from the plasmid
pMNenv2 was inserted into the HpaI site of pS2gpt-P2
resulting in the plasmid pP2-gp160MN (SEQ ID N0:69).
The chimeric viruses vP2-gp160~A and vP2-gp160",fl,~H
were constructed as follows: The SmaI-fragment consisting
of the P2-gp160 and P7.5-gpt-gene cassettes was inserted
by direct molecular cloning into the single SmaI site of
the host vaccinia strain vdTK (Example 4) resulting in the
chimeric viruses vP2-gp160~A and vP2-gp160i,Q,,B (Figure
CA 02515166 1992-08-25
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9.2). In particular, the vaccinia virus vdTK of Example
4 was cut at its single SmaI site and ligated with the
4.Okb SmaI fragment that contains, the P7.5-gpt-gene and
the P2-gp160-gene cassettes. Correspondingly, the
vaccinia strain WR6/2 was cut at its single SmaI (NotI)
site and ligated with the 4.Okb SmaI (NotI) fragment that
contains the P7.5-gpt-gene and the P2-gp160-gene
cassettes. The cloning procedures were carried out as
described in Example 1. In the virus vP2-gp160~A,.the
gp160-gene is transcribed in the same direction as the
genes clustered around the viral thymidine kinase gene; in
the virus vP2-gp160~B, the gp160-gene is transcribed in
the reverse direction. Since gene position effects can
influence expression levels in vaccinia constructs, the
SmaI (NotI)-fragment consisting of the P2-gp160 and P7.5-
gpt-gene cassettes was also inserted into the Smal (NotI)
site of the WR 6/2 strain. The in vivo packaging was done
as described in Example 3.
Structure of the chimeric viruses. To confirm the
theoretical structures of the_chimeric viruses (Figure
9.3), Southern blot analyses are carried out. DNAs of the
purified viruses are cleaved with PstI and resulting
fragments are separated on an agarose gel, transferred to
a nitrocellulose membrane and hybridized to a vaccinia
thymidine kinase (tk) gene and a gp160-gene probe. With
the tk-gene probe, in the case of vP2-gp160,,,n,~A, the
predicted 6.9 and the 14.3 kb fragments are visible, and
for vP2-gp160,"Q,,B, the predicted 8.7 and 12.5 kb fragments
are visible. With the gp160-probe (pNINenvl), the
predicted 14.3 kb of vP2-gp160~"n,,A and 8.7kb fragment of
vP2-gp160r"Q,,B are visible, confirming the integration of the
foreign gene cassettes in two different orientations.
Expression studies with the chimeric viruses vP2
gp160~A and vP2-gp160~,n,~H. Vero cells are . chosen for
expression studies. Growth of cells, infection with the
chimeric viruses and purification of the recombinant gp160
protein are carried out as described by Barrett et al.,
supra . _
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Westera blots of'gp160: The Western blots are done
essentially as described by Towbin et al., Proc. Natl.
Acad. Sci. USA 83: 6672-6676 (1979). The first antibody
is a mouse monoclonal anti-HIV-gp120 antibody (Du Pont,
Inc. #NEA9305) used at a 1:500 dilution. The second
antibody is a goat-anti-mouse IgG (H+L) coupled with
alkaline phosphatase (HioRad, Inc., #170-6520) used at a
1:1000 dilution. The reagents (HCIP and NBT) and staining
protocols are from Promega, Inc.
Construction of the plasmid pselP-gp160bQ1 and of the
ehimeric virus vselP-gp160",Q,,. The synthetic early/late
promoter self (SEQ ID N0:70) (S. Chakrabarti & B. Mass;
see European Patent Application No. 91 114 300.-6,
Recombinant Fowlpox Virus) which is one of the strongest
known vaccinia virus promoters, was used in this example
to express the gp160-gene of the HIV-1 MN strain. First,
the plasmid pselP-gpt-L2 was constructed (Fig. 9.4). This
plasmid includes the self-promoter followed by a multiple
cloning site for the insertion of foreign genes, as either
complete or incomplete open reading frames, and
translational stop codons in all reading frames followed
by the vaccinia virus early transcription stop signal,
TTTTTNT. Rohrmann et al., Cell 46:1029-1035 (1986). The
P7.5 gpt-gene cassette is located adjacent to the promoter
and serves as a dominant selection marker. Falkner et
al., J. virol. 62: 1849-1854 (1988). The selP-
promoter/marker gene cassettes are flanked by restriction
endonuclease cleavage sites that are unique in the
vaccinia virus genome (SfiI, NotI, RsrII) and can also be
excised as blunt ended fragments (for instance, by
cleavage with HpaI and SnaBI). To be able to insert the
gp160-gene into pselP-gpt-L2, an NcoI site was introduced
around the translational start codon. This mutation
results in the substitution of the amino acid arginine
(AGA) with alanine (GCC). This mutation in the second
amino acid of the signal peptide is not likely to
interfere with efficient expression of the gp160-gene.
The cloning procedure and the sequence around the wild-
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type and the modified gp160-gene is outlined in Fig. 9.5.
To introduce this mutation into the gp160-gene, a PCR
generated proximal fragment was exchanged. The
construction of the plasmids is described in more detail
below.
pL2: For the construction of pL2, the 0.6kb XbaI-
ClaI fragment of the plasmid pTM3 (Moss et al., Nature,
348: 91 (1990)) was substituted by an XbaI-ClaI adaptor
fragment consisting of the annealed oligonucleotidea o-542
(SEQ ID N0:52) 5'-CGA TTA CGT AGT TAA CGC GGC CGC GGC CTA
GCC GGC CAT AAA AAT-3' and o-544 (SEQ ID N0:53) 5'-CTA GAT
TTT TAT GGC CGG CTA GGC CGC GGC CGC GTT AAC TAC GTA AT-3'.
The intermediate plasmid resulting from this cloning step
was called pLl. The 0.84kb AatII-SphI fragment (parts of
noncoding gpt-sequences) were substituted by the AatII-
SphI adaptor fragment consisting of the annealed
oligonucleotides o-541 (SEQ ID N0:54: 5'-CTT TTT CTG CGG
CCG CGG ATA TGG CCC GGT CCG GTT AAC TAC GTA GAC GT-3') and
o - 5 4 3 ( S EQ ID NO : 5 5 : 5 ' - CTA CGT AGT TAA CCG GAC CGG GCC
2 0 ATA TAG GCC GCG GCC GCA GAA AAA GCA TG- 3 ' ) . The resul t ing
plasmid was called pL2.
pTZ-L2: The XbaI-SphI fragment (consisting of the
T7-promoter-EMC-T7-terminator segment, the multiple
cloning site and the P7.5-gpt gene cassette) was treated
with Klenow-polymerase and inserted between the PvuII
sites of the plasmid pTZl9R (Pharmacia, Inc.). The
resulting plasmid was called pTZ-L2 (SEQ ID N0:64).
Additional features of this plasmid are shown in the table
below.
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pTZ-L2 (4701 bp) (SEO ID N0:64)
Location Description
1 pTZl9R sequences (Pharmacia)
-
55
56 108 Linker I in rc orientation (STNT, NotI, SfiI,
-
RsrII, HpaI, SnaBI, AatII
110- 860 E. coli gpt sequences in rc orientation. The
gpt
open reading frame starts with a rc TAC start
codon at position 860 and ends with a rc ATT
stop
codon at pos 403
861 1338 Vaccinia Virus p7.5 promoter sequences in rc
-
orientation
1339 - 1344 HpaI site between bacteriophage T7
terminator and Vaccinia Virus p7.5 promoter
1345 - 1488 Bacteriophage T7 terminator sequences in rc
orientation (Studier et al.)
1489 - 1558 Multiple cloning site in rc
orientation (SalI, translation stop
codons for all three open reading
frames, StuI, XhoI, PstI, BamHI,
2 SpeI, SacI, SmaI, EcoRI, NcoI)
0
1559 - 2131 s a q a a n c a s f r o m t h a
Encephalomyocarditis Virus (EMC-
Virus) 5'untranslated region () in rc
orientation
2 2132 - 2187 Bacteriophage T7 promoter sequences
5
in rc orientation ()
2190 - 2242 Linker II in rc orientation (SnaBI,
HpaI, NotI, SfiI, STMT)
2243 - 4701 pTZl9R sequences (Pharmacia)
30 PTZseIP-L2 and pselP-gpt-L2: The 0.6kb ClaI-NcoI
fragment (the T7-promoter-EMC-sequence) was replaced with
a synthetic promoter fragment consisting of the annealed
oligonucleotides o-selPI (SEQ ID N0:56: 5'-CGA TAA AAA TTG
AAA TTT TAT TTT TTT TTT TTG GAA TAT AAA TAA GGC CTC-3'; 51
35 mer) and o-selPII (SEQ ID N0:57: 5'-CAT GGA GGC CTT ATT
TAT ATT CCA AAA AAA AAA AAT AAA ATT TCA ATT TTT AT 3').
The resulting intermediate plasmid pTZselP-L2 still
contains the T7-terminator and a HpaI site, that were
removed in the following cloning step thereby inserting a
40 vaccinia early transcription stop signal and reducing the
size of the P7.5 promoter fragment from 0.28 to O.lBkb.
The 239bp SaII-NdeI fragment waa substituted by the SaII-
NdeI adaptor consisting of the annealed oligonucleotides
0-830 (SEQ ID N0:58: 5'-TCG ACT TTT TAT CA-3') and o-857
45 (SEQ ID N0:59: 5'-TAT GAT AAA AAC-3'). The resulting
plasmid was called pselP-gpt-L2 (SEQ ID N0:65).
Additional features of this construct are shown in the
table below.
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pselP-crpt-L2 (3878 bp) (SEO ID N0:65)
Location Descr~tion
1 - 55 pTZl9R sequences (Pharmacia)'
56 - 108 Linker I in rc orientation (STMT, NotI, SfiI,
RsrII, HpaI, SnaBI, AatII)
110 -860 E.coli gpt sequences in rc orientation. The
gpt
open reading frame starts with a rc TAC start
codon at position 860 and ends with a rc ATT
stop
codon at position 403
861 - 1245 Vaccinia Virus p7.5 promoter sequences in rc
orientation starting with the p7.5 internal
NdeI site at position 1241
1246 - 1258 Vaccinia Virus early transcription stop signal
in
rc orientation flanked by a NdeI site (position
1245) and a SalI site (position 1253)
1259 - 1322 Multiple cloning site in rc orientation (SalI,
translation atop codons for all three reading
frames, StuI, XhoI, PstI, BamHI, SpeI, SacI,
SmaI,
EcoRI, NCOI)
2 0 1323 - Vaccinia Virus synthetic early late promoter
1374 in rc
orientation flanked by a NcoI site at position
1317 and a ClaI site at position 1370
1375 -1414 Linker II in rc orientation (SnaBI, HpaI, NotI,
SfiI, STMT)
1415 - 3878pTZl9R Sequences (Pharmacia)
pselP-gp160baT: The 3.lkb env gene containing the
EcoRI-PvuII fragment of pNINenvI was inserted into the
EcoRI and StuI cut plasmid pselP-gpt-L2 resulting in the
intermediate plasmid pselP-gp160.1. The 0.8kb NcoI-NsiI
fragment of pselP-gp160 was substituted by a PCR-generated
0.31kb NcoI-NsiI fragment resulting in the final plasmid
pselP-gp160,"Q,~ (SEQ ID N0:66) . Additional features of this
plasmid are shown in the table below.
gselP-gp160MN (6474 bp) (SEO ID N0:66)
Location Description
1 - 55 pTZl9R sequences (Pharmacia)
56 - 108 Linker I in rc orientation (STNT, SfiI, RsrII,
HpaI, SnaBI, AatII)
110 -860 E.coli gpt sequences in rc orientation. The gpt
4 0 open reading frame starts at position 860 with
a
rc TAC start codon and ends at position 403 with
a rc ATT stop codon
861 - 1245 Vaccinia Virus p7.5 promoter sequences in rc
orientation starting with the p7.5 internal NdeI
4 5 site at position 1241
1246 - 1258 Vaccinia Virus early transcription stop signal
in
rc orientation (position 1245-1252) flanked by
a
NdeI site at position 1241 and a SalI site at
position 1253.
50 1259 - 3916HIV-MN env gene in rc orientation. The ORF starts
at position 3916 with a rc TAC start codon and
ends at position 1348 with a rc ATT stop codon
3917 - 3970 Vaccinia Virus synthetic early late promoter
in rc
orientation flanked by a NcoI site (position
55 3913) and a ClaI site (position 3966)
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3971 - 4015 Linker II in rc orientation (SnaBI, HpaI, NotI,
Sfil, STNT)
4016 - 6474 pTZl9R sequences (Pharmacia)
The primers used for the PCR reaction were o-NcoI
(40mer) SEQ ID N0:60: 5'-GAG CAG AAG ACA GTG GCC ATG GCC
GTG AAG GGG ATC AGG A-3', and o-NsiI (30mer) SEQ ID N0:61:
5'-CAT AAA CTG ATT ATA TCC TCA TGC ATC TGT-3'. For
further cloning the PCR product was cleaved with NcoI and
NsiI.
Chimeric viruses vselP-gp160".n,,A vselP-gp160",Q,,H are
constructed as follows: The HpaI-fragment consisting of
the self-gp160 and P7.5-gpt-gene cassettes is inserted by
direct molecular cloning (Fig. 9.6) into the single SmaI
site of the vaccinia strain WR6/2 (Moss et al., J. Virol.
40: 387-395 (1981)] which is a highly attenuated vaccinia
virus strain. See Huller et al., Nature 317: 813-815
(1985). The vaccinia virus strain WR6/2 (Moss et al.,
supra) is cut at its single SmaI site and ligated with the
4.Okb HpaI fragment that contains the P7.5-gpt-gene and
the self-gp160-gene cassettes. The cloning procedures are
carried out as described in Example 1.
The resulting chimeric viruses, vselP-gp160,,,Q.1A and
vselP-gp160",flrB, are purified and further characterized. In
the virus vselP-gp160,,,Q,1A, the gp 160-gene is transcribed in
the same direction (left to right) as the genes clustered
around the insertion site (the A51R open reading frame;
Goebel et al., Virology 179: 247-266 (1990)]. In the
virus vaelP-gp160"Q,IH, the gp160-gene is transcribed in the
reverse direction. The in vivo packaging is done as
described in Example 3.
Structure of the chimeric viruses: To confirm the
theoretical structures of the chimeric viruses (Fig. 9.7),
Southern blot analyses are carried out . The DNA of the
purified viruses was cleaved with SalI and fragments are
separated on an agarose gel, transferred to a
nitrocellulose membrane and hybridized to vaccinia SalF-
fragment probe (pTZ-SalF) and a gp160-gene probe
(pMNenvl). With the SalF-fragment probe, for vselP-
CA 02515166 1992-08-25
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gp160".n,,A the predicted 6.8 and 10.7 kb fragments are
visible; and for vselP-gp160~B, the predicted 3.5 and 13.7
fragments are visible. With the'gp160-probe, the same
fragments are seen, but the 10.7 kb fragment in vselP-
gp160~''°''A and the 3.5kb fragment in vselP-gp160,,,Q,,B give less
intense signals, because only about 400 by of each total
fragment is homologous to the probe.
Since direct cloning also results in integration of
tandem multimer structures, the DNA of the viruses is also
digested with XbaI which does not cut the inserted DNA.
The XbaI wild-type fragment is 447bp in size. Integration
of one copy of the 3.8kb sized insert results in a
fragment of 4.3kb. In multimeric structures the size of
the 4.3kb fragment increases in increments of 3.8kb.
Expression studies with the chimerie viruses vselP
gp160"Q,,A and vselP-gp160",fl,,H: Vero cells are used for
expression studies. Growth of cells, infections with the
chimeric viruses and purification of the recombinant gp160
protein are carried out as described by Barrett et al.,
supra.
Example 10. Construction of novel chimeric
vaccinia viruses encoding human
protein S (vProtS) and expression of
recombinant protein S.
This example illustrates the construction of
recombinant protein S expressed by chimeric vaccinia
virus. Human protein S is a 70 kDa glycoprotein involved
in the regulation of blood coagulation. DiScipio et al.,
Biochemistry, 18: 89-904 (1979) The cDNA and the genomic
DNA of Protein S have been cloned and characterized.
Lundwall et al., Proc. Natl. Acad. Sci. USA, 83: 6716
(1986); Hoskins et al., Proc. Natl. Acad. Sci. USA, 84:
349 (1987); Edenbrandt et al., Biochemistry, 29: 7861
(1990); Schmidel et al., Biochemistry, 29: 7845 (1990)
Human protein S, normally synthesized as a 70 kDa
protein in liver and endothelial cells (DiScipio et al.,
supra), has been expressed in permanent cell lines derived
from human 293 and hamster AV12-664 cells (adenovirus
CA 02515166 1992-08-25
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transformed cell lines) at levels of up to 7 ~.g/106
cells/day (Grinnell et al., Elood, 76: 2546 (1990)) or in
mouse C127 cell/papilloma virus system at similar
expression levels. Malm et al., Eur. J. Eiochem., 187:
737 (1990) The protein derived from the latter cells was
larger than plasma-derived protein S probably due to
aberrant glycosylation.
The present expression of protein S uses a double
gene cassette consisting of the complementary DNA for the
human blood factor protein S and the gpt gene, each
controlled by a vaccinia promoter. This was cloned into
the unique NotI site and packaged in fowlpox helper virus-
infected mammalian cells. Human protein S was expressed
in infected Vero cells in levels of 4-6 ~,g per 106 cells.
For the cell screening, for optimal protein S
expression by the chimeric vaccinia virus, five different
host cell lines were used, WI 38 (human embryonal lung
fibroblast), CV-1 and Vero (monkey kidney cells), Chang
liver and SK Hepl. Protein S was indistinguishable from
plasma-derived protein S by several criteria: the
recombinant material derived from the infected cells of
this cell line showed the same electrophoretic migration
patterns and the same chromatographic elution profiles as
plasma-derived protein S. This indicates that the correct
post-translational modification of this complex
glycoprotein has occurred. The methods are described in
detail below.
Construction of the plasmid pN2-gptaProtS.
Single-stranded DNA prepared from the plasmid pBluescript
ProtS, comprising the cDNA coding for human protein S
(provided by R. T. A. McGillivray) was used to mutagenize
the region around the translational start codon of the
protein S coding region into an NcoI site (CCATGG). The
mutagenic primer, oProtSl (SEQ ID N0:68), has the sequence
5'-ACC CAG GAC CGC CAT GGC GAA GCG CGC-3'; the mutagenesis
was carried out as described in the mutagenesis protocol
(Amersham, Inc.). The signal peptide is mutated, with the
CA 02515166 1992-08-25
-125-
second amino acid changed from Arg to Ala (Figure 10.1 H
and SEQ ID NOs. 77 and 79) This introduces the NcoI site
required for further cloning and~brings the ATG start
codon into an optimal context for translation. This may
improve the secretion of protein S.
The protein S cDNA was subsequently excised as an
NcoI-NotI fragment and inserted into the vaccinia
insertion plasmid pTKgpt-self (Falkner et al., supra) a
plasmid providing a strong synthetic vaccinia promoter.
The promoter-protein S gene cassette was then excised as
a eglII-NotI fragment and inserted into the plaemid pN2-
gpta (Example 1) resulting in pN2-gptaProtS (SEQ ID
N0:67). Additional features of this construct are shown
in the following table.
pN2-cmtaProtS (6811 bp) !S$O ID N0:67)
Location Description
1 - 2217 Bluescript II SK- sequences (Stratagene)
2218 - 2225 NotI site 1
2226 - 4938 ProtS sequences in rc orientation. The open
reading frame starts at position 4938 with
a rc
TAC start codon and ends at position 2910
with a
rc ATT stop codon
4939 - 4992 Vaccinia Virus synthetic early late promoter
in rc
orientation flanked by a NcoI site (position
4935)
and a fused BglII/BamHI site (position 4987-4992)
.
The Ncol site harbors the Prot S rc start
codon
TAC
4993 - 5493 Vaccinia Virus p7.5 promoter sequences
5494 - 6127 fi.coli gpt sequences. The ORF starts at
position
3 0 5494 with ATG start codon and ends at position
5950 with a TAA stop codon.
6228 - 6235 NotI Bite 2
6236 - 6811 Bluescript II SK- sequences (Stratagene)
In this plasmid, the gpt-gene controlled by the
vaccinia virus P7.5 promoter and the protein S cDNA, is
transcribed divergently and flanked by NotI sites.
Insertion of the cDNA for human protein S into the
single NotI site of vaccinia virus to form vProtS. The
NotI-fragment consisting of the gpt gene and protein S
gene cassettes was ligated with the vaccinia vector arms
and transfected into FPV infected mammalian CV-1 cells.
Only packaged vaccinia virus multiplied under these
conditions. More particularly, vaccinia wild-type DNA of
CA 02515166 1992-08-25
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the WR strain (1 ~.g) was cut with NotI, the enzyme was
heat-inactivated for 30 min at 65°C. The vector was
ligated overnight with 1 ~.g of the 3.8kb gpt/Protein S
gene cassette (excised as a NotI fragment out of the
plasmid pN2gpta-ProtS) in 30 ~1 using 15 units of T4 DNA
ligase.
The crude virus stocks prepared after five days of
incubation were titrated in the presence and in the
absence of mycophenolic acid (MPA). This procedure
distinguished chimeric from back-ligated wild-type virus.
With MPA 4x104 and without the drug 6 x 103 gfu/106 host
cells were obtained. About 6-7% of the viral plaques were
chimeric viruses. Ten of the gpt-positive isolates were
plaque-purified twice, grown to small crude stocks and
were used to infect CV-1 cells. Total DNA was prepared,
cut with the restriction enzymes SacI and Notl and
subjected to Southern blot analysis (Fig. 10.2). The SacI
digest, hybridized with the cloned SacI-T fragment
(plasmid pTZ-Sacl; Example 4), allowed the determination
of the orientation of the inserted DNA because SacI cuts
the inserts asymmetrically. In all ten isolates the
inserts were in the 'a'-orientation (fragments of 6.3 and
4.6kb; see Fig. 10.2A and C), indicating that this
configuration is strongly preferred. The NotI fragments
were hybridized with the protein S probe. In this case
the 3.8kb NotI gene cassette was released (Fig. 10.2H).
Expression of human protein S by a chimeric vaccinia
virus. Crude stocks were grown from gpt-positive chimeric
viruses and used for infection of various mammalian cell
lines. Monolayers of 5x106 cells were infected with 0.1
pfu/cell in the presence of serum free medium (DMEM)
supplemented with 50 ~,g/ml vitamin K and incubated for 72
hours. Supernatants were collected and protein S antigen
was determined using an ELISA test kit from Hoehringer
Mannheim, FRG (Kit Nr. 1360264). Amounts of protein S
synthesized are given in Table 1 in milli-units (1 U
corresponds to 25 ~.g of protein S).
CA 02515166 1992-08-25
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Alternatively, 10 ~.1 of supernatant from Vero cells
were analyzed in a Western Hlot using 50 ng of human
plasma- derived protein S as a 'standard and a mouse
polyclonal serum specific for "hu Prot S" (Axell) (Fig. A
10.3). Blots were stained using an alkaline phosphatase
conjugated goat anti- mouse polyclonal serum (Dakopatts)
and NBT/BCIP ae a substrate.
Purification of recombinant protein S from cell
culture supernatants was performed as described, by
Grinnell et al., 1990.
Table 10.1
Cell line ATCC# mU huProtS per 106 cells
SK Hepl (HTB52) 750
Vero (CCL 81) 127
Chang Liver (CCL 13) 135
CV-1 (CCL 70) 450
WI 38 (CCL 75) 440
Example 11: Construction of novel chimeric vaccinia
viruses encoding human factor IX and
expression of recombinant factor IX.
A double gene cassette consisting of the
complementary DNA for the human blood factor IX and the
gpt gene, each controlled by a vaccinia promoter, was
cloned into the unique NotI site of the vaccinia virus WR
genome and packaged in fowlpox helper virus-infected
mammalian cells. Human factor IX was expressed in several
cell types.
Human clotting factor IX is a 56 kDa glycoprotein
involved in the regulation of blood coagulation. This
clotting factor undergoes complex post-translational
modifications: vitamin K dependent carboxylation of the
first 12 glutamic residues, glycosylation, 3-hydroxylation
of an aspartic acid and amino terminal protein processing.
Davie, E. W., "The Hlood Coagulation Factors: Their
cDNAs, Genes and Expression", Hemostasis and Thrombosis,
CA 02515166 1992-08-25
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Colman, R. W., Hirsh, J. Marder, V.J. and Salzman, E. W.,
eds., J.B. Lippincott Co. (1987). Hemophilia B, an X
chromosome-linked bleeding disorder, is caused by mutation
of factor IX. Patients with hemophilia are currently
treated by substitution with plasma-derived factor IX.
The cDNA and the genomic DNA of factor IX ( "FIX" )
have been cloned and characterized and FIX has been
expressed in permanent cell lines. Busby et al., Nature,
316: 271 (1985); Kaufman, et al., J. Biol. Chem., 261:
9622 (1986); Halland, et al., Eur. J. Biochem., 172: 565
(1922), and Lin et al., J. Biol. Chem., 265: 144 (1990).
Expression of factor IX in vaccinia recombinants has also
been described. de la Salle, et al., Nature, 316: 268
(1985).
Construction of plasmids.
pN2gpta-FIX: The FIX cDNA (kindly provided by R. T. A.
MacGillivray) was cut out from the plasmid pHluescript-FIX
with EcoRI and ligated with the EcoRI linearized plasmid
pTM3. Moss, et al., Nature, 348: 91 (1990) Single strand
DNA was isolated from a recombinant plasmid which
contained the FIX insert in the correct orientation and a
NcoI site (CCATGG) was introduced around the FIX ATG start
codon by oligonucleotide mediated site directed
mutagenesis using oligonucleotide oFIX.l (SEQ ID N0:71: 5'
-TCA TGT TCA CGS GCT CCA TGG CCG CGG CCG CAC C-3') and a
commercial mutagenesis kit (Amersham, Inc.; kit No. PPN
1523). Vector and FIX NcoI sites were fused, insert DNA
was isolated by NcoI and NotI digestion and ligated with
the NcoI/NotI cut vector pTKgpt-self. Falkner et al.,
supra The promoter/FIX cassette was cut out from this
plasmid with HgIII and NotI and ligated with the
BamHI/NotI linearized vector pN2-gpta (Example 1). From
this construct a NotI cassette containing the FIX cDNA
(under the control of the self promoter) and the gpt gene
(under the control of the vaccinia P7.5 promoter) was
isolated and used for in vitro molecular cloning and
packaging as described in Example 10.
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Additional characteristics of this plasmid are shown in
the table below.
pN2 qpta-FIX (5532bp) (SEO ID N0:72)
Location Description
1-2217 Bluescript II SK-sequences (Stratagene)
2218-2225 NotI site 1
2226-3659 FIX sequence in rc orientation. The open reading
frame starts at position 3659 with a rc TAC
start
codon and ends at position 2276 with a rc ATT
stop
codon
3660-3713 Vaccinia Virus synthetic early late promoter
in rc
orientation flanked by a NcoI site (position
3656
and fused BglII/BamHI site (position 3708-3713).
The Ncoi site harbors the FIX rc start codon
TAC
3714-4214 Vaccinia Virus P7.5 promoter sequences
4215-4848 fi.coli gpt sequences. The ORF starts at position
4215 with an ATG start codon and ends at position
4671 with a TAA stop codon.
4849-4856 NotI site 2
2 4857-5532 Bluescript II SK-sequences (Stratagene)
0
Insertion of the eDNA for human Factor IX into the
single NotI site of vaccinia virus. Prior to insertion of
the factor IX cDNA into vaccinia virus, this cDNA was
inserted into the plasmid pN2-gpta resulting in the
plasmid pN2gpta-FIX (Fig. il.lA, SEQ ID N0:72). To obtain
the optimal sequence context between the synthetic
vaccinia promoter and the factor IX coding region, the 5'
untranslated region of factor IX was deleted by
introduction of a novel NcoI site at the start codon of
factor IX and fusion of this NcoI site with the NcoI site
provided by the promoter. This mutation resulted in a
mutated signal peptide (Fig. 11.1B). In the wildtype
factor IX the second amino acid of the signal peptide is
a glutamine residue while in pN2gpta-FIX the second amino
acid is a glutamic acid residue.
The Notl fragment consisting of the gpt-gene and
factor IX gene cassettes was ligated with the vaccinia
vector arms and transfected into FPV infected mammalian
CV-1 cells. Only packaged vaccinia virus multiplied
under these conditions. The crude virus stocks prepared
after five days of incubation were titrated in the
presence and in the absence of mycophenolic acid (MPA).
This procedure distinguished chimeric from back ligated
wild-type virus. With MPA 5x104 and without the drug 5x106
CA 02515166 1992-08-25
-130-
pfu/106 host cells were obtained. In this example, about
1% of the viral plaques were chimeric viruses. Ten of the
gpt-positive isolates were plaque-purified twice, grown to
small crude stocks and were used to infect CV-1 cells. '
Total DNA was prepared from eight cell cultures infected
with the respective viral isolates, digested with the
restriction enzymes SfuI, Ndel and Notl and subjected to
Southern blot analysis.
The SfuI digest, hybridized with the factor IX
probe, allowed the determination of the orientation of the
inserted DNA because SfuI cuts the inserts asymmetrically.
In all eight isolates the inserts were in the 'a'
orientation ( f ragments of 6 . 3 and 4 . 6kb; see Fig . 11. 2 ) ,
indicating that this configuration is strongly preferred.
The NdeI (NotI) fragments were also hybridized with the
factor IX probe. In this case a fragment of 6.6kb (the
3.Skb NotI gene cassette) was released, proving the
predicted structure.
Expression Of Human Factor IX. Crude stocks were
grown from eight single plaque isolates and used for
infection of various mammalian cell lines. 5x106 cells in
a 10 cm petri dish were infected with a moi of 0.1
pfu/cell in the presence of serum free medium (DMEM) and
50 ~,g/ml vitamin K. Infected cells were incubated for 72
hours until cells started to detach from the bottom of the
petri dish. Supernatants were collected, cell fragments
were removed by centrifugation and FIX amounts were
determined using an ELISA test kit from Boehringer
Mannheim, FRG (Kit Nr. 1360299). Amounts of FIX antigen
and of factor IX activities are given in Table 11.1.
Alternatively, 10 ~1 of supernatant from Vero cells
were analyzed in a Western Hlot using 50 ng of human
plasma derived huFIX as a standard and a mouse polyclonal
serum specific for huFIX (Axell). Blots were stained
using an alkaline phosphatase conjugated goat-anti-mouse
polyclonal serum (Dakopatts) and NBT/BCIP as a substrate.
As shown in Fig. 11.3, the recombinant material migrated
as a broad band similar to the plasma-derived factor IX
CA 02515166 1992-08-25
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standard. Clotting assays of the partially purified Vero
cell derived factor IX showed that about 50% of the
material was active factor IX. 'The virus isolate #5,
designated vFIX#5, was grown to large scale and used for
further experiments.
As in the case of the protein S chimeric viruses
(Example 10), the factor IX expressing chimeras had
inserts in one preferred orientation.
The protein of transcription of the gene of interest
( factor IX and protein S ) was f rom right to lef t , i . a . the
same direction as the genes clustered around the NotI
site. It seems therefore, that strongly transcribed units
have to be aligned in the preferred transcriptional
direction when cloned into the NotI cluster. Viruses with
this configuration of the insert. are strongly preferred
and show the beat growth characteristics. The direction
of transcription of the second gene cassette, the P7.5 gpt
gene, was from the left to right. The P7.5 promoter
segment is therefore in an inverted repeat configuration
relative to the nearby endogenous gene coding for the 7.5
kDa protein, i.e. the expected stable configuration is
preferred. Since no chimeras with the reverse orientation
were found, the 'b' -orientation is probably unstable.
Insertion of the above mentioned gene cassettes in the
'b'-orientation by in vivo recombination would have
failed, leading to the misinterpretation that the NotI
intergenic region is essential for viral growth. This
situation illustrates one of the advantages of the direct
cloning approach: only 'allowed' are structures are
formed.
By insertion of simple small gene cassettes, both
orientations and multimers were obtained (Example 1) while
insertion of complex gene cassettes (divergently
transcribed double gene cassettes with homologies to
internal genes such as the P7.5 promoter segment)
preferred structures were formed.
The cell screening for optimal factor IX expression
showed that infection of CV-1 and SK Hepl cells resulted
CA 02515166 1992-08-25
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in the highest antigen levels. The material from CV-1
cells had the highest clotting activites (table 11.1),
indicating that this cell line possesses effective
post-translational modification systems. Factor IX
has been expressed previously in the conventional
vaccinia expression system using the P7.5 promoter and
HepG2 and BHK cells (de la Salle et al . , 1985 Nature
316:268). Cell lines with better growth
characteristics, like Vero and CV-1 cells, have been
shown to produce higher levels of expression with the
instant viruses, due to improved promoters and
methods. In addition, deletion of the 5'-untranslated
region of the factor IX cDNA and the modification of
the signal peptide seems to have positive effects on
secretion and expression levels.
Table 11.1.
Factor IX Expression in Different Cell Lines
cell line ATCC# antigen activity ratio
(mU/10°cells) * %
gR Hepi 183 22.5
(HT852) 810
Vero (CCL81) 500 282 56:4
Chang Liver (CCL13) 190 100 52.6
CV-1 (CCL70) 850 1290 151.8
RK13 (CCL37) 300 460 153.3
30
* 1 unit corresponds to 5 ~g FIX per ml human plasma
Example 12: Construction of the chimeric fowlpox
virus f-envIIIB and expression of
recombinant HIVIIIB envelope proteins
in chicken embryo fibroblasts.
The large scale production of gp160 in a vaccinia
virus-Velo cell expression system has been described
recently (Barrett et. a1. 1989 AIDS Res. Hum.
Retrovirus 6:159-171). Since vaccinia virus is still
pathogenic to many vertebrates including mammals and
fowlpox virus is host restricted to avian species we
CA 02515166 1992-08-25
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have developed an avipox based expression system (EP
0,338,809). Chimeric fowlpox viruses have now been
constructed by direct molecular cloning to express the
envelope gene of the HIV-1 IIIB isolate. In .this
recombinant
CA 02515166 1992-08-25
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virus the env gene is controlled by a strong synthetic
late promoter. For the production of envelope
glycoproteins, the chimeric fowlpox virus is used to
infect chicken embryo aggregate cell cultures. Mundt et
al., PCT/WO 91 709 937.
Construction and structure of the chimeric fowlpox
virus f-envIIIH. For construction of f-envIIIH (Fig.
12.1) a double gene cassette consisting of the P7.5-
promoter/gpt gene and the S4-promoter/gp160 gene were
excised as a NotI-fragment out of the plasmid pN2gpt-gp160
(Example 5). This cassette was ligated with NotI-cleaved
genomic DNA of the fowlpox virus f-TK2a (Example 2) and
chimeric virus was isolated as described in Materials and
Methods. Total DNA from chicken embryo fibroblasts
infected with twelve different plaques was digested with
SspI and further analyzed by Southern blotting and
hybridization with an isolated gp160 fragment as a probe
(Fig. 12.2A). The predicted fragments of 3.7, 1.0 and
0.8kb were found in 11 cases indicating that the gp160
gene had been integrated in the 'b'-orientation (Fig.
12.2H). One viral isolate, f-LF2e, did not hybridize to
the gp 160 probe.
The fact that one preferred orientation of the
insert exists, points to the possibility that the 'b'
orientation virus has growth advantages over the 'a'
orientation, the 'a' -orientation may even be unstable.
Letting the viral vector choose the best orientation may
be considered as an advantage of the direct cloning
approach.
Expression studies with the ehimeric virus f-
envIIIB. Expression studies were done in chicken embryo
fibroblasts (CEF). Confluent monolayers of CEFs were
infected with 0.1 pfu per cell of the different viral
crude stocks, grown for five days. Total cellular
proteins were separated on 10% polyacrylamide gels,
transferred onto nitrocellulose membranes and further
processed as described in Materials and Methods. A
Western blot showing the expression of gp160, gp120 and
CA 02515166 1992-08-25
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gp41 is shown in Figs. 12.3 and 12.4. All viral isolates,
except f-LF2e, induced expression of the env
glycoproteins. The virus f-LF2e was also negative in the
Southern blot and therefore does not carry the gp160 gene
sequences.
Construction of f-envIIIB. Two micrograms of DNA of
host virus vector f-Tk2a (Example 2) were cut with NotI
and ligated with 500 nanograms of the gene cassette
consisting of the P7.5-promoter/gpt gene and the.S4-
promoter/gp160 gene. The ligation was carried out in a
volume of 20 ~1 and 5U of ligase for four days at 12°C.
The ligation mixture was transfected into 6x 106 CEFs
infected with 0.5 pfu per cell of HP2, a fowlpox isolate
obtained by plaque-purification of HP1.441. After an
incubation period of five days a crude stock was prepared
(final volume 1 ml) which was amplified. The crude stock
was titrated on CEFs in six-well plates and grown for 5
days under gpt-selection (25 ~,g/ml mycophenolic acid, 125
ug xanthine). Cells on which the minimal dilution
resulted in a visible cytopathic effect, were harvested
and amplified twice according the same protocol. The
crude stock obtained from the second amplification from
the second amplification was titered on CEFs in the
presence of gpt-selection and 12 single plaques (f-LF2a-1)
were picked.
Western blots of gp160. The Western blots were done
essentially as described by Towbin et al., supra. For
gp160/gp120 detection, the first antibody was a mouse
monoclonal anti-HIV-gp120 antibody (Du Pont, Inc. #
NEA9305 used in a 1:500 dilution. For the gp41 detection
the human anti-HIV-gp41 3D6 Mab (provided by H. Katinger,
Universitat fur Hodenkultur, Inst. fur Angewandte
Mikrobiologie) was used a 1:500 dilution. The second
antibody was a goat-anti-mouse IgG (H+L) coupled with
alkaline phosphate (BioRad, Inc. #170-6520) used in a
1:1000 dilution. The reagents (BCIP and NBT) and staining
protocols are from Promega, Inc.
CA 02515166 1992-08-25
SEQUENCE LISTING
(1) GfiN'6RAL INFORMATION:
(i) APPLICANT: DORMER, F.
SCHEIFLINGER, F.
FALKNER, F. G.
PFLfiIDfiRER, M.
(ii) TITLE OF INVENTION: DIRECT MOLECULAR CLONING OF A MODIFIED
EUKARYOTIC CYTOPLASMIC DNA VIRUS GENOME
(iii) NUMBER OF SEQUENCES: 84
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEfi: Foley & Lardner
(B) STRfiET: 1800 Diagonal Road, Suite 500
(C) CITY: Alexandria
(D) STATfi: VA
(fi) COUNTRY: USA
(F) ZIP: 22313-0299
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBfiR: 07/750,080
(B) FILING DATE: August 26, 1991
(C) CLASSIFICATION: Unknown
(viii) ATTORNfiY/AGENT INFORMATION:
(A) NAME: 88NT, Stephen A.
(B) REGISTRATION NUMBER: 29,768 '
(C) REFERBNCfi/DOCKST NUMBER: 30472/106 IMMU
(ix) TELECOMMUNICATION INFORMATION:
(A) TfiLEPHONfi: (703) 836-9300
(B) TfiLEFAX: (703)683-4109
(C) TELEX: 899149
(2) INFORMATION FOR SEQ ID NO;l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPfi: nucleic acid
(C) STRANDFDNfiSS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCAAGCTTAT CGATACCGTC GCGGCCGCGA CCTCGAGGGG GGGCCCGG 48
-135-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1133 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) Il~'lEDIATE SOURCE:
(B) CLONE: pN2-gpta
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
CTAGAACTAG TGGATCCCCC AACTTAAGGG TACCGCCTCG ACATCTATAT 60
ACTATATAGT ~
AATACCAATA CTCAAGACTA CGAAACTGAT ACAATCTCTT ATCATGTGGG 120
TAATGTTCTC
GATGTCGAAT AGCCATATGC CGGTAGTTGC GATATACATA AACTGATCAC 180
TAATTCCAAA
CCCACCCGCT TTTTATAGTA AGTTTTTCAC CCATAAATAA TAAATACAAT 240
AATTAATTTC
TCGTAAAAGT AGAAAATATA TTCTAATTTA TTGCACGGTA AGGAAGTAGA 300
ATCATAAAGA
ACAGTGACGG ATGATCCCCA AGCTTGGACA CAAGACAGGC TTGCGAGATA 360
TGTTTGAGAA
TACCACTTTA TCCCGCGTCA GGGAGAGGCA GTGCGTAAAA AGACGCGGAC 420
TCATGTGAAA
TACTGGTTTT TAGTGCGCCA GATCTCTATA ATCTCGCGCA ACCTATTTTC 480
CCCTCGAACA
CTTTTTAAGC CGTAGATAAA CAGGCTGGGA CACTTCACAT GAGCGAAAAA 540
TACATCGTCA
CCTGGGACAT GTTGCAGATC CATGCACGTA AACTCGCAAG CCGACTGATG 600
CCTTCTGAAC
AATGGAAAGG CATTATTGCC GTAAGCCGTG GCGGTCTGGT ACCGGGTGCG 660
TTACTGGCGC
GTGAACTGGG TATTCGTCAT GTCGATACCG TTTGTATTTC CAGCTACGAT 720
CACGACAACC
AGCGCGAGCT TAAAGTGCTG AAACGCGCAG AAGGCGATGG CGAAGGCTTC 780
ATCGTTATTG
ATGACCTGGT GGATACCGGT GGTACTGCGG TTGCGATTCG TGAAATGTAT 840
CCAAAAGCGC
ACTTTGTCAC CATCTTCGCA AAACCGGCTG GTCGTCCGCT GGTTGATGAC 900
TATGTTGTTG
ATATCCCGCA AGATACCTGG ATTGAACAGC CGTGGGATAT GGGCGTCGTA 960
TTCGTCCCGC
CAATCTCCGG TCGCTAATCT TTTCAACGCC TGGCACTGCC GGGCGTTGTT 1020
CTTTTTAACT
TCAGGCGGGT TACAATAGTT TCCAGTAAGT ATTCTGGAGG CTGCATCCAT 1080
GACACAGGCA
AACCTGAGCG AAACCCTGTT CAAACCCCGC TTTGGGCTGC AGGAATTCGA 1133
TAT
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LBNGTH: 1133 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
-136-
CA 02515166 1992-08-25
(vii) IMMEDIATE SODRCE:
(B) CLONE: pN2-gptb
(xi) SEQUENCE DESCRTPTION: SEQ ID N0:3:
CTAGAACTAG TGGATCCCCC AAAGCGGGGT TTGAACAGGG TTTCGCTCAG 60
GTTTGCCTGT
GTCATGGATG CAGCCTCCAG AATACTTACT GGAAACTATT GTAACCCGCC 120
TGAAGTTAAA
AAGAACAACG CCCGGCAGTG CCAGGCGTTG AAAAGATTAG CGACCGGAGA 180
TTGGCGGGAC
GAATACGACG CCCATATCCC ACGGCTGTTC AATCCAGGTA TCTTGCGGGA 240
TATCAACAAC
ATAGTCATCA ACCAGCGGAC GACCAGCCGG TTTTGCGAAG ATGGTGACAA 300
AGTGCGCTTT
TGGATACATT TCACGAATCG CAACCGCAGT ACCACCGGTA TCCACCAGGT 360
CATCAATAAC
GATGAAGCCT TCGCCATCGC CTTCTGCGCG TTTCAGCACT TTAAGCTCGC ~
GCTGGTTGTC 420
GTGATCGTAG CTGGAAATAC AAACGGTATC GACATGACGA ATACCCAGTT 480
CACGCGCCAG
TAACGCACCC GGTACCAGAC CGCCACGGCT TACGGCAATA ATGCCTTTCC 540
ATTGTTCAGA
AGGCATCAGT CGGCTTGCGA GTTTACGTGC ATGGATCTGC AACATGTCCC 600
AGGTGACGAT
GTATTTTTCG CTCATGTGAA GTGTCCCAGC CTGTTTATCT ACGGCTTAAA 660
AAGTGTTCGA
GGGGAAAATA GGTTGCGCGA GATTATAGAG ATCTGGCGCA CTAAAAACCA 720
GTATTTCACA
TGAGTCCGCG TCTTTTTACG CACTGCCTCT CCCTGACGCG GGATAAAGTG 780
GTATTCTCAA
ACATATCTCG CAAGCCTGTC TTGTGTCCAA GCTTGGGGAT CATCCGTCAC 840
TGTTCTTTAT
GATTCTACTT CCTTACCGTG CAATAAATTA GAATATATTT TCTACTTTTA 900
CGAGAAATTA
ATTATTGTAT TTATTATTTA TGGGTGAAAA ACTTACTATA AAAAGCGGGT 960
GGGTTTGGAA
TTAGTGATCA GTTTATGTAT ATCGCAACTA CCGGCATATG GCTATTCGAC 1020
ATCGAGAACA
TTACCCACAT GATAAGAGAT TGTATCAGTT TCGTAGTCTT GAGTATTGGT 1080
ATTACTATAT
AGTATATAGA TGTCGAGGCG GTACCCTTAA GTTGGGCTGC AGGAATTCGA 1133
TAT
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECOLE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHindJ-2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CGCATTTTCT AACGTGATGG GATCCGTTAA CTCGCGAGAA TTCTGTAGAA AGTGTTACAT 60
CGACTC 66
-137-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPfi : nucleic acid
(C) STRANDEDNHSS: single
(D) TOPOLOGY: linear
(ii) MOLECULfi TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(8) CLONE: pHindJ-3
(xi) SEQUENCfi DfiSCRIPTION: SfiQ ID N0:5:
CGCATTTTCT AACGTGATGG GATCCGGCCG GCTAGGCCGC GGCCGCCCGG GTTTTTATCT ~ 60
CGAGACAAAA AGACGGACCG GGCCCGGCCA TATAGGCCCA ATTCTGTAGA AAGTGTTACA 120
TCGACTC 127
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTfiRISTICS:
(A) LENGTH: 115 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULfi TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pA0
(xi) SfiQUENCfi DESCRIPTION: SEQ ID N0:6:
AGGGAACAAA AGCTGGAGCT AGGCCGGCTA GGCCGCGGCC GCCCGGGTTT TTATCTCGAG 60
ACAAAAAGAC GGACCGGGCC CGGCCATATA GGCCAGTACC CAATTCGCCC TATAG 115
(2) INFORMATION FOR SfiQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 base pairs
(B) TYPfi: nucleic acid
(C) STRANDfiDNfiSS: single
(D) TOPOLOGY: linear
(ii) MOLECULfi TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATfi SOURCfi:
(B) CLONE: pAl
(xi) SEQUENCfi DESCRIPTION: SEQ ID N0:7:
CGGCCGCCCG GGTTTTTATC TCGACATATG CTGCAGTTAA CGAATTCCAT GGGGATCCGA 60
TATCAAGCTT AGGCCTGTCG ACGTCGAGAC AAAAAGACGG ACC 103
-138-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 base pairs
(B) TYPE: nucleic acid
(C) STRRNDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMi~sDIATE SOURCE:
(B) CLONE: pA2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
CGGCCGCCCG GGTTTTTATC TCGACGTCGA CAGGCCTAAG CTTGATATCG GATCCCCATG ~ 60
GAATTCGTTA ACTGCAGCAT ATGTCGAGAC AAAAAGACGG ACC 103
(2) INFORMATION FOR S8Q ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMI~DIATE SOURCE:
(B) CLONE: pAl-S1
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CCCGGGTTTT TATCTCGACA TACGGCTTGG TATAGCGGAC AACTAAGTAA TTGTAAAGAA 60
GAAAACGAAA CTATCAAAAC CGTTTATGAA ATGATAGAAA AAAGAATATA AATAATCCTG 120
TATTTTAGTT TAAGTAACAG TAAAATAATG AGTAGAAAAT ACTATTT1ZT ATAGCCTATA 180
AATCATGAAT TCGGATCCGA TATCAAGCTT AGG 213
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pA2-S1
-139-
CA 02515166 1992-08-25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CAGGCCTAAG CTTGATATCG GATCCGAATT CATGATTTAT AGGCTATAAA AAATAGTATT 60
TTCTACTCAT TATTTTACTG TTACTTAAAC TRAAATACAG GATTATTTAT ATTCTTTTTT 120
CTATCATTTC ATAAACGGTT TTGATAGTTT CGTTTTCTTC TTTACAATTA CTTAGTTGTC 180
CGCTATACCA AGCCGTATGT CGAGACAAAA AGACG 215
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 88 base pairs
(B) TYPB: nucleic acid
(C) STRANDEDNSSS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pAl-S2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TCTCGACATA TGCTGCAGTT GGGAAGCTTT TTTTTTTTTT T~TTGGC ATATAAATAG 60
GCTGCAGGAA.TTCCATGGGG ATCCGATA 88
(2) TNFnRMATTON FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECOLE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMBDIATE SOURCE:
(B) CLONE: pA2-S2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
TTGATATCGG ATCCCCATGG AATTCCTGCA GCCTATTTAT ATGCCAAAAA p~~FIAAAAAAA 60
AAAAAGCTTC CCAACTGCAG CATATGTCGA GA 92
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
-140-
CA 02515166 1992-08-25
(vi i ) IMMEDIATE SOURCE
(B) CLONE: pN2gpt-S3A (fig. 4.7)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
TACCCTTAAG TTGGGCTGCA GAAGCTTTTT TTTT'ITZ'TTT TTIZTGGCAT ATAAATGAAT 60
TCCATGGCCC GGGAAGGCCT CGGACCGGGC CCGGCCATAT AGGCCAGCGA TACCGTCGCG 120
GCCGCGA 127
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 134 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii ) IN~IBDIATE SOURCE
(B) CLONE: pN2gpt-S4
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
TACCCTTAAG TTGGGCTGCA GAAGCTTTTT TZ'ITTTTTTT TTITTGGCAT ATAAATCGTT 60
AACGAATTCC ATGGCCCGGG AAGGCCTCGG ACCGGGCCCG GCCATATAGG CCAGCGATAC 120
CGTCGCGGCC GCGA 134
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS: _.
(A) LENGTH: 1988 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii ) INB4fiDIATE SOiJRCE
(B) CLONE: pAlSl-PT
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
TTTTATAGCC TATAAATCAT GAATTCCGCG CACGTCCGAG GCTTGCAGCT GCCTGGCTGC 60
CTGGCCCTGG CTGCCCTGTG TAGCCTTGTG CACAGCCAGC ATGTGTTCCT GGCTCCTCAG 120
CAAGCACGGT CGCTGCTCCA GCGGGTCCGG CGAGCCAACA CCTTCTTGGA GGAGGTGCGC 180
AAGGGCAACC TAGAGCGAGA GTGCGTGGAG GAGACGTGCA GCTACGAGGA GGCCTTCGAG 240
GCTCTGGAGT CCTCCACGGC TACGGATGTG TTCTGGGCCA AGTACACAGC TTGTGAGACA 300
GCGAGGACGC CTCGAGATAA GCTTGCTGCA TGTCTGGAAG GTAACTGTGC TGAGGGTCTG 360
GGTACGAACT ACCGAGGGCA TGTGAACATC ACCCGGTCAG GCATTGAGTG CCAGCTATGG 420
-141-
CA 02515166 1992-08-25
AGGAGTCGCT ACCCACATAA GCCTGAAATC AACTCCACTA CCCATCCTGG 480
GGCCGACCTA
CAGGAGAATT TCTGCCGCAA CCCCGACAGC AGCAACACGG GACCATGGTG 540
CTACACTACA
GACCCCACCG TGAGGAGGCA GGAATGCAGC ATCCCTGTCT GTGGCCAGGA 600
TCAAGTCACT
GTAGCGATGA CTCCACGCTC CGAAGGCTCC AGTGTGAATC TGTCACCTCC 660
ATTGGAGCAG
TGTGTCCCTG ATCGGGGGCA GCAGTACCAG GGGCGCCTGG CGGTGACCAC 720
ACATGGGCTC
CCCTGCCTGG CCTGGGCCAG CGCACAGGCC AAGGCCCTGA GCAAGCACCA 780
GGACTTCAAC
TCAGCTGTGC AGCTGGTGGA GAACTTCTGC CGCAACCCAG ACGGGGATGA 840
GGAGGGCGTG
TGGTGCTATG TGGCCGGGAA GCCTGGCGAC TTTGGGTACT GCGACCTCAA 900
CTATTGTGAG
GAGGCCGTGG AGGAGGAGAC AGGAGATGGG CTGGATGAGG ACTCAGACAG 960
GGCCATCGAA
GGGCGTACCG CCACAAGTGA GTACCAGACT TTCTTCAATC CGAGGACCTT 1020
TGGCTCGGGA
GAGGCAGACT GTGGGCTGCG ACCTCTGTTC GAGAAGAAGT CGCTGGAGGA 1080
CAAAACCGAA
AGAGAGCTCC TGGAATCCTA CATCGACGGG CGCATTGTGG AGGGCTCGGA 1140
TGCAGAGATC
GGCATGTCAC CTTGGCAGGT GATGCTTTTC CGGAAGAGTC CCCAGGAGCT 1200
GCTGTGTGGG
GCCAGCCTCA TCAGTGACCG CTGGGTCCTC ACCGCCGCCC ACTGCCTCCT 1260
GTACCCGCCC
TGGGACAAGA ACTTCACCGA GAATGACCTT CTGGTGCGCA TTGGCAAGCA 1320
CTCCCGCACC
AGGTACGAGC GAAACATTGA AAAGATATCC ATGTTGGAAA AGATCTACAT 1380
CCACCCCAGG
TACAACTGGC GGGAGAACCT GGACCGGGAC ATTGCCCTGA TGAAGCTGAA 1440
GAAGCCTGTT
GCCTTCAGTG ACTACATTCA CCCTGTGTGT CTGCCCGACA GGGAGACGGC 1500
AGCCAGCTTG
CTCCAGGCTG GATACAAGGG GCGGGTGACA GGCTGGGGCA ACCTGAAGGA 1560
GACGTGGACA
GCCAACGTTG GTAAGGGGCA GCCCAGTGTC CTGCAGGTGG TGAACCTGCC 1620
CATTGTGGAG
CGGCCGGTCT GCAAGGACTC CACCCGGATC CGCATCACTG ACAACATGTT 1680
CTGTGCTGGT
TACAAGCCTG ATGAAGGGAA ACGAGGGGAT GCCTGTGAAG GTGACAGTGG 1740
GGGACCCTTT
GTCATGAAGA GCCCCTTTAA CAACCGCTGG TATCAAATGG GCATCGTCTC 1800
ATGGGGTGAA
GGCTGTGACC GGGATGGGAA ATATGGCTTC TACACACATG TGTTCCGCCT 1860
GAAGAAGTGG
ATACAGAAGG TCATTGATCA GTTTGGAGAG TAGGGGGCCA CTCATATTCT 1920
GGGCTCCTGG
AACCAATCCC GTGAAAGAAT TATI~'ITGTG TTTCTAAAAC TAGAATTCGG 1980
ATTCGATATC
AAGCTTAG 19 8
8
(2) INFORMATION FOR SfiQ ID N0:16:
(i) SEQUfiNCfi CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPfi: nucleic acid
(C) STRANDfiDNESS : single
(D) TOPOLOGY: linear
(ii) MOLfiCULfi TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
-142-
CA 02515166 1992-08-25
(vi i ) IMMEDIATE SOURCE
(B) CLONE: odNl
(xi) SEQUENCE DESCRIPTION: SfiQ ID N0:16:
GGCCAGGCCT TTTAAATTAA GATATC , 2 6
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2gpt-GPg
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
TTTTTGGCAT ATAAATCGTT CCAGTCCCAA AATGTAATTG GACGGGAGAC AGAGTGACGC 60
ACGCGGCCGC TCTAGAACTA GTGGATCCCC CAACGAATTC CATGGCCCGG G 111
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2296 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2gpt-LPg
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:18:
ATAAATCGTT AACGAATTCCATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTC 6
0
TGAAATCAGG TCAAGGAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACT 120
ACAGAGGGAC GATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTT 180
CTCCCCACAG ACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACT 240
ACTGCAGGAA TCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAA 300
AGAGATATGA CTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAG 360
AAAACTATGA CGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACT 420
CTCAGAGCCC ACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGA 480
AGAATTACTG TCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCA 540
ACAAGCGCTG GGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTC 600
-143-
CA 02515166 1992-08-25
CCACCTACCA GTGTCTGAAG GGAACAGGTG AAAACTATCG CGGGAATGTG 660
GCTGTTACCG
TTTCCGGGCA CACCTGTCAG CACTGGAGTG CACAGACCCC TCACACACAT 720
AACAGGACAC
CAGAAAACTT CCCCTGCAAA AATTTGGATG AAAACTACTG CCGCAATCCT 780
GACGGAAAAA
GGGCCCCATG GTGCCATACA ACCAACAGCC AAGTGCGGTG GGAGTACTGT 840
AAGATACCGT
CCTGTGACTC CTCCCCAGTA TCCACGGAAC AATTGGCTCC CACAGCACCA 900
CCTGAGCTAA
CCCCTGTGGT CCAGGACTGC TACCACGGTG ATGGACAGAG CTACCGAGGC 960
ACATCCTCCA
CCACCACCAC AGGAAAGAAG TGTCAGTCTT GGTCATCTAT GACACCACAC 1020
CGGCACCAGA
AGACCCCAGA AAACTACCCA AATGCTGGCC TGACAATGAA CTACTGCAGG 1080
AATCCAGATG
CCGATAAAGG CCCCTGGTGT TTTACCACAG ACCCCAGCGT CAGGTGGGAG 1140
TACTGCAACC
TGAAAAAATG CTCAGGAACA GAAGCGAGTG TTGTAGCACC TCCGCCTGTT ~ 1200
GTCCTGCTTC
CAGATGTAGA GACTCCTTCC GAAGAAGACT GTATGTTTGG GAATGGGAAA 1260
GGATACCGAG
GCAAGAGGGC GACCACTGTT ACTGGGACGC CATGCCAGGA CTGGGCTGCC 1320
CAGGAGCCCC
ATAGACACAG CATTTTCACT CCAGAGACAA ATCCACGGGC GGGTCTGGAA 1380
AAAAATTACT
GCCGTAACCC TGATGGTGAT GTAGGTGGTC CCTGGTGCTA CACGACAAAT 1440
CCAAGAAAAC
TTTACGACTA CTGTGATGTC CCTCAGTGTG CGGCCCCTTC ATTTGATTGT 1500
GGGAAGCCTC
AAGTGGAGCC GAAGAAATGT CCTGGAAGGG TTGTGGGGGG GTGTGTGGCC 1560
CACCCACATT
CCTGGCCCTG GCAAGTCAGT CTTAGAACAA GGTTTGGAAT GCACTTCTGT 1620
GGAGGCACCT
TGATATCCCC AGAGTGGGTG TTGACTGCTG CCCACTGCTT GGAGAAGTCC 1680
CCAAGGCCTT
CATCCTACAA GGTCATCCTG GGTGCACACC AAGAAGTGAA TCTCGAACCG 1740
CATGTTCAGG
AAATAGAAGT GTCTAGGCTG TTCTTGGAGC CCACACGAAA AGATATTGCC 1800
TTGCTAAAGC
TAAGCAGTCC TGCCGTCATC ACTGACAAAG TAATCCCAGC TTGTCTGCCA 1860
TCCCCAAATT
ATGTGGTCGC TGACCGGACC GAATGTTTCA TCACTGGCTG GGGAGAAACC 1920
CAAGGTACTT
TTGGAGCTGG CCTTCTCAAG GAAGCCCAGC TCCCTGTGAT TGAGAATAAA 1980
GTGTGCAATC
GCTATGAGTT TCTGAATGGA AGAGTCCAAT CCACCGAACT CTGTGCTGGG 2040
CATTTGGCCG
GAGGCACTGA CAGTTGCCAG GGTGACAGTG GAGGTCCTCT GGTTTGCTTC 2100
GAGAAGGACA
AATACATTTT ACAAGGAGTC ACTTCTTGGG GTCTTGGCTG TGCACGCCCC 2160
AATAAGCCTG
GTGTCTATGT TCGTGTTTCA AGGTTTGTTA CTTGGATTGA GGGAGTGATG 2220
AGAAATAATT
AATTGGACGG GAGACAGAGT GACGCACGCG GCCGCTCTAG AACTAGTGGA 2280
TCCCCCGGGA
AGGCCTCGGA CCGGGC 2296
-144-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vi i ) IMN~DIATE SOURCE
(B) CLONE: pN2gpt-gp160
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
TTTTTGGCAT ATAAATCGTT ATCCACCATG TAAGATAACG AATTCCATGG CCCGGG ~ 56
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 331 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pvWF
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
TTTTTTTTGG CATATAAATC GCGGCCGCGG GTGGTTGGTG GATGTCACAG CTTGGGCTTT 60
ATCTCCCCCA GCAGTGGGAT TCCACAGCCC CTGGGCTACA TAACAGCAAG ACAGTCCGGA 120
GCTGTAGCAG ACCTGATTGA GCCTTTGCAG CAGCTGAGAG CATGGCCTAG GGTGGGCGGC 180
ACCATTGTCC AGCAGCTGAG TTTCCCAGGG ACCTTGGAGA TAGCCGCAGC CCTCATTTGC 240
AGGGGAAGAT GTGAGGCTGC TGCAGCTGCA TGGGTGCCTG CTGCTGCCTG CCTTGGCCTG 300
ATGGCGGCCG CCCGGGTTTT TATCTCGAGA C 331
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pBcoK-dhr
-145-
CA 02515166 1992-08-25
(xi) SEQUfiNCfi DRSCRIPTION: SEQ ID N0:21:
ATTAGCGTCT CGTTTCAGAC GCGGCCGCGG TAATTAGATT CTCCCACATT 50
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LfiNGTH: 1209 base pairs
(B) TYPE: nucleic acid
(C) STRANDfiDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pdhr-gpt
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ATTAGCGTCT CGTTTCAGAC GCGGCCGCTC TAGAACTAGT GGATCCCCCA 60
ACTTAAGGGT
ACCGCCTCGA CATCTATATA CTATATAGTA ATACCAATAC TCAAGACTAC 120
GAAACTGATA
CAATCTCTTA TCATGTGGGT AATGTTCTCG ATGTCGAATA GCCATATGCC 180
GGTAGTTGCG
ATATACATAA ACTGATCACT AATTCCAAAC CCACCCGCTT TTTATAGTAA 240
GTTTTTCACC
CATAAATAAT AAATACAATA ATTAATTTCT CGTAAAAGTA GAAAATATAT 300
TCTAATTTAT
TGCACGGTAA GGAAGTAGAA TCATAAAGAA CAGTGACGGA TGATCCCCAA 360
GCTTGGACAC
AAGACAGGCT TGCGAGATAT GTTTGAGAAT ACCACTTTAT CCCGCGTCAG 420
GGAGAGGCAG
TGCGTAAAAA GACGCGGACT CATGTGAAAT ACTGGTTTTT AGTGCGCCAG 480
ATCTCTATAA
TCTCGCGCAA CCTATTTTCC CCTCGAACAC TTTTTAAGCC GTAGATAAAC 540
AGGCTGGGAC
ACTTCACATG AGCGAAAAAT ACATCGTCAC CTGGGACATG TTGCAGATCC 600
ATGCACGTAA
ACTCGCAAGC CGACTGATGC CTTCTGAACA ATGGAAAGGC ATTATTGCCG 660
TAAGCCGTGG
CGGTCTGGTA CCGGGTGCGT TACTGGCGCG TGAACTGGGT ATTCGTCATG 720
TCGATACCGT
TTGTATTTCC AGCTACGATC ACGACAACCA GCGCGAGCTT AAAGTGCTGA 780
AACGCGCAGA
AGGCGATGGC GAAGGCTTCA TCGTTATTGA TGACCTGGTG GATACCGGTG 840
GTACTGCGGT
TGCGATTCGT GAAATGTATC CAAAAGCGCA CTTTGTCACC ATCITCGCAA 900
AACCGGCTGG
TCGTCCGCTG GTTGATGACT ATGTTGTTGA TATCCCGCAA GATACCTGGA 960
TTGAACAGCC
GTGGGATATG GGCGTCGTAT TCGTCCCGCC AATCTCCGGT CGCTAATCTT 1020
TTCAACGCCT
GGCACTGCCG GGCGTTGTTC TITI'TAACTT CAGGCGGGTT ACAATAGTTT 10
CCAGTAAGTA 8
0
TTCTGGAGGC TGCATCCATG ACACAGGCAA ACCTGAGCGA AACCCTGTTC 1140
AAACCCCGCT
TTGGGCTGCA GGAATTCGAT ATCAAGCTTA TCGATACCGT CGCGGCCGCG 1200
GTAATTAGAT
TCTCCCACA 1209
-146-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid'
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: odN2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GGCCGATATC TTAATTTAAA AGGCCT . 26
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
( vi i ) IMMEDIATE SOURCE
(B) CLONE: odN3
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CCAATGTTAC GTGGGTTACA TCAG -. 24
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: I-SceI linker 1
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
TAGGGATAAC AGGGTAAT 18
-147-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) II~iEDIATE SOURCE:
(B) CLONE: I-SceI linker 2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
ATTACCCTGT TATCCCTA . 18
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: odS2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GTATAAAGTC CGACTATTGT TCT _. 23
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii ) II~iDIATE SOURCE
(B) CLONE: odS3
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
TCTGAGGCCT AATAGACCTC TGTACA 26
-148-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) NK)LECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) Il~'sDIATE SOURCE:
(B) CLONE: SfiI (1)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GGCCGGCTAG GCC . 13
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: SfiI(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GGCCATATAG GCC _. 13
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IN~EDIATE SOURCE:
(B) CLONE: odTKl
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GAGTCGATGT AACACTTTCT ACAGGATCCG TTAACTCGCG AGAATTCCAT CACGTTAGAA 60
AATGCG 66
-149-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMN~DIATE SOURCE:
(B) CLONE: P-J (1)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
GATCCGGCCG GCTAGGCCGC GGCCGCCCGG GTTTTTATCT CGAGACAAAA AGACGGACCG 60
GGCCCGGCCA TATAGGCCC 79
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-J(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
AATTGGGCCT ATATGGCCGG GCCCGGTCCG TC'ITITTGTC TCGAGATAAA AACCCGGGCG 60
GCCGCGGCCT AGCCGGCCG 79
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMNH;'DIATE SOURCE:
(B) CLONE: odTK2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
AGAAGCCGTG GGTCATTG 18
-150-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid'
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: odTK3
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
TACCGTGTCG CTGTAACTTA C 21
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQDENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-A(0.1)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
AGGCCGGCTA GGCCGCGGCC GCCCGGGTTT TTATCTCGAG ACAAAAAGAC GGACCGGGCC 60
CGGCCATATA GGCCA 75
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-A(0.2)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
GTACTGGCCT ATATGGCCGG GCCCGGTCCG TCTTTTTGTC TCGAGATAAA AACCCGGGCG 60
GCCGCGGCCT AGCCGGCCTA GCT 83
-151-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-artP(11)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
GGCCACGTTT TTATGGGAAG CTTTTTTTTT TTTTTIZTiT TGGCATATAA ATCGC . 5 5
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-artP(12)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
GGCCGCGATT TATATGCCAA AAAAAAAAAA AAAAAAAAGC TTCCCATAAA AACGT 55
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
s
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-artP(8)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
CGCTGGCCTA TATGGCCGGG CCCGGTCCGA GGCCTTCCCG GGCCATGGAA TTCATTTATA 60
TGCCAAAAAA AAAAAAAAAA AAAAGCTTCT GCA 93
-152-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-artP(10)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
CGCTGGCCTA TATGGCCGGG CGTCCGAGGC CTTCCCGGGC CATGGAATTC GTTAACGATT 60
TATATGCCAA AAAAAAAAAA ~'~AAAAAAAGC TTCTGCA 97
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: oligonucleotide P-hr(3)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
ATTAGCGTCT CGTTTCAGAC GCGGCCGCGG TAATTAGATT CTCCCACATT 50
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CTAGCCCGGG 10
-153-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ,
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATfi SOURCfi:
(B) CLONE: P-P2 5'(1)
(xi) SEQUfiNCfi DESCRIPTION: SEQ ID N0:44:
GTACGTACGG CTGCAGTTGT TAGAGCTTGG TATAGCGGAC AACTAAG . 47
(2) INFORMATION FOR SfiQ ID N0:45:
( i ) SfiQUENCfi CHARACTERISTICS
(A) LfiNGTH: 50 base pairs
(B) TYPfi: nucleic acid
(C) STRANDfiDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULfi TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATfi SOURCE:
(B) CLONfi: P-P2 3' (1)
(xi) SEQUENCfi DESCRIPTION: SEQ ID N0:45:
TCTGACTGAC GTTAACGATT TATAGGCTAT AAAAAATAGT ATTTTCTACT 50
(2) INFORMATION FOR SfiQ ID N0:46:
( i ) SfiQUENCfi CHARACTERISTICS
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLfiCULfi TYPfi: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCfi:
(B) CLONE: P-SM(2)
(xi) SEQUENCfi DfiSCRIPTION: SEQ ID N0:46:
GTCTTGAGTA TTGGTATTAC 2 0
-154-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMN~DIATE SOURCE:
(B) CLONE: P-SM(3)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
CGAAACTATC AAAACGCTTT ATG . 23
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNfiSS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-MN(1)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
AGCTAGCTGA ATTCAGGCCT CATGAGAGTG AAGGGGATCA GGAGGAATTA TCA 53
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMN~DIATE SOURCE:
(B) CLONE: P-MN(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
CATCTGATGC ACAAAATAGA GTGGTGGTTG 30
-155-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) II~9''sDIATE SOURCE:
(B) CLONE: P-Seq(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
CTGTGGGTAC ACAGGCTTGT GTGGCCC ~ 27
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: P-Seq(3)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
CAATTTITCT GTAGCACTAC AGATC _. 25
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: 0-542
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
CGATTACGTA GTTAACGCGG CCGCGGCCTA GCCGGCCATA AAAAT 45
-156-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: 0-544
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
CTAGATTTTT ATGGCCGGCT AGGCCGCGGC CGCGTTAACT ACGTAAT ~ 47
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: 0-541
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
CTTTTTCTGC GGCCGCGGAT ATGGCCCGGT CCGGTTAACT ACGTAGACGT 50
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: 0-543
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
CTACGTAGTT AACCGGACCG GGCCATATAG GCCGCGGCCG CAGAAAAAGC ATG 53
-157-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDBDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: o-selPI
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
CGATAAAAAT TGAAATTTTA TTTTTTTTTT TTGGAATATA AATAAGGCCT C ~ 51
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LfiNGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: o-selPII
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
CATGGAGGCC TTATTTATAT TCCAAAAAAA AAAAATAAAA TTTCAATTTT TAT 53
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: 0-830
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
TCGACTTTTT ATCA 14
-158-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQDENCE CHARACTERISTICS:
(A) LENGTH: 1,2 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMN~DIATE SOURCE:
(B) CLONE: 0-857
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
TATGATAAAA AC ~ 12
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: O-NCOI
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
GAGCAGAAGA CAGTGGCCAT GGCCGTGAAG GGGATCAGGA 40
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: o-NsiI
(xi) SEQUENCE DESCRIPTION; SEQ ID N0:61:
CATAAACTGA TTATATCCTC ATGCATCTGT 30
-159-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:62:
(i) SfiQUSNCfi CHARACTfiRISTICS:
(A) LENGTH: 4145 base pairs
(B) TYPfi: nucleic acid
(C) STRANDEDNSSS : single
(D) TOPOLOGY: linear
(ii) MOLfiCULfi TYPfi: Other nucleic acid; ,
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMfiDIATfi SOURCE:
(8) CLONE: pS2gpt-S4
(xi) SfiQUENCfi DESCRIPTION: SBQ ID N0:62:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATITITC 60
TAAATACATT .
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA 120
TATTGAAAAA
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTITt'IT 180
GCGGCATTTT
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT 240
GAAGATCAGT
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC 300
CTTGAGAGTT
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA 360
TGTGGCGCGG
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC 420
TATTCTCAGA
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC 480
ATGACAGTAA
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC 540
TTACTTCTGA
CAACGATCGG AGGACCGAAG GAGCTAACCG CTTITITGCA CAACATGGGG 600
GATCATGTAA
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC 660
GAGCGTGACA
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC 720
GAACTACTTA
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT 780
GCAGGACCAC
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA 840
GCCGGTGAGC
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC 900
CGTATCGTAG
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG 960
ATCGCTGAGA
TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA 1020
TATATACTTT
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC 10
CTTTTTGATA 8
0
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 1140
GACCCCGTAG
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC 1200
TGCTTGCAAA
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 1260
CCAACTCTTT
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 1320
CTAGTGTAGC
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 1380
GCTCTGCTAA
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG 1440
TTGGACTCAA
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 1500
TGCACACAGC
-160-
CA 02515166 1992-08-25
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 1560
CTATGAGAAA
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 1620
AGGGTCGGAA
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG_GAAACGCCTG GTATCTTTAT 1680
AGTCCTGTCG
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 1740
GGGCGGAGCC
TATGGAAAAA CGCCAGCAAC GCGGCCZTIT TACGGTTCCT GGCCTTTTGC 1800
TGGCCTTTTG
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT 1860
ACCGCCTTTG
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA 1920
GTGAGCGAGG
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG 1980
ATTCATTAAT
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC 2040
GCAATTAATG
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG ~ 2100
GCTCGTATGT
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC 2160
CATGATTACG
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC 2220
CGCGGTGGCG
GCCGCTCTAG CCCGGGCTAG AACTAGTGGA TCCCCCAAAG CGGGGTTTGA 2280
ACAGGGTTTC
GCTCAGGTTT GCCTGTGTCA TGGATGCAGC CTCCAGAATA CTTACTGGAA 2340
ACTATTGTAA
CCCGCCTGAA GTTAAAAAGA ACAACGCCCG GCAGTGCCAG GCGTTGAAAA 2400
GATTAGCGAC
CGGAGATTGG CGGGACGAAT ACGACGCCCA TATCCCACGG CTGTTCAATC 2460
CAGGTATCTT
GCGGGATATC AACAACATAG TCATCAACCA GCGGACGACC AGCCGGTTTT 2520
GCGAAGATGG
TGACAAAGTG CGCTTTTGGA TACATTTCAC GAATCGCAAC CGCAGTACCA 2580
CCGGTATCCA
CCAGGTCATC AATAACGATG AAGCCTTCGC CATCGCCTTC TGCGCGTTTC 2640
AGCACTTTAA
GCTCGCGCTG GTTGTCGTGA TCGTAGCTGG AAATACAAAC GGTATCGACA 2700
TGACGAATAC
CCAGTTCACG CGCCAGTAAC GCACCCGGTA CCAGACCGCC ACGGCTTACG 2760
GCAATAATGC
CTTTCCATTG TTCAGAAGGC ATCAGTCGGC TTGCGAGTTT ACGTGCATGG 2820
ATCTGCAACA
TGTCCCAGGT GACGATGTAT TTTTCGCTCA TGTGAAGTGT CCCAGCCTGT 2880
TTATCTACGG
CTTAAAAAGT GTTCGAGGGG AAAATAGGTT GCGCGAGATT ATAGAGATCT 2940
GGCGCACTAA
AAACCAGTAT TTCACATGAG TCCGCGTCTT TTTACGCACT GCCTCTCCCT 3000
GACGCGGGAT
AAAGTGGTAT TCTCAAACAT ATCTCGCAAG CCTGTCTTGT GTCCAAGCTT 3060
GGGGATCATC
CGTCACTGTT CTTTATGATT CTACTTCCTT ACCGTGCAAT AAATTAGAAT 312
ATATTTTCTA 0
CTTTTACGAG AAATTAATTA TTGTATTTAT TATTTATGGG TGAAAAACTT 318
ACTATAAAAA 0
GCGGGTGGGT TTGGAATTAG TGATCAGTTT ATGTATATCG CAACTACCGG 3240
CATATGGCTA
TTCGACATCG AGAACATTAC CCACATGATA AGAGATTGTA TCAGTTTCGT 3 3
AGTCTTGAGT 0
0
ATTGGTATTA CTATATAGTA TATAGATGTC GAGGCGGTAC CCTTAAGTTG 3360
GGCTGCAGAA
GCTTTZTI~T TTT~TiTIT TTGGCATATA AATCGTTAAC GAATTCCATG 3420
GCCCGGGAAG
GCCTCGGACC GGGCCCGGCC ATATAGGCCA GCGATACCGT CGCGGCCGCG 3480
ACCTCGAGGG
GGGGCCCGGT ACCCAATTCG CCCTATAGTG AGTCGTATTA CGCGCGCTCA 3540
CTGGCCGTCG
-161-
CA 02515166 1992-08-25
TTTTACAACG TCGTGACTGG GAAAACCCTG GCGTTACCCA ACTTAATCGC 3600
CTTGCAGCAC
ATCCCCCTTT CGCCAGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC 3660
CCTTCCCAAC
AGTTGCGCAG CCTGAATGGC GAATGGAAAT TGTAAGCGTT AATATTTTGT 3720
TAAAATTCGC
GTTAAATTTT TGTTAAATCA GCTCATITTT TAACCAATAG GCCGAAATCG 3780
GCAAAATCCC
TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT 3840
GGAACAAGAG
TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT 3900
ATCAGGGCGA
TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTT'IZTG GGGTCGAGGT 3960
GCCGTAAAGC
ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA 4020
AGCCGGCGAA
CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC 4080
TGGCAAGTGT
AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC ~
TACAGGGCGC 4140
GTCAG 4145
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4277 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pS2gpt-P2
(xi) SEQUENCE DESCRIPTION: SfiQ ID N0:63:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TAAATACATT 60
TTTATTTTTC
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT TATTGAAAAA 120
GCTTCAATAA
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT GCGGCATTTT IBO
TCCCTTTTTT
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT GAAGATCAGT 240
AAAAGATGCT
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CTTGAGAGTT 300
CGGTAAGATC
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA TGTGGCGCGG 360
AGTTCTGCTA
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG TATTCTCAGA 420
CCGCATACAC
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT ATGACAGTAA 480
TACGGATGGC
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TTACTTCTGA 540
TGCGGCCAAC
CAACGATCGG AGGACCGAAG GAGCTAACCG CTiTIZTGCA GATCATGTAA 600
CAACATGGGG
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT GAGCGTGACA 660
ACCAAACGAC
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT GAACTACTTA 720
ATTAACTGGC
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GCAGGACCAC 780
GGATAAAGTT
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA GCCGGTGAGC 840
TAAATCTGGA
-162-
CA 02515166 1992-08-25
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC 900
CGTATCGTAG
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG 960
ATCGCTGAGA
TAGGTGCCTC ACTGATTAAG CATTGGTAAC'TGTCAGACCA AGTTTACTCA 1020
TATATACTTT
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC 10
CTTTTTGATA 8
0
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 1140
GACCCCGTAG
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC 1200
TGCTTGCAAA
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 1260
CCAACTCTTT
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 1320
CTAGTGTAGC
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 1380
GCTCTGCTAA
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG ~
TTGGACTCAA 1440
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 1500
TGCACACAGC
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 1560
CTATGAGAAA
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 1620
AGGGTCGGAA
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT 1680
AGTCCTGTCG
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 1740
GGGCGGAGCC
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC 1800
TGGCCTTTTG
CTCACATGTT CTTTCCTGCG TTATCCCCTG AZTCTGTGGA TAACCGTATT 1860
ACCGCCTTTG
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA 1920
GTGAGCGAGG
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG 1980
ATTCATTAAT
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC 2040
GCAATTAATG
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG 2100
GCTCGTATGT
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC 2160
CATGATTACG
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC 2220
CGCGGTGGCG
GCCGCTCTAG CCCGGGCTAG AACTAGTGGA TCCCCCAAAG CGGGGTTTGA 2280
ACAGGGTTTC
GCTCAGGTTT GCCTGTGTCA TGGATGCAGC CTCCAGAATA CTTACTGGAA 2340
ACTATTGTAA
CCCGCCTGAA GTTAAAAAGA ACAACGCCCG GCAGTGCCAG GCGTTGAAAA 2400
GATTAGCGAC
CGGAGATTGG CGGGACGAAT ACGACGCCCA TATCCCACGG CTGTTCAATC 2460
CAGGTATCTT
GCGGGATATC AACAACATAG TCATCAACCA GCGGACGACC AGCCGGTTTT 2520
GCGAAGATGG
TGACAAAGTG CGCTTTTGGA TACATTTCAC GAATCGCAAC CGCAGTACCA 2580
CCGGTATCCA
CCAGGTCATC AATAACGATG AAGCCTTCGC CATCGCCTTC TGCGCGTTTC 2640
AGCACTTTAA
GCTCGCGCTG GTTGTCGTGA TCGTAGCTGG AAATACAAAC GGTATCGACA 2700
TGACGAATAC
CCAGTTCACG CGCCAGTAAC GCACCCGGTA CCAGACCGCC ACGGCTTACG 2760
GCAATAATGC
CTTTCCATTG TTCAGAAGGC ATCAGTCGGC TTGCGAGTTT ACGTGCATGG 2820
ATCTGCAACA
TGTCCCAGGT GACGATGTAT TTTTCGCTCA TGTGAAGTGT CCCAGCCTGT 2880
TTATCTACGG
-163-
CA 02515166 1992-08-25
CTTAAAAAGT GTTCGAGGGG AAAATAGGTT GCGCGAGATT ATAGAGATCT 2940
GGCGCACTAA
AAACCAGTAT TTCACATGAG TCCGCGTCTT TTTACGCACT GCCTCTCCCT 3000
GACGCGGGAT
AAAGTGGTAT TCTCAAACAT ATCTCGCAAG CCTGTCTTGT GTCCAAGCTT 3060
GGGGATCATC
CGTCACTGTT CTTTATGATT CTACTTCCTT ACCGTGCAAT AAATTAGAAT 312 0
ATATTTTCTA
CTTTTACGAG AAATTAATTA TTGTATTTAT TATTTATGGG TGAAAAACTT 3180
ACTATAAAAA
GCGGGTGGGT TTGGAATTAG TGATCAGTTT ATGTATATCG CAACTACCGG 3240
CATATGGCTA
TTCGACATCG AGAACATTAC CCACATGATA AGAGATTGTA TCAGTTTCGT 3300
AGTCTTGAGT
ATTGGTATTA CTATATAGTA TATAGATGTC GAGGCGGTAC CCTTAAGTTG 3360
GGCTGCAGTT
GTTAGAGCTT GGTATAGCGG ACAACTAAGT AATTGTAAAG AAGAAAACGA 3420
AACTATCAAA
ACCGTTTATG AAATGATAGA AAAAAGAATA TAAATAATCC TGTATTTTAG . 3480
TTTAAGTAAC
AGTAAAATAA TGAGTAGAAA ATACTATTTT TTATAGCCTA TAAATCGTTA 3540
ACGAATTCCA
TGGCCCGGGA AGGCCTCGGA CCGGGCCCGG CCATATAGGC CAGCGATACC 3600
GTCGCGGCCG
CGACCTCGAG GGGGGGCCCG GTACCCAATT CGCCCTATAG TGAGTCGTAT 3660
TACGCGCGCT
CACTGGCCGT CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC 3720
CAACTTAATC
GCCTTGCAGC ACATCCCCCT TTCGCCAGCT GGCGTAATAG CGAAGAGGCC 3780
CGCACCGATC
GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGAA ATTGTAAGCG 3840
TTAATATTTT
GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT 3 9 0
AGGCCGAAAT 0
CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG 3960
TTGTTCCAGT
TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC GTCAAAGGGC 4020
GAAAAACCGT
CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA TCAAGTTTTT 4080
TGGGGTCGAG
GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG 4140
CTTGACGGGG
AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG 4200
GCGCTAGGGC
GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA CCCGCCGCGC 4260
TTAATGCGCC
GCTACAGGGC GCGTCAG 4277
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4701 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vi i ) IMNISDIATE SOiIRCE
(H) CLONE: pTZ-L2
-164-
CA 02515166 1992-08-25
(xi) SEQUENCfi DfiSCRIPTION: SEQ ID N0:64:
AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA 60
TGCAGCTTTT
TCTGCGGCCG CGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCG I20
AGGATTTCGC
GTGGGTCAAT GCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATAC 180
CAGTGAGGGA
TGGTTTTACC ATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTA 240
AAGCGGCGGA
CTAGCGTCGA GGTTTCAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTTTCG 300
CTCAGGTTTG
CCTGTGTCAT GGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAAC 360
CCGCCTGAAG
TTAAAAAGAA CAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATTAGCGACC 420
GGAGATTGGC
GGGACGAATA CGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG 480
CGGGATATCA
ACAACATAGT CATCRACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGT ~
GACAAAGTGC 540
GCTTTTGGAT ACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC 600
CAGGTCATCA
ATAACGATGA AGCCTTCGCC ATCGCCTTCT GCGCGTTTCA GCACTTTAAG 660
CTCGCGCTGG
TTGTCGTGAT CGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC 720
CAGTTCACGC
GCCAGTAACG CACCCGGTAC CAGACCGCCA CGGCTTACGG CAATAATGCC 780
TTTCCATTGT
TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT 840
GTCCCAGGTG
ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC 900
TTAAAAAGTG
TTCGAGGGGA AAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA 960
AACCAGTATT
TCACATGAGT CCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA 1020
AAGTGGTATT
CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC 1080
GTCACTGTTC
TTTATGATTC TACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTAC 114
TTTTACGAGA 0
AATTAATTAT TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG 1200
CGGGTGGGTT
TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGGCTAT 1260
TCGACATCGA
GAACATTACC CACATGATAA GAGATTGTAT CAGTTTCGTA GTCTTGAGTA 13
TTGGTATTAC 2
0
TATATAGTAT ATNNNNNNGG TAACNNNNP1N NNNNN1~1NNNN NNNNNNNNNN13
NNNNNNNNiNN 8
0
NNNN1JNNAGA TCTCGATCCG GATATAGTTC CTCCTTTCAG CAAAAAACCC 1440
CTCAAGACCC
GTTTAGAGGC CCCAAGGGGT TATGCTAGTT ATTGCTCANN NNNNNNNNGT 1500
CGACTTAATT
AATTAGGCCT CTCGAGCTGC AGGGATCCAC TAGTGAGCTC CCCGGGGAAT 1560
TCCCATGGTA
TTATCGTGTT TTTCAAAGGA AAAAAACGTC CCGTGGTTCG GGGGGCTCTN 1620
NNNNNiJtJNNN
NNNNNNNNLJN NNNNNNNNNN NNNNNNNNNN NNNNNP1NNNN NNNNNNNNNN 16
NNNNN1.JNNPTN 8
0
NNNNNNNNNN NNNNNNNNNN NNNNL~JNNNN NNNbTNNNNNN NNNNNNNPTNN1740
NNNNNNNNNN
NNNNITNNNNN NNNNNN1~JNN NNNNNNt1l111N NNNNNNt~JNN NNNNNNNNTt~ILJ18
NNNNNP1NNNN 0
0
NNNNNNNNNN NNNNNPINbTI~TN NNNNNNNNZ1N NNNt~ NNNNNNNNNN 18
NNNNPTNNNNN 6
0
NNNNNZ11~RJNN NNNNNNNNNN NNNNNNLIZ~iN NNNNNNNNNN NNNNNNZiNNN19
NNNNITlILdNNN 2
0
NNNI.dNNNNNN NZ~iNNNNI>lNLdN NNNNNNNNNN NNNNNNNNNN NNNNNNP1NNN19
NNNN1.JNNNNN 8
0
-165-
CA 02515166 1992-08-25
NNNNNNNNNZQ NNNN1>1NNNNN NNNNNNNNNN NNNNNN1JNP1N NNNNNP11.JNNN2 0 4
NNNNNNNNNN 0
NNNNNNNNNN NNNNI.dNNNNN NNNNNNNNNN NNNNLJZJNNNN NNNNNNNNP1N210 0
NNNNNNNNNN
NNNNNNNNNN NNNNNNNNNN Nf~TNNNNNt~11.~1tJ NCCGCTAGAG GGAAACCGTT2160
GTGGTCTCCC
TATAGTGAGT CGTATTAATT TCGCGGGATC GATCGATTAC GTAGTTAACG 2220
CGGCCGCGGC
CTAGCCGGCC ATAAAAATCT AGCTGGCGTA ATAGCGAAGA GGCCCGCACC 2280
GATCGCCCTT
CCCAACAGTT GCGCAGCCTG AATGGCGAAT GGGAAATTGT AAACGTTAAT 2340
ATTTTGTTAA
AATTCGCGTT AAATTTTTGT TAAATCAGCT CATTT'ITTAA CCAATAGGCC 2400
GAAATCGGCA
AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT 2460
CCAGTTTGGA
ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA 2520
ACCGTCTATC
AGGGCGATGG CCCACTACGT GAACCATCAC CCTAATCAAG TTTTITGGGG 2580
TCGAGGTGCC
GTAAAGCACT AAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA 2640
CGGGGAAAGC
CGGCGAACGT GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT 2700
AGGGCGCTGG
CAAGTGTAGC GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT 2760
GCGCCGCTAC
AGGGCGCGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT 2820
TGTTTATTTT
TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA 2880
ATGCTTCAAT
AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT 2940
ATTCCCTTTT
TTGCGGCATT TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA 3000
GTAAAAGATG
CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC 3060
AGCGGTAAGA
TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT 3120
AAAGTTCTGC
TATGTGGCGC GGTATTATCC CGTGTTGACG CCGGGCAAGA GCAACTCGGT 3180
CGCCGCATAC
ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT 3240
CTTACGGATG
GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC 3300
ACTGCGGCCA
ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG 3360
CACAACATGG
GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC 3420
ATACCAAACG
ACGAGCGTGA CACCACGATG CCTGCAGCAA TGGCAACAAC GTTGCGCAAA 3480
CTATTAACTG
GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG 3540
GCGGATAAAG
TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT 3600
GATAAATCTG
GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT 3660
GGTAAGCCCT
CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA 3720
CGAAATAGAC
AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC 3780
CAAGTTTACT
CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC 3 84
TAGGTGAAGA 0
TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC 3900
CACTGAGCGT
CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG 3960
CGCGTAATCT
GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG 4020
GATCAAGAGC
-166-
CA 02515166 1992-08-25
TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC 4080
GCAGATACCA AATACTGTCC
TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC CCTACATACC 4140
TGTAGCACCG
TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG TGTCTTACCG 4200
CGATAAGTCG
GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG ACGGGGGGTT 4260
GTCGGGCTGA
CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA CTACAGCGTG 4320
ACTGAGATAC
AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC CCGGTAAGCG 4380
GGACAGGTAT
GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG TGGTATCTTT 4440
GGGAAACGCC
ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG TGCTCGTCAG 4500
ATTTTTGTGA
GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT CTGGCCTTTT 4560
TTTACGGTTC
GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC GATAACCGTA ~
TGATTCTGTG 4
62
0
TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG CGCAGCGAGT 4680
AACGACCGAG
CAGTGAGCGA GGAAGCGGAA G 4701
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3878 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vi i ) IMMEDIATE SOURCE
(B) CLONE: pselP-gpt-L2
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:65:
AGCGCCCAATACGCAAACCGCCTCTCCCCG CGCGTTGGCCGATTCATTAATGCAGCTTTT 60
TCTGCGGCCGCGGCCTATATGGCCCGGTCC GGTTAACTACGTAGACGTCGAGGATTTCGC 120
GTGGGTCAATGCCGCGCCAGATCCACATCA GACGGTTAATCATGCGATACCAGTGAGGGA 180
TGGTTTTACCATCAAGGGCCGACTGCACAG GCGGTTGTGCGCCGTGATTAAAGCGGCGGA 240
CTAGCGTCGAGGTTTCAGGATGTTTAAAGC GGGGTTTGAACAGGGTTTCGCTCAGGTTTG 300
CCTGTGTCATGGATGCAGCCTCCAGAATAC TTACTGGAAACTATTGTAACCCGCCTGAAG 360
TTAAAAAGAACAACGCCCGGCAGTGCCAGG CGTTGAAAAGATTAGCGACCGGAGATTGGC 420
GGGACGAATACGACGCCCATATCCCACGGC TGTTCAATCCAGGTATCTTGCGGGATATCA 480
ACAACATAGTCATCAACCAGCGGACGACCA GCCGGTTTTGCGAAGATGGTGACAAAGTGC 540
GCTTTTGGATACATTTCACGAATCGCAACC GCAGTACCACCGGTATCCACCAGGTCATCA 600
ATAACGATGAAGCCTTCGCCATCGCCTTCT GCGCGTTTCAGCACTTTAAGCTCGCGCTGG 660
TTGTCGTGATCGTAGCTGGAAATACAAACG GTATCGACATGACGAATACCCAGTTCACGC 720
GCCAGTAACGCACCCGGTACCAGACCGCCA CGGCTTACGGCAATAATGCCTTTCCATTGT 780
-167-
CA 02515166 1992-08-25
TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT 840
GTCCCAGGTG
ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC 900
TTAAAAAGTG
TTCGAGGGGA AAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA 960
AACCAGTATT
TCACATGAGT CCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA 1020
AAGTGGTATT
CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC 1080
GTCACTGTTC
TTTATGATTC TACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTAC 114
TTTTACGAGA 0
AATTAATTAT TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG 12
CGGGTGGGTT 0
0
TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGATAAA 1260
AAGTCGACTT
AATTAATTAG GCCTCTCGAG CTGCAGGGAT CCACTAGTGA GCTCCCCGGG 1320
GAATTCCCAT
GGAGGCCTTA TTTATATTCC F~14AAAAAAAA AATAAAATTT CAATT'ITTAT.
CGATTACGTA 13
8
0
GTTAACGCGG CCGCGGCCTA GCCGGCCATA AAAATCTAGC TGGCGTAATA 1440
GCGAAGAGGC
CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT GGCGAATGGG 1500
AAATTGTAAA
CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA ATCAGCTCAT 1560
TTTTTAACCA
ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA TAGACCGAGA 1620
TAGGGTTGAG
TGTTGTTCCA GTTTGGAACA AGAGTCCACT ATTAAAGAAC GTGGACTCCA 1680
ACGTCAAAGG
GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCT 1740
AATCAAGTTT
TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCC 1800
CCCGATTTAG
AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA GGGAAGAAAG 1860
CGAAAGGAGC
GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT CACGCTGCGC GTAACCACCA 1920
CACCCGCCGC
GCTTAATGCG CCGCTACAGG GCGCGTCAGG TGGCACTTTT CGGGGAAATG 1980
TGCGCGGAAC
CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA 2040
GACAATAACC
CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 2100
ATTTCCGTGT
CGCCCTTATT CCCTTTTITG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 2160
CAGAAACGCT
GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 2220
TCGAACTGGA
TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 2280
CAATGATGAG
CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT GTTGACGCCG 2340
GGCAAGAGCA
ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 2400
CAGTCACAGA
AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 2460
TAACCATGAG
TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 2520
AGCTAACCGC
TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 2580
CGGAGCTGAA
TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GCAGCAATGG 2640
CAACAACGTT
GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT 2700
TAATAGACTG
GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 2760
CTGGCTGGTT
TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 2820
CAGCACTGGG
-168-
CA 02515166 1992-08-25
GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 2880
AGGCAACTAT
GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 2940
ATTGGTAACT
GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 3000
TTTAATTTAA
AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 3060
AACGTGAGTT
TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 3120
GAGATCCTTT
TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG 3180
CGGTGGTTTG
TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA 3240
GCAGAGCGCA
GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 3300
AGAACTCTGT
AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 3360
CCAGTGGCGA
TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG .
CGCAGCGGTC 3420
GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 3480
ACACCGAACT
GAGATACCTA CAGCGTGAGC ATTGAGAAAG CGCCACGCTT CCCGAAGGGA 3540
GAAAGGCGGA
CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 3600
TTCCAGGGGG
AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 3660
AGCGTCGATT
TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 3720
CGGCCTTTTT
ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 3780
TATCCCCTGA
TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 3840
GCAGCCGAAC
GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAG 3878
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6474 base pairs
(B) TYPB: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) 1~20LBCULE TYPE; Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pselP-gp160MN
(xi) SEQOENCE DESCRIPTION:
SEQ ID N0:66:
AGCGCCCAAT ACGCAAACCGCCTCTCCCCG CGCGTTGGCCGATTCATTAA TGCAGCTTTT60
TCTGCGGCCG CGGCCTATATGGCCCGGTCC GGTTAACTACGTAGACGTCG AGGATTTCGC120
GTGGGTCAAT GCCGCGCCAGATCCACATCA GACGGTTAATCATGCGATAC CAGTGAGGGA180
TGGTTTTACC ATCAAGGGCCGACTGCACAG GCGGTTGTGCGCCGTGATTA AAGCGGCGGA240
CTAGCGTCGA GGTTTCAGGATGTTTAAAGC GGGGTTTGAACAGGGTTTCG CTCAGGTTTG300
CCTGTGTCAT GGATGCAGCCTCCAGAATAC TTACTGGAAACTATTGTAAC CCGCCTGAAG360
TTAAAAAGAA CAACGCCCGGCAGTGCCAGG CGTTGAAAAGATTAGCGACC GGAGATTGGC420
-169-
CA 02515166 1992-08-25
GGGACGAATA CGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG 480,
CGGGATATCA
ACAACATAGT CATCAACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGT 540
GACAAAGTGC
GCTTTTGGAT ACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC 600
CAGGTCATCA
ATAACGATGA AGCCTTCGCC ATCGCCTTCT GCGCGTTTCA GCACTTTAAG 660
CTCGCGCTGG
TTGTCGTGAT CGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC 720
CAGTTCACGC
GCCAGTAACG CACCCGGTAC.~:GAGACCGCCA CGGCTTACGG CAATAATGCC 780
TTTCCATTGT
TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT 840
GTCCCAGGTG
ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC 900
TTAAAAAGTG
TTCGAGGGGA AAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA 960
AACCAGTATT
TCACATGAGT CCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA 1020
AAGTGGTATT
CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC 1080
GTCACTGTTC
TTTATGATTC TACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTAC 114
TTTTACGAGA 0
AATTAATTAT TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG 12
CGGGTGGGTT 0
0
TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGATAAA 1260
AAGTCGACTT
AATTAATTAG GCTGGTTCAG CTCGTCTCAT TCTTTCCCTT ACAGTAGGCC 13
ATCCAGTCAC 2
0
ACGTTTTGAC CATTTGCCAC CCATCTTATA GCAAAGCCCT TTCCAAGCCC 1380
TGTCTTATTC
TTGTAGGTAT GTGGAGAATA GCTCTACCAG CTCTTTGCAG TACTTCTATA 1440
ACCCTATCTG
TCCCCTCAGC TACTGCTATA GCTGTGGCAT TAAGCAAGCT AACAGCACTA 1500
CTCTTTAGTT
CCTGACTCCA ATACTGTAGG AGATTCCACC AATATTTGAG GACTTCCCAC 1560
CCCCTGCGTC
CCAGAAGTTC CACAATCCTC GCTGCAATCA AGAGTAAGTC TCTGTGGTGG 1620
TAGCTGAAGA
GGAACAGGCT CCGCAGGTCG ACCCAGATAA TTGCTAAGAA TCCATGCACT 1680
AATCGACCGG
ATGTGTCTCT GTCTCTCTCT CCACCTTCTT CTTCGATTCC TTCGGGCCTG 1740
TCGGGTCCCC
TCGGAACTGG GGGGCGGGTC TGCAACGACA ATGGTGAGTA TCCCTGCCTA 1800
ACTCTATTCA
CTATAGAAAG TACAGCAAAA ACTATTCTTA AACCTACCAA GCCTCCTACT 1860
ATCATTATGA
ATATTTTTAT ATACCACAGC CAATTTGTTA TGTCAAACCA ATTCCACAAA 1920
CTTGCCCATT
TATCCAATTC CAATAATTCT TGTTCATTCT TTTCTTGTTG GGTTTGCGAT 19
TTTTCTAGTA 8
0
ATGAGTATAT TAAGCTTGTG TAATTGTCAA TTTCTCTTTC CCACTGCATC 2040
CAGGTCATGT
TATTCCAAAT ATCATCCAGA GATTTATTAC TCCAACTAGC ATTCCAAGGC 2100
ACAGTAGTGG
TGCAAATGAG TTTTCCAGAG CAACCCCAAA ACCCCAGGAG CTGTTGATCC 2160
TTTAGGTATC
TTTCCACAGC CAGGACTCTT GCCTGGAGCT GCTTGATGCC CCAGACTGTG 2220
AGTTGCAACA
TATGCTGTTG CGCCTCAATG GCCCTCAGCA AATTGTTCTG CTGTTGCACT 2280
ATACCAGACA
ATAATAGTCT GGCCTGTACC GTCAGCGTCA CTGACGCTGC GCCCATAGTG 2340
CTTCCTGCTG
CTCCTAAGAA CCCAAGGAAC AGAGCTCCTA TCGCTGCTCT TTTTTCTCTC 2400
TGCACCACTC
TTCTCTTTGC CTTGGTGGGT GCTACTCCTA ATGGTTCAAT TGTTACTACT 2460
TTATATTTAT
-170-
CA 02515166 1992-08-25
ATAATTCACT TCTCCAATTG TCCCTCATAT CTCCTCCTCC AGGTCTGAAG 2520
ATCTCGGTGT
CGTTCGTGTC CGTGTCCTTA CCACCATCTC TTGTTAATAG TAGCCCTGTA 2580
ATATTTGATG
AACATCTAAT TTGTCCTTCA ATGGGAGGGG CATACATTGC TTTTCCTACT 2640
TCCTGCCACA
TGTTTATAAT TTGTTTTATT TTGCATTGAA GTGTGATATT GTTATTTGAC 2 7 0
CCTGTAGTAT 0
TATTCCAAGT ATTATTACCA TTCCAAGTAC TATTAAACAG TGGTGATGTA 2760
TTACAGTAGA
AAAATTCCCC TCCACAATTA' ~1.1AACTGTGCA TTACAATTTC TGGGTCCCCT2 82
CCTGAGGATT 0
GATTAAAGAC TATTGTTTTA TTCTTAAATT GTTCTTTTAA TTTGCTAACT 2 8 8
ATCTGTCTTA 0
AAGTGTCATT CCATTTTGCT CTACTAATGT TACAATGTGC TTGTCTTATA 2940
GTTCCTATTA
TATTTTTTGT TGTATAAAAT GCTCTCCCTG GTCCTATATG TATCCTTTTT 3000
CTTTTATTGT
AGTTGGGTCT TGTACAATTA ATTTGTACAG ATTCATTCAG ATGTACTATG , 3060
ATGGTTTTAG
CATTATCAGT GAAATTCTCA GATCTAATTA CTACCTCTTC TTCTGCTAGA 3120
CTGCCATTTA
ACAGCAGTTG AGTTGATACT ACTGGCCTAA TTCCATGTGT ACATTGTACT 3180
GTGCTGACAT
TTTTACATGA TCCTTTTCCA CTGAACTTTT TATCGTTACA TTTTAGAATC 3240
GCAAAACCAG
CCGGGGCACA ATAGTGTATG GGAATTGGCT CAAAGGATAT CTTTGGACAA 3300
GCTTGTGTAA
TGACTGAGGT ATTACAACTT ATCAACCTAT AGCTGGTACT ATCATTATCT 3360
ATTGATACTA
TATCAAGTTT ATAAAGAAGT GCATATTCTT TCTGCATCTT ATCTCTTATG 3420
CTTGTGGTGA
TATTGAAAGA GCAGTTTTTC ATTTCTCCTC CCTTTATTGT TCCCTCGCTA 3480
TTACTATTGT
TATTAGCAGT ACTATTATTG GTATTAGTAG TATTCCTCAA ATCAGTGCAA 3 54
TTTAAAGTAA 0
CACAGAGTGG GGTTAATTTT ACACATGGCT TTAGGCTTTG ATCCCATAAA 3600
CTGATTATAT
CCTCATGCAT CTGTTCTACC ATGTTATTTT TCCACATGTT AAAATTTTCT 3 6 6
GTCACATTTA 0
CCAATTCTAC TTCTTGTGGG TTGGGGTCTG TGGGTACACA GGCTTGTGTG 3720
GCCCAAACAT
TATGTACCTC TGTATCATAT GCTTTAGCAT CTGATGCACA AAATAGAGTG 3780
GTGGTTGCTT
CTTTCCACAC AGGTACCCCA TAATAGACTG TGACCCACAA TTTTTCTGTA 3840
GCACTACAGA
TCATTAATAA CCCAAGGAGC ATCGTGCCCC ATCCCCACCA GTGCTGATAA 3900
TTCCTCCTGA
TCCCCTTCAC GGCCATGGAG GCCTTATTTA TATTCCAAAA AAAAAAAATA 3960
AAAT'ITCAAT
TTTTATCGAT TACGTAGTTA ACGCGGCCGC GGCCTAGCCG GCCATAAAAA 4020
TCTAGCTGGC
GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 4080
CTGAATGGCG
AATGGGAAAT TGTAAACGTT AATATTTTGT TAAAATTCGC GTTAAATTTT 414 0
TGTTAAATCA
GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA 4200
AAAGAATAGA
CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA 4260
AAGAACGTGG
ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA 4320
CGTGAACCAT
CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC ACTAAATCGG 4380
AACCCTAAAG
GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA CGTGGCGAGA 4440
AAGGAAGGGA
AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG 4500
CTGCGCGTAA
-171-
CA 02515166 1992-08-25
CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTCAGGTGGC 4560
ACTTTTCGGG
GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT 4620
ATGTATCCGC
TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATT'G AAAAAGGAAG 4680
AGTATGAGTA
TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT 4740
CCTGTTTTTG
CTCACCCAGA AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT 4800
GCACGAGTGG
GTTACATCGA ACTGGATCTC"'AACAGCGGTA AGATCCTTGA GAGTTTTCGC 4860
CCCGAAGAAC
GTTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA 4920
TCCCGTGTTG
ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC 4980
TTGGTTGAGT
ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA 5040
TTATGCAGTG
CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG . 5100
ATCGGAGGAC
CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC 5160
CTTGATCGTT
GGGAACCGGA GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG 5220
ATGCCTGCAG
CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA 5280
GCTTCCCGGC
AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG 5340
CGCTCGGCCC
TTCCGGCTGG CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG 5400
TCTCGCGGTA
TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC 5460
TACACGACGG
GGAGTCAGGC AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT 5520
GCCTCACTGA
TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT 5580
GATTTAAAAC
TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC 5640
ATGACCAAAA
TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG 5700
ATCAAAGGAT
CTTCT1'GAGA TCCTI~TIT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA 5760
AAACCACCGC
TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG 5820
AAGGTAACTG
GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG 5880
TTAGGCCACC
ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG 5940
TTACCAGTGG
CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA 6000
TAGTTACCGG
ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC 6060
TTGGAGCGAA
CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCATTG AGAAAGCGCC 6120
ACGCTTCCCG
AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA 6180
GAGCGCACGA
GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT 6240
CGCCACCTCT
GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG 6300
AAAAACGCCA
GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC 6360
ATGTTCTTTC
CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA 6420
GCTGATACCG
CTCGCCGCAG CCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG 6474
GAAG
-172-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6811 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2-gpta ProtS
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC 60
TAAATACATT
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA 120
TATTGAAAAA
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTITITT 180
GCGGCATTTT
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT 240
GAAGATCAGT
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC 300
CTTGAGAGTT
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA 360
TGTGGCGCGG
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC 420
TATTCTCAGA
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC 480
ATGACAGTAA
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC 540
TTACTTCTGA
CAACGATCGG AGGACCGAAG GAGCTAACCG CTTT'TTTGCA CAACATGGGG 600
GATCATGTAA
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC 660
GAGCGTGACA
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC 720
GAACTACTTA
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT 780
GCAGGACCAC
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA 840
GCCGGTGAGC
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC 900
CGTATCGTAG
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG 960
ATCGCTGAGA
TAGGTGCCTC.ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA 1020
TATATACTTT
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC 1080
CTTTTTGATA
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 1140
GACCCCGTAG
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTZTCTGCG CGTAATCTGC 1200
TGCTTGCAAA
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 1260
CCAACTCTTT
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 1320
CTAGTGTAGC
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 1380
GCTCTGCTAA
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG 1440
TTGGACTCAA
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 1500
TGCACACAGC
-173-
CA 02515166 1992-08-25
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 1560
CTATGAGAAA
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 1620
AGGGTCGGAA
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT 1680
AGTCCTGTCG
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 1740
GGGCGGAGCC
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC 1800
TGGCCTTTTG
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT 1860
ACCGCCTTTG
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA 1920
GTGAGCGAGG
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG 1980
ATTCATTAAT
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC 2040
GCAATTAATG
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG ~
GCTCGTATGT 2100
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC 2160
CATGATTACG
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC 2220
CGCGGTGGCG
GCCGCGTCGA CAGAAAAATT AATTAATTAT GGCCTCTCGA GCTGCAGCTG 2280
CCAAGAAGAA
GATTCCTGTG CTGCTCTCAG GAAAATATGT CCCACTTGTT TTCTAATTCA 2340
ATAAAGATAC
TGGTTTAAAT GTGAAGCCAC ACAAGAGAAA GATGAAGCCA AAGCTGGTCC 2400
CCCTGAGGAA
TTGTTTTGAA ATAAGGCATT AGGACCCTCC ATTCAATTCA TATTTAATAG 2460
ACCACCATCT
CTTCTGCCTT CATCAGGAAA AAAACAAAAA CATAAACAAA ATAGTATCTG 2520
CCTATGATTA
ATAGTATTTA ATTACACGCA CTTTTGTTTG AGTTTACTTC CTTGCTTTCT 2
GAAAAAAACA 5
8
0
TAGGTATTTA GACACTAGTT CATGATGATA AAATTAAAAA TTTAGTTTTA 2
CAAACAAAAA 6
4
0
TTGAAACTGT CATTTGTAGG AAAAAAATTC AAATTTAAAA TTGTTATTTT 2
TCACTATTCT 7
0
0
TAGATAGCAA GAGAAGTAAG AATTTCTTTA CTGTGATTTA TATCACAACA 2760
GAATTTTTTT
CCTTGACAAA GGACCTTTTA AAAATCCCAG GAAAGGACCA CAAAATAATC 2820
AAAGACTGCA
CATTGTAAAT AAAACCCTTC AGCTGTTATT GAAACATAAG TATAATTACA 2880
CACAAGGAAA
AGGTATTATA AGCAGAGAAA AGATGCCTTA AGAATTCTTT GTCTTTTTCC 2940
AAACTGATGG
ACATGAGTGA GCTCTAATAT CATTATGTTT AGAAATGGCT TCATCCAGAT 3000
CCAACTGTAC
ACCATTAATA TTCACTTCCA TGCAGCCATT ATAAAAGGCA TTCACTGGTG 3060
TGGCACTGAA
TGGAACATCT GGAAGGCCAC CCAGGTATGT GGCCACTTTT GCTTTCATTG 3120
CTTTGTCCAA
GACGGCAAGT TGTCTTTGAA GGTCTTCATG GGAGATGGTT TCTATTTTAA 3180
GTGGTGTCGA
CAACTCCAGA TTGTTTCTGT TGACTCTAAA TTCCAGATGA GATTGTTGAT 3
CGGAACATAG 2
4
0
ACTTAGGGCC TGTATCCGAT ATATTACAGT ATTTTCAACA GATAACAGAA 3300
TATCCTGTGA
TTTTTCAGAG GTGGAGTCCA CCAAGGACAC AGCAAAGGGC ACTGTGTTGT 3360
TACCAGAAAC
CAAGGCAAGC ATAACACCAG TGCCCGTGGA TGGACGAATA TTCAAGGTCA 3420
CATTTACATG
CCAACCCTCA GCACTGGATA CATTATTATA ATCTATGTGA AATTGAGCAA 3480
TTCCAGAACC
AGGATAGTAG GAGCCCTTCT CCACAGTAAC CAGGCAATGC TTATTTTGTT 3
TTTCTTGAAT 54
0
-174-
CA 02515166 1992-08-25
AATTTCCTTT ATTCCAGAAG CTCCTTGCTT CATCAAATTC CAGCTTCGTA 3600
TACATCCATC
TAGACGAGGG TTAATCGGTT TAATGAGTTC ACTTTCCACT TTCCGAGGGA 3660
ATCCTGCAAA
GTATACTTTG GTTTCCAGCA ATCCATTTTC CGGCTTAAAA AGGGGTCCAG 3720
GTTTATTTAT
ATCCATCACA GCTTCTTTAG CTATTTTAAT GCTAATACTA TGTTCTAATT 3780
CTTCCACAGA
CACCATATTC CATAGACCAT TATTAATAAC ATCACCTCCA GTTGTGATTT 3840
TGGATGTATG
TTCATTCTTA AGCTGAACTT CAATCTTTCC ACCACGAAGT GCAATCAGGA 3900
GCCACGCTGA
GTGATCGATA GATTCTGCGT ACAGTATCAC GCCTTCTGAA TCATATGTCC 3960
GGAAATCAAA
TTCTGCTGAA AATCTGCTGA TTTCTGGCAA ACGAAATTTT AAATATAAAA 4020
CAACCCCTGC
AAACTGCTCC GCCAAGTAAA GTAATTCATA CTTTGTGTCA AGGTTCAAGG 4080
GAAGGCACAC
TGAAACAACC TCACAACTCT TCTGATCTTG GGCAAGTTTG AATCCTTTCT 4140
TCCCATCACA
ATAGCAAGTG TAACCTCCAG GGTAATTGAC ACAAAGCTGA GCACACATGT 4200
TCTCAGAGCA
TTCATCTATA TCTTCACAAG ACTTTGATTT GAGATTATAT CTGTAGCCTT 4 2
CGGGGCATTC 6
0
ACATTCAAAA TCTCCTGGGA TGTTCTTGCA CACAGCTGTG CCACAAATGC 4320
TTGGCTTCAA
AGAGCATTCA TCCACATCTT TACAATCTTT CTTATTTGAA AGCATAACAA 4 3
AACCATTTTT 8
0
ACAGGAACAG TGGTAACTTC CAGGTGTATT ATCACAAATT TGACTGCAAC 4440
CTCCATTTAT
ATTTGAGGGA TCTTTGCATT CATTTATGTC AAATTCACAC TTTTCTCCTT 4 5
GCCARCCTGG 0
0
TTTACAAGTG CAAGTAAAAG AAGCTTTTCC ATCTTTGCAG CTCATATATC 4560
CATCTTCATT
GCATGGCAGA GGACTACACT GGTCTGGAAT GGCATTGACA CAGCTTCTTA 4620
GGTCAGGATA
AGCATTAGTT GACTGACGTG CAGCAGTGAA TAACCCAGTT TGAAAAGAGC 4680
GAAGACAAAC
TAAGTATTTT GGATAAAAAT AATCCGTTTC CGGGTCATTT TCAAAGACCT 4740
CCCTGGCTTC
TTCTTTATTG CACAGTTCTT CGATGCATTC TCTTTCAAGA TTACCCTGTT 4800
TGGTTTCTTC
AAGTAAAGAA TTTGCACGAC GCTTCCTAAC CAGGACTTGT GAAGCCTGTT 4860
GCTTTGACAA
AAAGTTTGCC TCTGAGACGG GAAGCACTAG GAGGAGACAC GCCAGCAACG 4920
CCCCGCAGCG
CCCACCCAGG ACCCCCATGG AGGCCTTATT TATATTCCAA TAAAATTTCA 4980
ATTTTTAGAT CCCCCAACTT AAGGGTACCG CCTCGACATC TATATACTAT 5040
ATAGTAATAC
CAATACTCAA GACTACGAAA CTGATACAAT CTCTTATCAT GTGGGTAATG 5100
TTCTCGATGT
CGAATAGCCA TATGCCGGTA GTTGCGATAT ACATAAACTG ATCACTAATT 5160
CCAAACCCAC
CCGCTTTTTA TAGTAAGTTT TTCACCCATA AATAATAAAT ACAATAATTA 5220
ATTTCTCGTA
AAAGTAGAAA ATATATTCTA ATTTATTGCA CGGTAAGGAA GTAGAATCAT 5280
AAAGAACAGT
GACGGATGAT CCCCAAGCTT GGACACAAGA CAGGCTTGCG AGATATGTTT 5340
GAGAATACCA
CTTTATCCCG CGTCAGGGAG AGGCAGTGCG TAAAAAGACG CGGACTCATG 5400
TGAAATACTG
GTTTTTAGTG CGCCAGATCT CTATAATCTC GCGCAACCTA TTTTCCCCTC 5460
GAACACTTTT
TAAGCCGTAG ATAAACAGGC TGGGACACTT CACATGAGCG AAAAATACAT 5520
CGTCACCTGG
GACATGTTGC AGATCCATGC ACGTAAACTC GCAAGCCGAC TGATGCCTTC 5580
TGAACAATGG
-175-
CA 02515166 1992-08-25
AAAGGCATTA TTGCCGTAAG CCGTGGCGGT CTGGTACCGG G'TGCGTTACT 5640
GGCGCGTGAA
CTGGGTATTC GTCATGTCGA TACCGTTTGT ATTTCCAGCT ACGATCACGA 5700
CAACCAGCGC
GAGCTTAAAG TGCTGAAACG CGCAGAAGGC GATGGCGAAG GCTTCATCGT 5760
TATTGATGAC
CTGGTGGATA CCGGTGGTAC TGCGGTTGCG ATTCGTGAAA TGTATCCAAA 5820
AGCGCACTTT
GTCACCATCT TCGCAAAACC GGCTGGTCGT CCGCTGGTTG ATGACTATGT 5880
TGTTGATATC
CCGCAAGATA CCTGGATTGA ACAGCCGTGG GATATGGGCG TCGTATTCGT 5940
CCCGCCAATC
TCCGGTCGCT AATCTTTTCA ACGCCTGGCA CTGCCGGGCG TTGTTCTTTT 6000
TAACTTCAGG
CGGGTTACAA TAGTTTCCAG TAAGTATTCT GGAGGCTGCA TCCATGACAC 6060
AGGCAAACCT
GAGCGAAACC CTGTTCAAAC CCCGCTTTGG GCTGCAGGAA TTCGATATCA 6120
AGCTTATCGA
TACCGTCGCG GCCGCGACCT CGAGGGGGGG CCCGGTACCC AATTCGCCCT ~
ATAGTGAGTC 6180
GTATTACGCG CGCTCACTGG CCGTCGTTTT ACAACGTCGT GACTGGGAAA 6240
ACCCTGGCGT
TACCCAACTT AATCGCCTTG CAGCACATCC CCCTTTCGCC AGCTGGCGTA 6300
ATAGCGAAGA
GGCCCGCACC GATCGCCCTT CCCAACAGTT GCGCAGCCTG AATGGCGAAT 6360
GGAAATTGTA
AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 6420
ATTTTTTAAC
CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 6480
GATAGGGTTG
AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC 6540
CAACGTCAAA
GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC 6600
CTAATCAAGT
TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG 6660
CCCCCGATTT
AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA 6720
AGCGAAAGGA
GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC 6780
CACACCCGCC
GCGCTTAATG CGCCGCTACA GGGCGCGTCA G 6811
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMNBSDIATE SOURCE:
(B) CLONE: oProtSi
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
ACCCAGGACC GCCATGGCGA AGCGCGC 27
-176-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6926 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vi i ) IMMEDIATE SOURCE
(B) CLONE: pP2-gp160MN
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC 60
TAAATACATT
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA 120
TATTGAAAAA
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTIZTtT 180
GCGGCATTTT
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT 240
GAAGATCAGT
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC 300
CTTGAGAGTT
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA 360
TGTGGCGCGG
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC 420
TATTCTCAGA
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC 480
ATGACAGTAA
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC 540
TTACTTCTGA
CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG 600
GATCATGTAA
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC 660
GAGCGTGACA
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC 720
GAACTACTTA
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT 780
GCAGGACCAC
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA 840
GCCGGTGAGC
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC 900
CGTATCGTAG
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG 960
ATCGCTGAGA
TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA 1020
TATATACTTT
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC 1080
CTTTITGATA
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 1140
GACCCCGTAG
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC 1200
TGCTTGCAAA
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 1260
CCAACTCTTT
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 1320
CTAGTGTAGC
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 1380
GCTCTGCTAA
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG 1440
TTGGACTCAA
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 1500
TGCACACAGC
-177-
CA 02515166 1992-08-25
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 1560
CTATGAGAAA
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 1620
AGGGTCGGAA
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT 1680
AGTCCTGTCG
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 1740
GGGCGGAGCC
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC 1800
TGGCCTTTTG
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT 1860
ACCGCCTTTG
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA 1920
GTGAGCGAGG
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG 1980
ATTCATTAAT
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC 2040
GCAATTAATG
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG 2100
GCTCGTATGT
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC 2160
CATGATTACG
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC 2220
CGCGGTGGCG
GCCGCTCTAG CCCGGGCTAG AACTAGTGGA TCCCCCAAAG CGGGGTTTGA 2280
ACAGGGTTTC
GCTCAGGTTT GCCTGTGTCA TGGATGCAGC CTCCAGAATA CTTACTGGAA 2340
ACTATTGTAA
CCCGCCTGAA GTTAAAAAGA ACAACGCCCG GCAGTGCCAG GCGTTGAAAA 2400
GATTAGCGAC
CGGAGATTGG CGGGACGAAT ACGACGCCCA TATCCCACGG CTGTTCAATC 2460
CAGGTATCTT
GCGGGATATC AACAACATAG TCATCAACCA GCGGACGACC AGCCGGTTTT 2520
GCGAAGATGG
TGACAAAGTG CGCTTTTGGA TACATTTCAC GAATCGCAAC CGCAGTACCA 2580
CCGGTATCCA
CCAGGTCATC AATAACGATG AAGCCTTCGC CATCGCCTTC TGCGCGTTTC 2640
AGCACTTTAA
GCTCGCGCTG GTTGTCGTGA TCGTAGCTGG AAATACAAAC GGTATCGACA 2700
TGACGAATAC
CCAGTTCACG CGCCAGTAAC GCACCCGGTA CCAGACCGCC ACGGCTTACG 2760
GCAATAATGC
CTTTCCATTG TTCAGAAGGC ATCAGTCGGC TTGCGAGTTT ACGTGCATGG 2820
ATCTGCAACA
TGTCCCAGGT GACGATGTAT TTTTCGCTCA TGTGAAGTGT CCC:AGCCTGT 2880
TTATCTACGG
CTTAAAAAGT GTTCGAGGGG AAAATAGGTT GCGCGAGATT ATAGAGATCT 2940
GGCGCACTAA
AAACCAGTAT TTCACATGAG TCCGCGTCTT TTTACGCACT GCCTCTCCCT 3000
GACGCGGGAT
AAAGTGGTAT TCTCAAACAT ATCTCGCAAG CCTGTCTTGT GTCCAAGCTT 3060
GGGGATCATC
CGTCACTGTT CTTTATGATT CTACTTCCTT ACCGTGCAAT AAATTAGAAT 3120
ATATTTTCTA
CTTTTACGAG AAATTAATTA TTGTATTTAT TATTTATGGG TGAAAAACTT 3180
ACTATAAAAA
GCGGGTGGGT TTGGAATTAG TGATCAGTTT ATGTATATCG CAACTACCGG 3240
CATATGGCTA
TTCGACATCG AGAACATTAC CCACATGATA AGAGATTGTA TCAGTTTCGT 3
AGTCTTGAGT 3
0
0
ATTGGTATTA CTATATAGTA TATAGATGTC GAGGCGGTAC CCTTAAGTTG 3360
GGCTGCAGTT
GTTAGAGCTT GGTATAGCGG ACAACTAAGT AATTGTAAAG AAGAAAACGA 3420
AACTATCAAA
ACCGTTTATG AAATGATAGA AAAAAGAATA TAAATAATCC TGTATTTTAG 3480
TTTAAGTAAC
AGTAAAATAA TGAGTAGAAA ATACTATTTT TTATAGCCTA TAAATCGTTC 3540
CTCATGAGAG
-178-
CA 02515166 1992-08-25
TGAAGGGGAT CAGGAGGAAT TATCAGCACT GGTGGGGATG GGGCACGATG 3600
CTCCTTGGGT
TATTAATGAT CTGTAGTGCT ACAGAAAAAT TGTGGGTCAC AGTCTATTAT 3660
GGGGTACCTG
TGTGGAAAGA AGCAACCACC ACTCTATTTT GTGCATCAGA TGCTAAAGCA 3720
TATGATACAG
AGGTACATAA TGTTTGGGCC ACACAAGCCT GTGTACCCAC AGACCCCAAC 3780
CCACAAGAAG
TAGAATTGGT AAATGTGACA GAAAATTTTA ACATGTGGAA AAATAACATG 3840
GTAGAACAGA
TGCATGAGGA TATAATCAGT TTATGGGATC AAAGCCTAAA GCCATGTGTA 3900
AAATTAACCC
CACTCTGTGT TACTTTAAAT TGCACTGATT TGAGGAATAC TACTAATACC 3960
AATAATAGTA
CTGCTAATAA CAATAGTAAT AGCGAGGGAA CAATAAAGGG AGGAGAAATG 4020
AAAAACTGCT
CTTTCAATAT CACCACAAGC ATAAGAGATA AGATGCAGAA AGAATATGCA 4080
CTTCTTTATA
AACTTGATAT AGTATCAATA GATAATGATA GTACCAGCTA TAGGTTGATA 4140
AGTTGTAATA
CCTCAGTCAT TACACAAGCT TGTCCAAAGA TATCCTTTGA GCCAATTCCC 4200
ATACACTATT
GTGCCCCGGC TGGTTTTGCG ATTCTAAAAT GTAACGATAA AAAGTTCAGT 4260
GGAAAAGGAT
CATGTAAAAA TGTCAGCACA GTACAATGTA CACATGGAAT TAGGCCAGTA 4320
GTATCAACTC
AACTGCTGTT AAATGGCAGT CTAGCAGAAG AAGAGGTAGT AATTAGATCT 4380
GAGAATTTCA
CTGATAATGC TAAAACCATC ATAGTACATC TGAATGAATC TGTACAAATT 4440
AATTGTACAA
GACCCAACTA CAATAAAAGA AAAAGGATAC ATATAGGACC AGGGAGAGCA 4500
TTTTATACAA
CAAAAAATAT AATAGGAACT ATAAGACAAG CACATTGTAA CATTAGTAGA 4560
GCAAAATGGA
ATGACACTTT AAGACAGATA GTTAGCAAAT TAAAAGAACA ATTTAAGAAT 4620
AAAACAATAG
TCTTTAATCA ATCCTCAGGA GGGGACCCAG AAATTGTAAT GCACAGTTTT 4680
AATTGTGGAG
GGGAATTTTT CTACTGTAAT ACATCACCAC TGTTTAATAG TACTTGGAAT 4740
GGTAATAATA
CTTGGAATAA TACTACAGGG TCAAATAACA ATATCACACT TCAATGCAAA 4800
ATAAAACAAA
TTATAAACAT GTGGCAGGAA GTAGGAAAAG CAATGTATGC CCCTCCCATT 4860
GAAGGACAAA
TTAGATGTTC ATCAAATATT ACAGGGCTAC TATTAACAAG AGATGGTGGT 4920
AAGGACACGG
ACACGAACGA CACCGAGATC TTCAGACCTG GAGGAGGAGA TATGAGGGAC 4980
AATTGGAGAA
GTGAATTATA TAAATATAAA GTAGTAACAA TTGAACCATT AGGAGTAGCA 5040
CCCACCAAGG
CAAAGAGAAG AGTGGTGCAG AGAGAAAAAA GAGCAGCGAT AGGAGCTCTG 5100
TTCCTTGGGT
TCTTAGGAGC AGCAGGAAGC ACTATGGGCG CAGCGTCAGT GACGCTGACG 5160
GTACAGGCCA
GACTATTATT GTCTGGTATA GTGCAACAGC AGAACAATTT GCTGAGGGCC 5220
ATTGAGGCGC
AACAGCATAT GTTGCAACTC ACAGTCTGGG GCATCAAGCA GCTCCAGGCA 5280
AGAGTCCTGG
CTGTGGAAAG ATACCTAAAG GATCAACAGC TCCTGGGGTT TTGGGGTTGC 5340
TCTGGAAAAC
TCATTTGCAC CACTACTGTG CCTTGGAATG CTAGTTGGAG TAATAAATCT 5400
CTGGATGATA
TTTGGAATAA CATGACCTGG ATGCAGTGGG AAAGAGAAAT TGACAATTAC 5460
ACAAGCTTAA
TATACTCATT ACTAGAAAAA TCGCAAACCC AACAAGAAAA GAATGAACAA 5520
GAATTATTGG
AATTGGATAA ATGGGCAAGT TTGTGGAATT GGTTTGACAT AACAAATTGG 5580
CTGTGGTATA
-179-
CA 02515166 1992-08-25
TAAAAATATT CATAATGATA GTAGGAGGCT TGGTAGGTTT AAGAATAGTT 5640
TTTGCTGTAC
TTTCTATAGT GAATAGAGTT AGGCAGGGAT ACTCACCATT GTCGTTGCAG 5700
ACCCGCCCCC
CAGTTCCGAG GGGACCCGAC AGGCCCGAAGIGAATCGAAGA AGAAGGTGGA 5760
GAGAGAGACA
GAGACACATC CGGTCGATTA GTGCATGGAT TCTTAGCAAT TATCTGGGTC 5820
GACCTGCGGA
GCCTGTTCCT CTTCAGCTAC CACCACAGAG ACTTACTCTT GATTGCAGCG 5880
AGGATTGTGG
AACTTCTGGG ACGCAGGGGG TGGGAAGTCC TCAAATATTG GTGGAATCTC 5940
CTACAGTATT
GGAGTCAGGA ACTAAAGAGT AGTGCTGTTA GCTTGCTTAA TGCCACAGCT 6000
ATAGCAGTAG
CTGAGGGGAC AGATAGGGTT ATAGAAGTAC TGCAAAGAGC TGGTAGAGCT 6060
ATTCTCCACA
TACCTACAAG AATAAGACAG GGCTTGGAAA GGGCTTTGCT ATAAGATGGG 6120
TGGCAAATGG
TCAAAACGTG TGACTGGATG GCCTACTGTA AGGGAAAGAA TGAGACGAGC ~
TGAACCAGAA 6180
CGAATTCCAT GGCCCGGGAA GGCCTCGGAC CGGGCCCGGC CATATAGGCC 6240
AGCGATACCG
TCGCGGCCGC GACCTCGAGG GGGGGCCCGG TACCCAATTC GCCCTATAGT 6300
GAGTCGTATT
ACGCGCGCTC ACTGGCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCCT 6360
GGCGTTACCC
AACTTAATCG CCTTGCAGCA CATCCCCCTT TCGCCAGCTG GCGTAATAGC 6420
GAAGAGGCCC
GCACCGATCG CCCTTCCCAA CAGTTGCGCA GCCTGAATGG CGAATGGAAA 6480
TTGTAAGCGT
TAATATTZTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT 6540
TTAACCAATA
GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG 6600
GGTTGAGTGT
TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG 6660
TCAAAGGGCG
AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCTAAT 6720
CAAGT'ITITT
GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC 6780
GATTTAGAGC
TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA 6840
AAGGAGCGGG
CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC 6900
CCGCCGCGCT
TAATGCGCCG CTACAGGGCG CGTCAG 6926
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUfiNCfi CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDFsDNESS: single
(D) TOP07~OGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMN~DIATE SOORCE:
(B) CLONE: self promoter
(xi) SfiQUENCE DESCRIPTION: SEQ ID N0:70:
TATGAGATCT AAAAATTGAA ATTTTATTTT TTt~ITGG AATATAAAT 49
-180-
CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide "
(vii) Il~'DIATE SOURCE:
(B) CLONE: oFIX.l
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
TCATGTTCAC GCGCTCCATG GCCGCGGCCG CACC ~ 34
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5532 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECOLE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2gpta-FIX
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC 60
TAAATACATT
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA 120
TATrGAAAAA
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTZTIT 180
GCGGCATTTT
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT 240
GAAGATCAGT
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC 300
CTTGAGAGTT
TTCGCCCCGA AGAACGT'ITT CCAATGATGA GCACTTTTAA AGTTCTGCTA 360
TGTGGCGCGG
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC 420
TATTCTCAGA
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC 480
ATGACAGTAA
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC 540
TTACTTCTGA
CAACGATCGG AGGACCGAAG GAGCTAACCG CTiTITTGCA CAACATGGGG 600
GATCATGTAA
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC 660
GAGCGTGACA
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC 720
GAACTACTTA
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT 780
GCAGGACCAC
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA 840
GCCGGTGAGC
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC 900
CGTATCGTAG
-181-
CA 02515166 1992-08-25
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG 960
ATCGCTGAGA
TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA 1020
TATATACTTT
AGATTGATTT AAAACTTCAT TTTTAATTTA ''AAAGGATCTA GGTGAAGATC1080
CT~IT~GATA
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 1140
GACCCCGTAG
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC 1200
TGCTTGCAAA
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 1260
CCAACTCTTT
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 1320
CTAGTGTAGC
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 1380
GCTCTGCTAA
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG 1440
TTGGACTCAA
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 1500
TGCACACAGC
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 1560
CTATGAGAAA
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 1620
AGGGTCGGAA
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT 1680
AGTCCTGTCG
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 1740
GGGCGGAGCC
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC 1800
TGGCCTTTTG
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT 1860
ACCGCCTTTG
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA 1920
GTGAGCGAGG
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG 1980
ATTCATTAAT
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC 2040
GCAATTAATG
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG 2100
GCTCGTATGT
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC 2160
CATGATTACG
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC 2220
CGCGGTGGCG
GCCGCTTGTT AATTTTCAAT TCCAATGAAT TAACCTTGGA AATCCATCTT 2 2
TCATTAAGTG 8
0
AGCTTTGTTT TTTCCTTAAT CCAGTTGACA TACCGGGATA CCTTGGTATA 2340
TATTCCATAT
TTGCCTTTCA TTGCACACTC TTCACCCCAG CTAATAATTC CAGTTAAGAA 2400
ACTGGTCCCT
TCCACTTCAG TAACATGGGG TCCCCCACTA TCTCCTTGAC ATGAATCTCT 2460
ACCTCCTTCA
TGGAAGCCAG CACAGAACAT GTTGTTATAG ATGGTGAACT TTGTAGATCG 2520
AAGACATGTG
GCTCGGTCAA CAAGTGGAAC TCTAAGGTAC TGAAGAACTA AAGCTGATCT 2580
CCCTTTGTGG
AAGACTCTTC CCCAGCCACT TACATAGCCA GATCCAAATT TGAGGAAGAT 2640
GTTCGTGTAT
TCCTTGTCAG CAATGCAAAT AGGTGTAACG TAGCTGTTTA GCACTAAGGG 2700
TTCGTCCAGT
TCCAGAAGGG CAATGTCATG GTTGTACTTA TTAATAGCTG CATTGTAGTT 2760
GTGGTGAGGA
ATAATTCGAA TCACATTTCG CTTTTGCTCT GTATGTTCTG TCTCCTCAAT 2820
ATTATGTTCA
CCTGCGACAA CTGTAATTTT AACACCAGTT TCAACACAGT GGGCAGCAGT 2880
TACAATCCAT
TTTTCATTAA CGATAGAGCC TCCACAGAAT GCATCAACTT TACCATTCAA 2940
AACAACCTGC
-182-
CA 02515166 1992-08-25
CAAGGGAATT GACCTGGTTT GGCATCTTCT CCACCAACAA CCCGAGTGAA 3000
GTCATTAAAT
GATTGGGTGC TTTGAGTGAT GTTATCCAAA ATGGTTTCAG CTTCAGTAGA 3 0 6
ATTTACATAG 0
TCCACATCAG GAAAAACAGT CTCAGCACGG GTGAGCTTAG AAGTTTGTGA 3120
AACAGAAACT
CTTCCACATG GAAATGGCAC TGCTGGTTCA CAGGACTTCT GGTTTTCTGC 3180
AAGTCGATAT
CCCTCAGTAC AGGAGCAAAC CACCTTGTTA TCAGCACTAT TTTTACAAAA 3240
CTGCTCGCAT
CTGCCATTCT TAATGTTACA TGTTACATCT AATTCACAGT TCTTTCCTTC 3300
AAATCCAAAG
GGACACCAAC ATTCATAGGA ATTAATGTCA TCCZTGCAAC TGCCGCCATT 3360
TAAACATGGA
TTGGACTCAC ACTGATCTCC ATCAACATAC TGCTTCCAAA ATTCAGTTGT 3420
TCTTTCAGTG
TTTTCAAAAA CTTCTCGTGC TTCTTCAAAA CTACACTTTT CTTCCATACA 3 4 8
TTCTCTCTCA 0
AGGTTCCCTT GAACAAACTC TTCCAATTTA CCTGAATTAT ACCTC'I~TGG ~ 3540
CCGATTCAGA
ATTTTGTTGG CGTTTTCATG ATCAAGAAAA ACTGTACATT CAGCACTGAG 3600
TAGATATCCT
AAAAGGCAGA TGGTGATGAG GCCTGGTGAT TCTGCCATGA TCATGTTCAC 3660
GCGCTCCATG
GAGGCCTTAT TTATATTCCA AAAAAAAAAA ATAAAATTTC AATTTTTAGA 3720
TCCCCCAACT
TAAGGGTACC GCCTCGACAT CTATATACTA TATAGTAATA CCAATACTCA 3780
AGACTACGAA
ACTGATACAA TCTCTTATCA TGTGGGTAAT GTTCTCGATG TCGAATAGCC 3840
ATATGCCGGT
AGTTGCGATA TACATAAACT GATCACTAAT TCCAAACCCA CCCGCTTTTT 3900
ATAGTAAGTT
TTTCACCCAT AAATAATAAA TACAATAATT AATTTCTCGT AAAAGTAGAA 3960
AATATATTCT
AATTTATTGC ACGGTAAGGA AGTAGAATCA TAAAGAACAG TGACGGATGA 4020
TCCCCAAGCT
TGGACACAAG ACAGGCTTGC GAGATATGTT TGAGAATACC ACTTTATCCC 4080
GCGTCAGGGA
GAGGCAGTGC GTAAAAAGAC GCGGACTCAT GTGAAATACT GGTTTTTAGT 4140
GCGCCAGATC
TCTATAATCT CGCGCAACCT ATTTTCCCCT CGAACACTTT TTAAGCCGTA 4200
GATAAACAGG
CTGGGACACT TCACATGAGC GAAAAATACA TCGTCACCTG GGACATGTTG 4260
CAGATCCATG
CACGTAAACT CGCAAGCCGA CTGATGCCTT CTGAACAATG GAAAGGCATT 4320
ATTGCCGTAA
GCCGTGGCGG TCTGGTACCG GGTGCGTTAC TGGCGCGTGA ACTGGGTATT 4380
CGTCATGTCG
ATACCGTTTG TATTTCCAGC TACGATCACG ACAACCAGCG CGAGCTTAAA 4440
GTGCTGAAAC
GCGCAGAAGG CGATGGCGRA GGCTTCATCG TTATTGATGA CCTGGTGGAT 4500
ACCGGTGGTA
CTGCGGTTGC GATTCGTGAA ATGTATCCAA AAGCGCACTT TGTCACCATC 4560
TTCGCAAAAC
CGGCTGGTCG TCCGCTGGTT GATGACTATG TTGTTGATAT CCCGCAAGAT 4620
ACCTGGATTG
AACAGCCGTG GGATATGGGC GTCGTATTCG TCCCGCCAAT CTCCGGTCGC 4680
TAATCTTTTC
AACGCCTGGC ACTGCCGGGC GTTGTTCTTT TTAACTTCAG GCGGGTTACA 4740
ATAGTTTCCA
GTAAGTATTC TGGAGGCTGC ATCCATGACA CAGGCAAACC TGAGCGAAAC 4800
CCTGTTCAAA
CCCCGCTTTG GGCTGCAGGA ATTCGATATC AAGCTTATCG ATACCGTCGC 4860
GGCCGCGACC
TCGAGGGGGG GCCCGGTACC CAATTCGCCC TATAGTGAGT CGTATTACGC 4920
GCGCTCACTG
GCCGTCGTTT TACAACGTCG TGACTGGGAA AACCCTGGCG TTACCCAACT 4980
TAATCGCCTT
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CA 02515166 1992-08-25
GCAGCACATC CCCCTTTCGC CAGCTGGCGT AATAGCGAAG AGGCCCGCAC CGATCGCCCT 5040
TCCCAACAGT TGCGCAGCCT GAATGGCGAA TGGAAATTGT AAGCGTTAAT ATTTTGTTAA 5100
AATTCGCGTT AAATTTTTGT TAAATCAGCT CATTTTTTAA CCAATAGGCC GAAATCGGCA 5160
AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT CCA1GTTTGGA 5220
ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA ACCGTCTATC 5280
AGGGCGATGG CCCACTACGT GAACCATCAC CCTAATCAAG TTTTTTGGGG TCGAGGTGCC 5340
GTAAAGCACT AAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA CGGGGAAAGC 5400
CGGCGAACGT GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG 5460
CAAGTGTAGC GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT GCGCCGCTAC 5520
AGGGCGCGTC AG 5532
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTfiRISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCfi:
(B) CLONE: mild-type gp160MN
( ix) FfiATURE
(A) NAME/KEY: CDS
(B) LOCATION: 3..14
(xi) SfiQUENCfi DfiSCRIPTION: SEQ ID N0:73:
CA ATG AGA GTG AAG 14
Met Arg Val Lys
1
(2) INFORMATION FOR SEQ ID N0:74:
( i ) SEQiJfiNCE CHARACTERISTICS
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
( i i ) MOLECULfi TYPfi : prote in
(xi) SfiQUENCfi DfiSCRIPTION: SfiQ ID N0:74:
Met Arg Val Lye
1
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CA 02515166 1992-08-25
( 2 ) INFORMATION 'FOR SEQ ID NO: 75
(i) SEQUfiNCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNSSS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vi i ) II~sDIATE SOURCE
(B) CLONE: gp160 in vselP-gp160 virus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..14
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
CC ATG GCC GTG AAG 14
Met Ala Val Lys
1
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DfiSCRIPTION: SEQ ID N0:76:
Met Ala Val Lys _-
1
(2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vi i ) IN~DIATE SOURCE
(B) CLONE: wild-type Protein S
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4..18
(xi) SfiQUENCE DESCRIPTION: SEQ ID N0:77:
GAA ATG AGG GTC CTG GGT 18
Met Arg Val Leu Gly
1 5
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CA 02515166 1992-08-25
(2) INFORMATION FOR SfiQ ID N0:78:
(i) SfiQUENCE CHARACTERISTICS:
(A) LfiNGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DfiSCRIPTION: SEQ ID N0:78:
Met Arg Val Leu Gly
1 5
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPfi: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULfi TYPfi: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IN~I6DIATE SOURCE
(B) CLONE: Protien S in the chimeras
( ix) FfiATURfi
(A) NAMfi/KEY: CDS
(B) LOCATION: 4..18
(xi) SEQUENCE DESCRIPTION: SfiQ ID N0:79:
GCC ATG GCG GTC CTG GGT 18
Met Ala Val Leu Gly
1 5 _.
(2) INFORMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE:-amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
Met Ala Val Leu Gly
1 5
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CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMMEDIATE SOURCE:
(B) CLONE: wild-type factor IX
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..17
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:81:
TT ATG CAG CGC GTG AAC 17
Met Gln Arg Val Asn
1 5
(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:82:
Met Gln Arg Val Asn
1 5
(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IM~DIATE SOURCE:
(B) CLONE: factor IX vFIX#5
(ix) FEATURE:
(A1 NAME/KEY: CDS
(B) LOCATION: 3..17
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:
CC ATG GAG CGC GTG AAC 17
Met Glu Arg Val Asn
1 5
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CA 02515166 1992-08-25
(2) INFORMATION FOR SEQ ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
Met Glu Arg Val Asn
1 5
-las-
