Note: Descriptions are shown in the official language in which they were submitted.
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DIRECT MOLECULAR CLONING OF
A MODIFIED EUKARYOTIC CYTOPLA.9MIC DNA VIRUS GENOME
This application is a division of application 2,515,166
filed on September 9, 2005, which is a division of
application 2,076,839 filed on August 25, 1992.
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 modif i ed 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 f oreign
DNA fragment comprising a desired gene is inserted
directly into a genomic poxvirus DNA at ayrestriction
endon.uclease 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
porvirus.
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 iridoviirus
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) recombiiiation.
The use of intracellular recombination was first
described as a process of "marker rescue" with.subgenomic
fragments of viral DNA by Sam & Dumbell, Ann. Virol.
(Institut Pasteur) 132E: 135 (1981) . These authors
demonstrated that a temperature- sensitive -vaccinia virus
mutant could be "rescued" by intracellular recombination
with a subgenomic DNA fragment of a rabb~i.t 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'l Acad. Sci.
U.S.A. 79: 4927-4931 (1982) ; Mackett, et al., Proc. Nat'l
Acad. Sci. U.S.A. 79: 7415-7419 (1982); and U.S. patent
No. 4,769,330. More specifically, the extant technology
for producing recombinant poxviruses involves two steps.
First, a DNA fragment is p/repared that has regions of
homology to the poxvirus genome surrounding a foreign
gene. Alternatively, an "insertion" plasmid is
constructed by in vitro (extracellular) recombination of
a foreign gene with a Blasmid. This plasmid comprises
short viral DNA sequeuces 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 sitL flanked by the viral DNA sequences
and, typically, downstream of a poxvirus promoter that
will control transcription 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 tne 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. A difficulty
with this approach is that a new insertion plasmid is
required f or 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 hosc cell.
Another problem with extant methodology in this
regard is a low yield of recombi,mant genomes, which can
necessitate screening hundreds of individual viruses to
find a single desired recombigant. The poor yield is a
function of the low frequency uf individual intracellular
recombination events, compounded by the requirement for
multiple events of this sort to achieve integration of
the insertion plasmid into a viral genome. As a result,
the majority of viral geiiomes 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 othet desired sequence, to permit ready
detection of the required rare recombinants without the
need of characterizing isolated DNAs from numerous
individual virus clones.
Purified DNAEs of eukaryotic cytoplasmic DNA viruses
are incapable of replicating when introduced into
susceptible host cells using methods that initiate
infections wit'h viral DNAs that replicate in the nucleus.
This lack of infectivity of DNAs of cytoplasmic DNA
viruses resuits from the fact that viral transcription
must be inittiated 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 i.1: 185-
201 (1960). In 1981 Sam and Dumbell demonstrated that
isolated, noninfectious genomic DNA of a f u'rst poxvirus
could be packaged into infectious poxvirus virions in
cells infected with a second, genetlcally distinct
poxvirus. Sam & Dumbell, Ann. Virol. kInstitut Pasteur)
132E: 135 (1981). This packaging of naked poxvirus DNA
was first demonstrated by transf ectzon of unmodified DNA
comprising a first wildtype vrthopoxvirus genome,
isolated from virions or infected cells, into cells
infected with a second naturall)~ occurring orthopoxvirus
genome. However, heterologou,s 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 intrdcellular 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 poxvirus 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
poxviruses because poxvirus DNA is not infectious. F.
FENNER, R. WITTEK & K.R. DUMBELL, THE POXVIRUSES
(Academi~c Press, 1989). Others working in the area have
likewise discounted endonucleolytic cleavage and
religation 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).
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Summary of the Invention
In one aspect, the invention provides a plasmid
for producing a modified chordopoxvirus by direct molecular
cloning of a gene of interest, wherein the 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, wherein the direct
molecular cloning of the gene of interest into the genome of
the chordopoxvirus is carried out through the cleavage of
NotI, and wherein the gene of interest is cloned into the
sequence-specific endonuclease cleavage site that is unique
in the plasniid.
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In an embodiment, the DNA segment further
comprises a selective marker gene under transcriptional
control of a chordopoxvirus promoter.
In an embodiment, the plasmid is as shown in
Figure 1.3, and is selected from the group of plasmids
designated pN2-gpta comprising the sequence of
SEQ. ID. NO. 2, and pN2-gptb comprising the sequence of
SEQ. ID. NO. 3.
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In an embodiment, the DNA segment further
comprises a second chordopoxvirus promoter operatively
linked to a DNA sequence comprising a restriction
endonuclease cleavage site.
In an embodiment, the plasmid is as shown in
Figure 4.7, designated pN2gpt-S4, and comprises the sequence
of SEQ. ID. NO. 14.
In an embodiment, the plasmid comprises a
translation start codon operatively linked to the DNA
sequence comprising a restriction endonuclease cleavage
site.
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In an embodiment, the plasmid is as shown in
Figure 4.7, designated pN2gpt-S3A, and comprises the
sequence of SEQ. ID. NO. 13.
In an embodiment, the DNA segment further
comprises a second chordopoxvirus promoter operatively
linked to a DNA sequence encoding human plasminogen.
In an embodiment, the plasmid is selected from the
group of plasmids: pN2gpt-GPg, as shown in Figure 5.2,
encoding human glu-plasminogen and comprising the sequence
of SEQ. ID. NO. 17, and pN2gpt-LPg, as shown in Figure 5.3,
encoding lys-plasminogen and comprising a sequence of
SEQ. ID. NO. 18.
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In an embodiment, the DNA segment further
comprises a second chordopoxvirus promoter operatively
linked to a DNA sequence encoding human immunodeficiency
virus (HIV) gp160.
In an embodiment, the plasmid is as shown in
Figure 5.4, designated pN2gpt-gpl6O, and comprises the
sequence of SEQ. ID. NO. 19.
In an embodiment, the plasmid is as shown in
Figure 9.1, designated jpP2-gpl6OMN, and comprises the
sequence of SEQ. ID. NO. 69.
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In an embodiment, the DNA segment further
comprises a second chordopoxvirus promoter operatively
linked to a DNA sequence encoding human protein S.
In an embodiment, the plasmid is as shown in
Figure 10.1, designated pN2-gptaProtS, encodes human
protein S and comprises the sequence SEQ. ID. NO. 67.
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In an embodiment, the DNA segment further
comprises a second chordopoxvirus promoter operatively
linked to a DNA sequence encoding human factor IX.
In an embodiment, the plasmid is as shown in
Figure 11.1, designated pN2-gpta-FIX, encodes human
factor IX and comprises the sequence of SEQ. ID. NO. 72.
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In another aspect, the invention provides a
plasmid for producing a modified chordopoxvirus, wherein the
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,
wherein the DNA segment containing a gene of interest is
cloned into the sequence-specific endonuclease cleavage site
that is unique in the plasmid, wherein the DNA segment
containing the gene of interest is cloned into the genome of
the chordopoxvirus through the cleavage of NotI and wherein
said plasmid is as shown in Figure 1.3, designated pN2 and
comprising the sequences of SEQ. ID. NO. 1.
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In another aspect, the invention provides use of a
plasmid as described above for producing a modified
chordopoxvirus by direct molecular cloning of a gene of
interest, wherein a DNA segment containing the gene of
interest is cloned into the sequence-specific endonuclease
cleavage site that is unique in the plasmid, and wherein the
direct molecular cloning of the gene of interest into the
genome of the chordopoxvirus is carried out through the
cleavage of NotI.
Other objects, features and advantages of the
present invention will become apparent from the following
detailed description. It should be understood, however,
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that the detailed description and the specific examples,
while indicating preferred embodiments of the invent ion,
are given by way of illustration only, since var-ious
changes and modifications within the spirit and scope of
the invention will become apparent to those skille d in
the art from this detailed description.
Brief Description of the Drawings
Figure 1.1 illustrates expression of marker genies 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#l-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, vaccinia
recombinant vPgD (source of packaged DNA); lanes 4-7 and
11-14, 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.
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Arrows indicate the directions of transcription fron-v the
promoters.
Figure 1.4 demonstrates that poxvirus gerxomes
produced by direct molecular cloning contain the gpt
marker gene cassette inserted at a unique (NotI) cle3vage
site, as shown by Southern blot analyses of plaque-
purified viral DNAs digested with the Hindlil
endonuclease using a gpt-gene probe. Lane 1, marker DNAs
(HindIII digested phage X DNA); lanes 2 and 3, wilc3type
vaccinia virus (WR) DNA cut with HindIII (500 and 100 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 maLrker
in kilobasepairs.
Figure 1.5. further illustrates structures of
modified poxvirus DNAs using Southern blots of Notl-
digested DNAs of cells infected with various isolates and
hybridized with a gpt-gene probe. Lane 1, marker DNAs
(HindIII digested phage X 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 cells 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, HindIIl digests of WT and vp7; lanes 5 and 6,
HindIII and NotI combined digests of WT and vp7; lanes 7
and 8, PstI digests of WT and vp7; lanes 9 and 10, Pstl
and Notl 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 X DNA; and phage OX
cut with HaeIII). Arrows on the left indicate sizes of
fragments (in kilobasepairs) of NotI digest of vaccinia
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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 thc? gel
shown in Figure 1.6 using a gpt-gene probe. A3=-rows
indicate marker sizes.
Figure 1.8 presents Southern blot analyse~ 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 phage ~
DNA; and phage OX 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 uninf ected
CV-1 host cells; lane 14, vaccinia WT DNA; lane 15,
marker DNAs (HindIII digested phage X DNA; and phage OX
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 pstl,
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 X DNA; and phage OX 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 lt agarose
gel. Lane 5, marker (phage X H.indiII fragments, uncut
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phage X and vaccinia WR); lanes 1 and 2, fowlpox virus
HP1.441 DNA, uncut and cut with NotI; lanes 3 arnd 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 molecular
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 fibrobl asts.
Figure 3.1 illustrates a process for construction of
modified poxviruses by extracellular genome engineering
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 "left arm"
(la) of vaccinia virus DNA cleaved at a unique site with
the endonuclease SmaI. Packaging is done by the fowipox
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
X 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,
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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 1tes.
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 300bp in
size) separated by an internal HindIII site from the gpt
sequences (about 800 bp). Arrows indicate the dire ction
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 = wi l dtype
(WT) Western Reserve (WR) strain of vaccinia virus (VV).
Panel A shows a method using only direct mol e cular
modification of the vaccinia virus genome, inc luding
deletion of undesired NotI and SmaI sites. Panel B
outlines an alternative approach for deletion of a Notl
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-
Si and pA2-Sl) comprised of gene expression cassettes
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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 f orced
cloning. The S1 promoter is present in diff erent
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 diff erent
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 (pA1S1-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 (pN29pt-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
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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 lng of
modified vaccinia viruses made by direct mol ecular
cloning based on concurrent insertion of a marke r, gene
(the E. coli lacZ gene) which confers a vi sually
distinctive phenotype ("blue" plaque compared to normal
"white" plaques of viruses lacking a lacZ gene).
Figure 5.6 illustrates the construction of pl asmids
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 (vvWF) for expression of von-Will ebrand
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 fowipox 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
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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 plasmids
pS2gpt-P2 and pP2gpl6OMN. 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-
gp160NIN-B.
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 pse1P-
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 B) shows sequences around
translational start codons of wild-type (SEQ ID NO:73)
and modified gp160-genes (SEQ ID NO:75).
Figure 9.6 is a schematic outline of construction of
the chimeric vaccinia viruses vse1P-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 vse1P-gp160NIlVA and vP2-
gp160MNB. 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 (selP) is flanked by NotI
restriction sites. Figure 10.1 B) shows sequences around
translational start codons of wild-type Protein S gene
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(SEQ ID NO:77) and the Protein S gene in the chimeras
(SEQ ID NO: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. B) 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 ligati.on 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 l) of SK Hepi 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 (selP) 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
pBluescript-FIX). In all eight isolates (#1-6, 9 and 10)
the insert had the 'a'-orientation; m = marker; VV-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 l)
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 orient ations
of env gene inserts. Lanes 1-12, viral isolates f-LFa-1;
lane 13 and 14, HP1,441 and f-TK2a (negative cont rols);
lane 15, SspI digest (10 ng) of pN2gpt-gpl6O (positive
control). Panel B) shows restriction maps of SspI
fragments of inserts in the two possible orientat ions in
the chimeric fowipox virus. Numbers indicate si 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; lanes 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 gp4l 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 cytoplasmic 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, B. 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
(fowlpox) ; 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 invention, 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 ligating 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 religation 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 ligation 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,
CA 02558864 1992-08-25
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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 /3-
galactosidase. As described in Example 2 (avipox),
insertion of a DNA sequence into this site disrupts the
lacZ coding sequence and thereby prevents production of
S-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
CA 02558864 1992-08-25
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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 cytopla9mic 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
poxviruses to enhance expression of an inserted DNA
sequence. See, for example, Fuerst, T. R. et al., J.
Mol. Biol. 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
CA 02558864 1992-08-25
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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 B. .
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 first 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 0-
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-
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
modif ied viral genomic DNAs f rom 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 arm, 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 f irst 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, BamHI,
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-gpth 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 pAO
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 for
insertion of DNA segments, for instance, synthetic or
natural promoter fragments and were constructed by
inserting into the Xhol site of pAO 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).
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-Sl 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 pA1-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 pAlSl-PT (Figure 5.1) in which
a modified prothrombin cDNA is inserted into the single
EcoRI site of the plasmid pAl-Sl.
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 K 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 rnicleotides
ATG = translational start codon
EMBL ID = Identifier in EMBL DATABANK
EXAMPLE 1. Direct molecular cloning of foreign DNA
comprising a selective marker gene (the
gpt gene of E. coli) into a unique (NotI)
cleavage site in the genome of an
orthopoxvirus (vaccinia)
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.
Viro1. 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 (Al, A4, Cl 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
105 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 Sl
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-Bellard et
al., Eur. J. Biochem. 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 ligase 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, 10% 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.M _
Glover, DNA Cloning: A Practical Approach, 191-2Z 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 & Coupar, 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 NotI
sites.
More particularly, the multiple cloning site of pN2
consists of the following sites in the stated order:
NotI, XbaI, SpeI, BamHI, 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 pBluescript 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-gpth: The
1.1 kb HpaI-DraI fragment (containing the P7.5 promoter-
gpt gene cassette) was isolated from the plasmid pTKgpt-
F1s (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 -gpt.b. 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
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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 975. (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 DNA: 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. Viro1. 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 Pl 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 1t 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
HindilI-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 (l0s) 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 (Al-4 and C1-4) and 4 small
plaques (El-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
(Al-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 0.6t of the
viral genome and contains only 300 bp 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 El, 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 normal-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, Cl, C2, C4, E3 and E4 (Figure 1.9, lanes 1, 4, 5, 6,
8, 11 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 El-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 Al, A4, Cl 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 El, 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 (E. coli gpt) into a unique
(Notl) cleavage site of a modified avipoxvirus
genome (fowlpox virus clone f-TK2a)
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
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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-Bellard et al., Eur. J. Biochem. 36:
32-38 (1973).
Construction of a fowlpox virus vector (f-TK2a)
having a unique (NotI) cleavage site in an inserted DNA
segment: The vaccinia virus tk-gene, together with the
E. coli lacZ gene was inserted into the intergenic region
between the tk-gene and the 3' -orf of fowlpox virus. The
plasmids pTKm-VVtka and pTKm-VVtkb were constructed by
cloning the functional vaccinia virus tk-gene into the
intermediate plasmid pTKm-sPll. Upon intracellular
recombination of pTKm-VVtka and pTKm-VVtkb 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, 10o 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 Approach 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 (Scheif linger 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 NotI
cleavage site. This Notl site of the vector is in
the coding region of a lacZ gene, which serves as a
color screening marker that is inactivated upon
gene insertion. Thus, lacZ-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 fibroblasts. Total cell DNA is isolated
and the separated NotI fragments are subjected to
Southern blotting with 32P-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 CIaI 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 (fowipox) helper virus and
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 vivo 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
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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 5x103 to
1x104 pfu per 6xl 06 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
1x10a pfu per 6x106 chicken embryo f ibroblasts . The large
excess of fowipox 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 Sx106 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 vaccinia 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 ligations 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
ligated 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 jil 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 fibroblasts (6x106) infected with the
helper virus (0.5 pfu/cell of HP1.441) and
incubated for 2 h. Two gg 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. Virol 62:1849-1854) and ligated
directly into the unique Smal 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 Smal 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
fibroblasts 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 6xl 06 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 0.7t
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 (vdTlt) 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 Notl, 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 SinaI 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 polymerase
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 vaccinia (WR) virust 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 pg of vaccinia virus WR wild
type DNA is cut with NotI and ligated with one pg of the
double-stranded NotI-deletion adaptor. The adaptor
consists of two partially complementary strands: odNi
(SEQ. ID. NO. 16) and odN2 (SEQ. ID. NO. 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 Sacl "I" fragment is isolated from low
melting point agarose and cloned into the SacI site of a
suitable plasmid, such as pTZ19R (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.. Rotwal, 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
Sacl fragment prepared from the plasmid
pTZ-SacIdN is transfected into the cells. The cells are
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 t 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 bp downstream of
the first primer sequence. The template is total
DNA from 1 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 SaII, the SalI 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-Sa1F, has two SmaI sites, one in a multiple
cloning site and the other in the vaccinia sequences
(Figure 4.1, panel C). pTZ-SaIF is partially digested
with SmaI and I-SceI linkers are added, as f ollows :. first
strand, I-SceI linker 1 (SEQ. ID. NO. 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 SmaI
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 18mer
site and, therefore, cuts random DNA sequences extremely
infrequently. For instance, I-SceI cuts the yeast genome
only once. Thierry, A., Perrin, A., Boyer, 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-SceI site is
introduced into a vector having no 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-SceI 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 B. Isolates vdN/A1 #6.1111 and vdN/Al #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# 7.11 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 vdN
A) Titer after first amplification of six viral
vdN-isolates (pfu/ml crude 9tock):
vdN/A1# 2.1111 1.0 x 10, pfu/ml
vdN/A1# 4.1111 1.3 x 10s pfu/ml
vdN/A1# 6.1111 9.0 x 107 pfu/ml
vdN/A1# 8.1111 8.0 x 107 pfu/ml
vdN/A1# 10.1111 4.0 x 107 pfu/ml
vdN/Al# 12.1111 1.1 x 108 pfu/ml
B) Titer after second amplification:
vdN/A1# 2.1111 3.6 x 10s pfu/ml
vdN/A1# 4.1111 2.5 x 10g pfu/ml
vdN/A1# 6.1111 5.9 x 10g pfu/ml
vdN/A1# 8.1111 4.2 x 108 pfu/ml
vdN/A1# 10.1111 4.3 x 10a pfu/ml
vdN/A1# 12.1111 2.2 x 108 pfu/ml
WR-WT 5.4 x 10g pfu/ml
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Table 4.2
Viability 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 107 pfu/ml
vdS# 3.11 6.5 x 10' pfu/ml
vdS# 4.11 8.0 x 107 pfu/ml
vdS# 5.11 2.7 x 107 pfu/ml
vdS# 7.11 4.7 x 107 pfu/ml
B) Titer after second amplification
vdS# 2.11 1.6 x 108 pfu/ml
vdS# 3.11 1.4 x 108 pfu/ml
vdS# 4.11 8.0 x 107 pfu/ml
vdS# 5.11 1.3 x 108 pfu/ml
vdS# 7.11 1.7 x 10g pfu/ml
WR-WT 2.8 x 10a pfu/ml
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 bp upstream of the SmaI site,
while that of oligonucleotide odS3 is located about 340
bp 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 bp 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 SmaI site
is designated vdSN.
Deletion of the coding region of the thymidine kinase
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 from 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: Sfil(1), GGCCGGCT'AGGCC (SEQ. ID. NO. 29) and
Sfil(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
UV-light and prepared according to standard techniques.
The fragment was inserted into the single HindIII site of
the plasmid pTZ19R (Pharmacia, Inc.) resulting in pHindJ-
1.
Construction 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 M13KO7 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 BamHI,
HpaI, NruI and EcoRI. The mutagenesis 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. & Moss 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 (pTZ19R) 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. NO. 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 BamHI, 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 pHindJ-3: Plasmid
pHindJ-2 is digested with BamHI 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. NO. 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-i
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 143B 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
(vdTK) 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 bp upstream of the
tk-gene. The second primer, odTK3 (SEQ. ID. NO. 35), is
located about 220 bp 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 vdTK vector: The plasmid
pAO 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 pAO 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 SacI and Asp718. The large
vector fragment was ligated with an adaptor consisting of
the annealed oligonucleotides P-A(0.1) (SEQ. ID. NO. 36)
and P-A(0.2) (SEQ. ID. NO. 37).
The multiple cloning site of pAO (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 pAO).
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
pAO 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 pAO 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 pAO 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 pAl).
The multiple cloning site of pA2 (corresponding to
the oligonucleotide P-A(l.2)) and twenty bases of the 5'
and 3'-ends of pA2 are shown in SEQ. ID. NO. 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-Sl).
Construction of plasmids pAl-S1 and pA2-S1: Plasmids
pAl-Sl and pA2-S1 provide the strong synthetic poxvirus
promoter Si, 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 Si is a modified version of a strong
poxvirus late promoter designated P2.
Plasmids pAl-Si and pA2-S1 are obtained by inserting
a first double-stranded promoter fragment into the NdeI
and BamHI site of pAl 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-Si).
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 31-flanking regions of pA2
are shown in SEQ. ID. NO. 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-Sl).
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. Biol. 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 pAl-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 plasmid 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. NO. 11. The insert sequence starts
at position 21 and ends at position 68. (The first "T"
residue at the 51 -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 pAl-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 (pN2ggpt-S3A and pN2gpt-
S4): Besides plasmids designed for forced cloning,
described hereinabove, two additional plasmids were
constructed for transferring genes into one unique (NotI)
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 NotI alone or with two enzymes, for
example, NotI and SmaI (or RsrII or ApaI). The excised
fragment is then inserted into the corresponding site(s)
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-gpth: See
Example 1 and Figure 4.6
Construction of plasmid pN2gpt-S3As 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. Expresaion of polypeptides in a vaccinia
virus vector (vdTK) by direct molecular
insertion of gene expression cassettes
This example demonstrates the facility with which
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 vdTK 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 prothrombin: 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 pTKgpt-PTHBb, and
inserted into the single EcoRI site of the plasmid pAl - Si
(Example 4, Figure 4.4) resulting in the plasmid pA1Sl-PT
(Figure 5.1). In the expression cassette of this
plasmid, the prothrombin cDNA is driven by the synthetic
poxvirus promoter 91 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.
Biochemistry 22: 2087-2097 (1983). This sequence is
accessible in the EMBO Data Library under the Identifier
(ID) HSTHRI. The sequence in ID HSTHRI is not complete;
it lacks the first 19 bp of the prothrombin coding
region. The present inventors have sequenced the missing
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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 Sl promoter and 20 bases of plasmid
flanking sequences is shown in SEQ. ID. NO. 15.
By 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 pA1S1-PT corresponds to base
#2394 and the last base to #4381 of the full sequence of
plasmid pA1S1-PT).
For transfer into the vaccinia virus vector vdTK, the
cassette is excised from the plasmid pA1S1-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 fowipox 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 vPT1. 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 vaccinia 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 BalI-Smai fragment. The sequence
of human plasminogen has been published by Forsgren M,
Raden B, Israelsson M, Larsson K & Heden L-O. FEBS
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-plasminogens A sequence encoding lys-
plasminogen was prepared by deletion of the 231 bp coding
region for the first 77 amino acids (Glul 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 pN2grpt-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.
Biochem. 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 Nco2 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,
oNcol 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
SmaI. 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. NO. 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
C'V-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 g/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 inmunodeficiency virus glycoprotein 160
(HIV 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. Biol. 7: 2538-2544
(1987)]. This fragment is inserted into the plasmid
pN2ggpt-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 EMBO Data
Library (GenBank) under the Identifier (ID) HIVH3BH8.
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. Biol. 7:
2538-2544 (1987). The sequence continues with the stop
codon (base #31 this listing; base #2779 in ID HIVH3BH8)
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-gpl6O.
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 vaccinia virus vector providing for
screening for modified viruses carrying insertions by
coinsertion 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 pTZ19R (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
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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 virusvector 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 lacZ
fragment was excised from plasmid placZN*, which was
constructed by digesting the plasmid pFP-Zsart (European
Patent Application No. 91 114 300.-6, Recombinant Fowipox
Virus) with NotI and ligating pFP-Zsart with the
oligonucleotide P-NotI" (5'-GGCCAT-3'). This 3.3 kb
SnrnaI-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 ggpt marker, but lacZ
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, B.
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 permitting
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: Bonthron, D. et al., Nucl. Acids Res.
14: 7125-7128 1986). The sequence is accessible in the
EMBO Data Library under the Identifier (ID) HSVWFR1.
SEQ. ID. NO. 20 shows the junction in the virus genome of
vvWF 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 vvWF.
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 bp
upstream of the tk-gene; the reverse primer ovWFl is
located in the vWF gene about 50 bp 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. & Berg, 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 as 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
(fowipox) 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. Besides
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-1 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 102
48 4.6 x 10
72 5.0 x 105
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 102 pfu/ml at 24 hours to about 2 x 107 after
120 hours. Incubation times in the range of 48 to 72
hours produced convenient levels of packaged vaccinia
virus (between 10 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 mammalian 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
g) 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
e t. insert (ng) titers chimeras
(pfu x10"2/6x106 cells) A.)
- 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 0.1t 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-EcoKi. 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-tk18i 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-tklO.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 (GenBank) under
the Accession Number M35027. The sequence of the vaccinia
virus host range gene KiL 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
(GenBank) 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 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 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 vaccinia
viruses encoding HIV gp160 (vP2-gp160mNA,
vP2-gp160mNB and vselP-gp160MN) and
expression of recombinant gp160mN in 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-lm,,
isolate. Production of the gp160 of the HIV., isolate
described by Ratner et al., Nature 313: 277-284 (1985),
using a conventionally constructed vaccinia virus
expression vector, has been described by Barrett et al.,
AIDS Research and Human Retroviruses 5: 159-171 (1989).
The HIV= 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 HIVmN isolate.
Construction of the plasmid pP2-gp160mN and of the
chimeric viruses vP2-gpt160mNA and vP2-gp160mNB: 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 fowipox virus P2-promoter (European Patent
CA 02558864 1992-08-25
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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 vdTK (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 NO:62). Subsequently
the S4-promoter was exchanged by the P2-promoter
resulting in the plasmid pS2gpt-P2 (SEQ ID NO: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-
gp160mQ,i (Figure 9.1, SEQ ID NO:69). Additional
characteristics of the plasmid are shown in the following
table.
pP2gp160mn (6926bn) (SEO ID NO:69)
Location Description
1 - 3529 pS2gpt-P2 sequences
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
3358 - 3526 P2 promoter sequence according to EP application
Avipox "intergenic region".
3534 - 6001 CDS of the HIV-1 strain MN gp160 sequence (EMBL ID
REHIVMNC)
3534 A of the initiation codon ATG of the gp160NIN
6102 T of the stop codon TAA of the gp160NIld
6173 - 6926 pS2gpt-P2 sequences
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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
NO:43: 5'-CTAGCCCGGG-3') inactivating the XbaI and
creating a SmaI site. The resulting plasmid was
designated pS2gpt-S4 (SEQ ID NO:62). Additional
characteristics of this plasmid are shown in the following
table.
pS2rn:)t-S4 (4145bU) (SEO ID NO:62)
Location Description
1 - 2226 pN2gpt-S4 sequences of SEQ ID NO:14. Position 1
corresponds to the first nucleotide G'5-TGGCACTTT
TCGGGGAAAT-31.
2227 - 2236 SmaI-adaptor 51-CTAGCCCGGG-31.
2396 - 2851 rcCDS of E. coli gpt gene
2851 T of rc initiation codon TAC of the gpt gene
2395 A of the re 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 bp PstI-HpaI 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 NO:44)
is: 5'-GTACGTACGG CTGCAGTTGT TAGAGCTTGG TATAGCGGAC
AACTAAG-3'; the sequence of P-P2 3'(1) (SEQ ID NO: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 NO:63). The sequence primers used were
P-SM(2) (SEQ ID NO:46), 5'-GTC TTG AGT ATT GGT ATT AC-3'
and P-SM(3) (SEQ ID NO:47), 5'-CGA AAC TAT CAA AAC GCT TTA
TG-3'. Additional characteristics of the plasmid pS2gpt-
P2 are shown in the following table.
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pS2gnt-P2 (4277bu) (SEQ ID NO:63)
Location Description
1 - 3357 pS2gpt-S4 sequences
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
3358 - 3526 P2 promoter sequence according to EP application
Avipox "intergenic region".
3527 - 4277 pS2gpt-S4 sequences
pMNevn2: The plasmid pMNenvi 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 pNIIJenvl 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-
NIN(1) introduced, in addition, a StuI site 1 bp upstream
of the start codon. The sequence of P-MN(1) (SEQ ID
NO:48) is 5'-AGCTAGCTGA ATTCAGGCCT CATGAGAGTG AAGGGGATCA
GGAGGAATTA TCA-3'; the sequence of P-MN(2) (SEQ ID NO:49)
is 5'-CATCTGATGC ACAAAATAGA GTGGTGGTTG-3'. The resulting
plasmid was designated pMNenv2. To exclude mutations the
PCR generated fragment in this plasmid was sequenced with
the primers P-Seq (2) (SEQ ID NO:50) 5'-CTG TGG GTA CAC
AGG CTT GTG TGG CCC-3' and P-Seq(3) (SEQ ID NO:51) 5'-CAA
TTT TTC TGT AGC ACT ACA GAT C-3'.
pP2-gp160NN: 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-gpl6OMN (SEQ ID NO:69).
The chimeric viruses vP2-gp160mNA and vP2-gpl60mvB
were constructed as follows: The SmaI-fragment consisting
of the P2-gpl6O 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-gp160t,II,iA and vP2-gp160mNB (Figure
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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
4.0kb SmaI fragment that contains the P7.5-gpt-gene and
the P2-gpl6O-gene cassettes. Correspondingly, the
vaccinia strain WR6/2 was cut at its single SmaI (NotI)
site and ligated with the 4.0kb SmaI (NotI) fragment that
contains the P7.5-gpt-gene and the P2-gpl6O-gene
cassettes. The cloning procedures were carried out as
described in Example 1. In the virus vP2-gp160mQ,,A,.the
gp160-gene is transcribed in the same direction as the
genes clustered around the viral thymidine kinase gene; in
the virus vP2-gpl60mNB, the gpl60-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-gpl6O and P7.5-
gpt-gene cassettes was also inserted into the SmaI (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-gpl60mNA, the
predicted 6.9 and the 14.3 kb fragments are visible, and
for vP2-gpl60,,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-gp160mNA and 8.7kb fragment of
vP2-gp160t,NB are visible, confirming the integration of the
foreign gene cassettes in two different orientations.
Expression studies with the chimeric viruses vP2-
gpl60mQ,iA and vP2-gp160mNB. 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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Western blots of gpl6O: 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-gpl2O 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 (BioRad, Inc., #170-6520) used at a
1:1000 dilution. The reagents (BCIP and NBT) and staining
protocols are from Promega, Inc.
Construction of the plasmid pselP-gp160NIId and of the
chimeric virus vselP-gp160MI,,. The synthetic early/late
promoter selP (SEQ ID NO:70) (S. Chakrabarti & B. Moss;
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 se1P-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 oligonucleotides o-542
(SEQ ID NO:52) 51-CGA TTA CGT AGT TAA CGC GGC CGC GGC CTA
GCC GGC CAT AAA AAT-3' and o-544 (SEQ ID NO: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 pLi. The 0.84kb AatII-SphI fragment (parts of
noncoding gpt-sequences) were substituted by the AatII-
SphI adaptor fragment consisting of the annealed
oligonucleotide9 o-541 (SEQ ID NO: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
ATA TAG GCC GCG GCC GCA GAA AAA GCA TG-3'). The resulting
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 pTZ19R (Pharmacia, Inc.). The
resulting plasmid was called pTZ-L2 (SEQ ID NO:64).
Additional features of this plasmid are shown in the table
below.
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pTZ-L2 (4701 bp) (SEO ID NO:64)
Location Description
1 - 55 pTZ19R sequences (Pharmacia)
56 - 108 Linker I in rc orientation (5TNT, NotI, Sfil,
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,
SpeI, SacI, SmaI, EcoRI, NcoI)
1559 - 2131 s e q u e n c e s f rom the
Encephalomyocarditis Virus (EMC-
Virus) 5`untranslated region () in rc
orientation
2132 - 2187 Bacteriophage T7 promoter sequences
in rc orientation ()
2190 - 2242 Linker II in rc orientation (SnaBI,
HpaI, NotI, SfiI, 5TNT)
2243 - 4701 pTZ19R sequences (Pharmacia)
PTZse1P-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 NO:56: 5'-CGA TAA AAA TTG
AAA TTT TAT TTT TTT TTT TTG GAA TAT AAA TAA GGC CTC-3'; 51
mer) and o-se1PII (SEQ ID NO: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
vaccinia early transcription stop signal and reducing the
size of the P7.5 promoter fragment from 0.28 to 0.18kb.
The 239bp SaII-NdeI fragment was substituted by the SaiI-
NdeI adaptor consisting of the annealed oligonucleotides
0-830 (SEQ ID NO:58: 5'-TCG ACT TTT TAT CA-3') and o-857
(SEQ ID NO:59: 5'-TAT GAT AAA AAC-3'). The resulting
plasmid was called pse1P-gpt-L2 (SEQ ID NO:65).
Additional features of this construct are shown in the
table below.
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pselP-crpt-L2 (3878 bp) (SEO ID NO:65)
Location Description
1 - 55 pTZ19R sequences (Pharmacia)'
56 - 108 Linker I in rc orientation (5TNT, 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 stop codons for all three reading
frames, StuI, XhoI, PstI, BamHI, SpeI, SacI, SmaI,
EcoRI, NcoI)
1323 - 1374 Vaccinia Virus synthetic early late promoter in rc
orientation flanked by a NcoI site at position
1317 and a C1aI site at position 1370
1375 -1414 Linker II in rc orientation (SnaBI, HpaI, NotI,
SfiI, STNT)
1415 - 3878 pTZ19R Sequences (Pharmacia)
pse1P-gp160NN: The 3.lkb env gene containing the
EcoRI-PvuII fragment of pMNenvI was inserted into the
EcoRI and StuI cut plasmid pse1P-gpt-L2 resulting in the
intermediate plasmid pse1P-gpl60.1. 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
pse1P-gp160mn,7 (SEQ ID N0:66) . Additional features of this
plasmid are shown in the table below.
pse1P-qp160MN (6474 bA) (SEO ID NO:66)
Location Description
1 - 55 pTZ19R sequences (Pharmacia)
56 - 108 Linker I in rc orientation (5TNT, SfiI, RsrII,
HpaI, SnaBI, AatII)
110 -860 E.coli gpt sequences in rc orientation. The gpt
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
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.
1259 - 3916 HIV-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
3913) and a ClaI site (position 3966)
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3971 - 4015 Linker II in rc orientation (SnaBI, HpaI, NotI,
SfiI, 5TNT)
4016 - 6474 pTZ19R sequences (Pharmacia)
The primers used for the PCR reaction were o-NcoI
5(40mer) SEQ ID NO:60: 5'-GAG CAG AAG ACA GTG GCC ATG GCC
GTG AAG GGG ATC AGG A-3', and o-NsiI (30mer) SEQ ID NO:61:
5'-CAT AAA CTG ATT ATA TCC TCA TGC ATC TGT-3'. For
further cloning the PCR product was cleaved with NcoI and
Nsil.
Chimeric viruses vse1P-gp160mNA vselP-gp160mNB are
constructed as follows: The HpaI-fragment consisting of
the selP-gpl60 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 Buller 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
4.0kb HpaI fragment that contains the P7.5-gpt-gene and
the se1P-gpl60-gene cassettes. The cloning procedures are
carried out as described in Example 1.
The resulting chimeric viruses, vse1P-gp160rõQ,,A and
vse1P-gp160mQ,,B, are purified and further characterized. In
the virus vselP-gp160,,Q,,A, 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 vselP-gp1601,Q,,B, 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 Sa1F-
fragment probe (pTZ-SalF) and a gp160-gene probe
(pMNenvl). With the Sa1F-fragment probe, for vselP-
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gp160mNA the predicted 6.8 and 10.7 kb fragments are
visible; and for vse1P-gp160mNB, 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-
gp160mQ'A and the 3.5kb fragment in vselP-gp160mNB give less
intense signals, because only about 400 bp 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 chimeric viruses vselP-
gp160mNA and vse1P-gp160,,H: Vero cells are used for
expression studies. Growth of cells, infections with the
chimeric viruses and purification of the recombinant gpl60
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
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transformed cell lines) at levels of up to 7 g/106
cells/day (Grinnell et al., Blood, 76: 2546 (1990)) or in
mouse C127 cell/papilloma virus system at similar
expression levels. Malm et al., Eur. J. Biochem., 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 Hepi. 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 NO: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
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second amino acid changed from Arg to Ala (Figure 10.1 B
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-selP (Falkner et al., supra) a
plasmid providing a strong synthetic vaccinia promoter.
The promoter-protein S gene cassette was then excised as
a.Bg1II-NotI fragment and inserted into the plasmid pN2-
gpta (Example 1) resulting in pN2-gptaProtS (SEQ ID
NO:67). Additional features of this construct are shown
in the following table.
pN2-crptaProtS (6811 bp) (Sfi0 ID NO: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 Bg1II/BamHI site (position 4987-4992) .
The NcoI site harbors the Prot S rc start codon
TAC
4993 - 5493 Vaccinia Virus p7.5 promoter sequences
5494 - 6127 E.coli gpt sequences. The ORF starts at position
5494 with ATG start codon and ends at position
5950 with a TAA stop codon.
6228 - 6235 NotI site 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
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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 l 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 105 pfu/106 host
cells were obtained. About 6-7W 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 NotI and
subjected to Southern blot analysis (Fig. 10.2). The SacI
digest, hybridized with the cloned SacI-I fragment
(plasmid pTZ-SacI; 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. 10.2B).
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 Boehringer
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 02558864 1992-08-25
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Alternatively, 10 l of supernatant from Vero cells
were analyzed in a Western Blot using 50 ng of human
plasma- derived protein S as a standard and a mouse
polyclonal serum specific for "hu Prot S" (Axell) (Fig.
10.3). Blots were stained using an alkaline phosphatase
conjugated goat anti- mouse polyclonal serum (Dakopatts)
and NBT/BCIP as 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 Blood Coagulation Factors: Their
cDNAs, Genes and Expression", Hemostasis and Thrombosis,
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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); Balland, 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 pBluescript-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 NO:71: 51
-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-selP. Falkner et al.,
supra The promoter/FIX cassette was cut out from this
plasmid with BgIII 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 selP 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.
pN2gpta-FIX (5532bp) (SEO ID NO: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 E.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
4857-5532 Bluescript II SK-sequences (Stratagene)
Insertion of the cDNA 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. 11.1A, SEQ ID NO: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 NotI 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 02558864 1992-08-25
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pfu/106 host cells were obtained. In this example, about
it 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 NotI 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 (fragments 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
3.8kb 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 l of supernatant from Vero cells
were analyzed in a Western Blot 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
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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 from right to left, i.e. 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 best 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 02558864 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# anticzen activity ratio
(mU/lObcells) * t
SK Hepl (HTB52) 810 183 22.5
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
* 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. al. 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
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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
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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-envIIIB. For construction of f-envIIIB (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.
12.2B). 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 chimeric 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
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gp4l 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-gpl2O antibody (Du Pont, Inc. #
NEA9305 used in a 1:500 dilution. For the gp4l detection
the human anti-HIV-gp4l 3D6 Mab (provided by H. Katinger,
Universitat fur Bodenkultur, 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 02558864 1992-08-25
-- == .~
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: DORNER, F.
SCHEIFLINGER, F.
FALKNER, F. G.
PFLEIDERER, 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) ADDRESSEE: Foley & Lardner
(B) STREET: 1800 Diagonal Road, Suite 500
(C) CITY: Alexandria
(D) STATE: VA
(E) 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 NUMBER: 07/750,080
(B) FILING DATE: August 26, 1991
(C) CLASSIFICATION: Unknown
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: BENT, Stephen A.
(B) REGISTRATION NUMBER: 29,768
(C) REFERENCE/DOCKET NUMBER: 30472/106 IMMU
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (703)836-9300
(B) TELEFAX: (703) 683-4109
(C) TELEX: 899149
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCAAGCTTAT CGATACCGTC GCGGCCGCGA CCTCGAGGGG GGGCCCGG 48
-135-
CA 02558864 1992-08-25
in
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: pN2-gpta
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTAGAACTAG TGGATCCCCC AACTTAAGGG TACCGCCTCG ACATCTATAT ACTATATAGT 60
AATACCAATA CTCAAGACTA CGAAACTGAT ACAATCTCTI' ATCATGTGGG TAATGTTCTC 120
GATGTCGAAT AGCCATATGC CGGTAGTTGC GATATACATA AACTGATCAC TAATTCCAAA 180
CCCACCCGCT TTTTATAGTA AGTTTTTCAC CCATAAATAA TAAATACAAT AATTAATTTC 240
TCGTAAAAGT AGAAAATATA TTCTAATT'TA TTGCACGGTA AGGAAGTAGA ATCATAAAGA 300
ACAGTGACGG ATGATCCCCA AGCTTGGACA CAAGACAGGC TTGCGAGATA TGTTTGAGAA 360
TACCACTTTA TCCCGCGTCA GGGAGAGGCA GTGCGTAAAA AGACGCGGAC TCATGTGAAA 420
TACTGG'IT'IT TAGTGCGCCA GATCTCTATA ATCTCGCGCA ACCTATTTT C CCCTCGAACA 480
CTTTTTAAGC CGTAGATAAA CAGGCTGGGA CACTTCACAT GAGCGAAAAA TACATCGTCA 540
CCTGGGACAT GTTGC.AGATC CATGCACGTA AACTCGCAAG CCGACTGATG CCTTCTGAAC 600
AATGGAAAGG CATTATTGCC GTAAGCCGTG GCGGTCTGGT ACCGGGTGCG TTACTGGCGC 660
GTGAACTGGG TATTCGTCAT GTCGATACCG TTTGTATTTC CAGCTACGAT CACGACAACC 720
AGCGCGAGCT TAAAGTGCTG AAACGCGCAG AAGGCGATGG CGAAGGCTTC ATCGTTATTG 780
ATGACCTGGT GGATACCGGT GGTACTGCGG TTGCGATTCG TGAAATGTAT CCAAAAGCGC 840
ACTTT GTCAC CATCZTCGCA AAACCGGCTG GTCGTCCGCT GGTTGATGAC TATGTTGTTG 900
ATATCCCGCA AGATACCTGG ATTGAACAGC CGTGGGATAT GGGCGTCGTA TTCGTCCCGC 960
CAATCTCCGG TCGCTAATCT TZTCAACGCC TGGCACTGCC GGGCGTTGTT CTTZTrAACT 1020
TCAGGCGGGT TAC.AATAGTT TCCAGTAAGT ATTCTGGAGG CTGCATCCAT GACACAGGCA 1080
AACCTGAGCG AAACCCTGTT CAAACCCCGC TTTGGGCTGC AGGAATTCGA TAT 1133
(2) INFORMATION FOR SEQ ID NO:3:
(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
-136-
CA 02558864 1992-08-25
.~:. ~
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2-gptb
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTAGAACTAG TGGATCCCCC AAAGCGGGGT TTGAACAGGG TTTCGCTCAG GTTTGCCTGT 60
GTCATGGATG CAGCCTCCAG AATACTTACT GGAAACTATT GTAACCCGCC TGAAGTTAAA 120
AAGAACAACG CCCGGCAGTG CCAGGCGTTG AAAAGATTAG CGACCGGAGA TTGGCGGGAC 180
GAATACGACG CCCATATCCC ACGGCTGTTC AATCCAGGTA TCTTGCGGGA TATCAACAAC 240
ATAGTCATCA ACCAGCGGAC GACCAGCCGG TTTTGCGAAG ATGGTGACAA AGTGCGCTTT 300
TGGATACATT TCACGAATCG CAACCGCAGT ACCACCGGTA TCCACCAGGT CATCAATAAC 360
GATGAAGCCT TCGCCATCGC CTTCTGCGCG TTTCAGCACT TTAAGCTCGC GCTGGTTGTC 420
GTGATCGTAG CTGGAAATAC AAACGGTATC GACATGACGA ATACCCAGZT CACGCGCCAG 480
TAACGCACCC GGTACCAGAC CGCCACGGCT TACGGCAATA ATGCCTT'TCC ATTGTTCAGA 540
AGGCATCAGT CGGCTTGCGA GTTTACGTGC ATGGATCTGC AACATGTCCC AGGTGACGAT 600
GTATTTTTCG CTCATGTGAA GTGTCCCAGC CTGTTTATCT ACGGCTTAAA AAGTGTTCGA 660
GGGGAAAATA GGTTGCGCGA GATTATAGAG ATCTGGCGCA CTAAAAACCA GTATTTCACA 720
TGAGTCCGCG TCTTTTI'ACG CACTGCCTCT CCCTGACGCG GGATAAAGTG GTATTCTCAA 780
ACATATCTCG CAAGCCTGTC TTGTGTCCAA GCTTGGGGAT CATCCGTCAC TGTTCTZTAT 840
GAZTCTACTT CCTTACCGTG CAATAAATTA GAATATATTT TCTACTTTTA CGAGAAATTA 900
ATTATTGTAT TTATTATTTA TGGGTGAAAA ACTTACTATA AAAIGCGGGT GGGTZTGGAA 960
TTAGTGATCA GTTTATGTAT ATCGCAACTA CCGGCATATG GCTATTCGAC ATCGAGAACA 1020
TTACCCACAT GATAAGAGAT TGTATCAGTT TCGTAGTCTT GAGTATTGGT ATTACTATAT 1080
AGTATATAGA TGTCGAGGCG GTACCCTTAA GTTGGGCTGC AGGAATTCGA TAT 1133
(2) INFORMATION FOR SEQ ID NO:4:
(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) IMMEDIATE SOURCE:
(B) CLONE: pHindJ-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CGCATTTTCT AACGTGATGG GATCCGTTAA CTCGCGAGAA TTCTGTAGAA AGTGZTACAT 60
CGACTC 66
-137-
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(2) INFORMATION FOR SEQ ID NO:5:
(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
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHindJ-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CGCATTTTCT AACGTGATGG GATCCGGCCG GCTAGGCCGC GGCCGCCCGG GZTITTATCT 60
CGAGACAAAA AGACGGACCG GGCCCGGCCA TATAGGCCCA ATTCTGTAGA AAGTGZTACA 120
TCGACTC 127
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 115 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: pAO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AGGGAACAAA AGCTGGAGCT AGGCCGGCTA GGCCGCGGCC GCCCGGGTTT TTATCTCGAG 60
ACAAAAAGAC GGACCGGGCC CGGCC.ATATA GGCCAGTACC CAATTCGCCC TATAG 115
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 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: pAl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CGGCCGCCCG GGTTTTTATC TCGACATATG CTGCAGTTAA CGAATTCCAT GGGGATCCGA 60
TATCAAGCTT AGGCCTGTCG ACGTCGAGAC AAAAAGACGG ACC 103
-138-
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(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 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: pA2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGGCCGCCCG GGTTTTTATC TCGACGTCGA CAGGCCTAAG CTTGATATCG GATCCCCATG 60
GAATTCGTTA ACTGCAGCAT ATGTCGAGAC AAAAAGACGG ACC 103
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: pAl-S1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCCGGGTTT'T TATCTCGACA TACGGCTTGG TATAGCGGAC AACTAAGTAA TTGTAAAGAA 60
GAAAACGAAA CTATCAAAAC CGTTTATGAA ATGATAGAAA AAAGAATATA AATAATCCTG 120
TATTTTAGTT TAAGTAACAG TAAAATAATG AGTAGAAAAT ACTATTT'IIT 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 02558864 1992-08-25
itm
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CAGGCCTAAG CTTGATATCG GATCCGAATT CATGATTTAT AGGCTATAAA AAATAGTATT 60
TTCTACTCAT TATTTTACTG TTACTTAAAC TAAAATACAG GATTATTTAT ATTCT.rTTZT 120
CTATCATZTC ATAAACGGTT TTGATAGTTT CGTTTTCTTC TTTACAATI'A CTTAGZTGTC 180
CGCTATACCA AGCCGTATGT CGAGACAAAA AGACG 215
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 88 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: pA1-S2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TCTCGACATA TGCTGCAGTT GGGAAGCTTT TTTI'i"ITITT TTTiTITGGC ATATAAATAG 60
GCTGCAGGAA.TTCCATGGGG ATCCGATA 88
(2) TNFnR.MATTON FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 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-S2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TTGATATCGG ATCCCCATGG AATTCCTGCA GCCTATTTAT ATGCCAAAAA AAAAAAAAAA 60
AAAAAGCTTC CCAACTGCAG CATATGTCGA GA 92
(2) INFORMATION FOR SEQ ID NO: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 02558864 1992-08-25
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2gpt-S3A (fig. 4.7)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TACCCTTAAG TTGGGCTGCA GAAGCT'ITTT TPITPITITT TTTTTGGCAT ATAAATGAAT 60
TCCATGGCCC GGGAAGGCCT CGGACCGGGC CCGGCCATAT AGGCCAGCGA TACCGTCGCG 120
GCCGCGA 127
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: pN2gpt-S4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TACCCTTAAG TTGGGCTGCA GAAGCTTTTT TITITTITIT TTTPTGGCAT ATAAATCGTT 60
AACGAATTCC ATGGCCCGGG AAGGCCTCGG ACCGGGCCCG GCCATATAGG CCAGCGATAC 120
CGTCGCGGCC GCGA 134
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: pAlSl-PT
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
TT'I'TATAGCC 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 GCAZTGAGTG CCAGCTATGG 420
-141-
CA 02558864 1992-08-25
AGGAGTCGCT ACCCACATAA GCCTGAAATC AACTCCACTA CCCATCCTGG GGCCGACCTA 480
CAGGAGAATI' TCTGCCGCAA CCCCGACAGC AGCAACACGG GACCATGGTG CTACACTACA 540
GACCCCACCG TGAGGAGGCA GGAATGCAGC ATCCCTGTCT GTGGCCAGGA TC.AAGTCACT 600
GTAGCGATGA CTCCACGCTC CGAAGGCTCC AGTGTGAATC TGTCACCTCC ATTGGAGCAG 660
TGTGTCCCTG ATCGGGGGCA GCAGTACCAG GGGCGCCTGG CGGTGACCAC ACATGGGCTC 720
CCCTGCCTGG CCTGGGCCAG CGCACAGGCC AAGGCCCTGA GCAAGCACCA GGACZTCAAC 780
TCAGCTGTGC AGCTGGTGGA GAACTTCTGC CGCAACCCAG ACGGGGATGA GGAGGGCGTG 840
TGGTGCTATG TGGCCGGGAA GCCTGGCGAC TTTGGGTACT GCGACCTCAA CTAZTGTGAG 900
GAGGCCGTGG AGGAGGAGAC AGGAGATGGG CTGGATGAGG ACTCAGACAG GGCCATCGAA 960
GGGCGTACCG CCACAAGTGA GTACCAGACT TTCTTCAATC CGAGGACCTT TGGCTCGGGA 1020
GAGGCAGACT GTGGGCTGCG ACCTCTGTTC GAGAAGAAGT CGCTGGAGGA CAAAACCGAA 1080
AGAGAGCTCC TGGAATCCTA CATCGACGGG CGCATTGTGG AGGGCTCGGA TGCAGAGATC 1140
GGCATGTCAC CZTGGCAGGT GATGCTTTTC CGGAAGAGTC CCCAGGAGCT GCTGTGTGGG 1200
GCCAGCCTCA TCAGTGACCG CTGGGTCCTC ACCGCCGCCC ACTGCCTCCT GTACCCGCCC 1260
TGGGACAAGA ACZTCACCGA GAATGACCTT CTGGTGCGCA TTGGCAAGCA CTCCCGCACC 1320
AGGTACGAGC GAAACATTGA AAAGATATCC ATGTTGGAAA AGATCTACAT CCACCCCAGG 1380
TACAACTGGC GGGAGAACCT GGACCGGGAC ATTGCCCTGA TGAAGCTGAA GAAGCCTGZT 1440
GCCTTCAGTG ACTACATTCA CCCTGTGTGT CTGCCCGACA GGGAGACGGC AGCCAGCTTG 1500
CTCCAGGCTG GATACAAGGG GCGGGTGACA GGCTGGGGCA ACCTGAAGGA GACGTGGACA 1560
GCCAACGTTG GTAAGGGGCA GCCCAGTGTC CTGCAGGTGG TGAACCTGCC CATTGTGGAG 1620
CGGCCGGTCT GCAAGGACTC CACCCGGATC CGCATCACTG ACAACATGTT CTGTGCTGGT 1680
TACAAGCCTG ATGAAGGGAA ACGAGGGGAT GCCTGTGAAG GTGACAGTGG GGGACCCTTT 1740
GTCATGAAGA GCCCCTTTAA CAACCGCTGG TATCAAATGG GCATCGTCTC ATGGGGTGAA 1800
GGCTGTGACC GGGATGGGAA ATATGGCTTC TACACACATG TGTTCCGCCT GAAGAAGTGG 1860
ATACAGAAGG TCATTGATCA GTTTGGAGAG TAGGGGGCCA CTCATATTCT GGGCTCCTGG 1920
AACCAATCCC GTGAAAGAAT TA2TITTGTG TTTCTAAAAC TAGAATTCGG ATTCGATATC 1980
AAGCTTAG 1988
(2) INFORMATION FOR SEQ ID NO:16:
(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
-142-
CA 02558864 1992-08-25
It
(vi i ) IMMEDIATE SOURCE :
(B) CLONE: odNl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GGCCAGGCCT TTTAAATTAA GATATC 26
(2) INFORMATION FOR SEQ ID NO: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) I14MDIATE SOURCE:
(B) CLONE: pN2gpt-GPg
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TTZTTGGCAT ATAAATCGTT CCAGTCCCAA AATGTAATTG GACGGGAGAC AGAGTGACGC 60
ACGCGGCCGC TCTAGAACTA GTGGATCCCC CAACGAATTC CATGGCCCGG G ill
(2) INFORMATION FOR SEQ ID NO:18:
( i ) SEQUENCE CEiARACTERISTICS :
(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 NO:18:
ATAAATCGTT AACGAATTCC ATGGAACATA AGGAAGTGGT TCTTCTACTT CTTTTATTTC 60
TGAAATCAGG TCAAGGAAAA GTGTATCTCT CAGAGTGCAA GACTGGGAAT GGAAAGAACT 120
ACAGAGGGAC GATGTCCAAA ACAAAAAATG GCATCACCTG TCAAAAATGG AGTTCCACTT 180
CTCCCCACAG ACCTAGATTC TCACCTGCTA CACACCCCTC AGAGGGACTG GAGGAGAACT 240
ACTGCAGGAA TCCAGACAAC GATCCGCAGG GGCCCTGGTG CTATACTACT GATCCAGAAA 300
AGAGATATGA CTACTGCGAC ATTCTTGAGT GTGAAGAGGA ATGTATGCAT TGCAGTGGAG 360
AAAACTATGA CGGCAAAATT TCCAAGACCA TGTCTGGACT GGAATGCCAG GCCTGGGACT 420
CTCAGAGCCC ACACGCTCAT GGATACATTC CTTCCAAATT TCCAAACAAG AACCTGAAGA 480
AGAATTACTG TCGTAACCCC GATAGGGAGC TGCGGCCTTG GTGTTTCACC ACCGACCCCA 540
ACAAGCGCTG GGAACTTTGC GACATCCCCC GCTGCACAAC ACCTCCACCA TCTTCTGGTC 600
-143-
CA 02558864 1992-08-25
CCACCTACCA GTGTCTGAAG GGAACAGGTG AAAACTATCG CGGGAATGTG GCTGTTACCG 660
TTTCCGGGCA CACCTGTCAG CACTGGAGTG CACAGACCCC TCACACACAT AACAGGACAC 720
CAGAAAACTT CCCCTGCAAA AATTTGGATG AAAACTACTG CCGCAATCCT GACGGAAAAA 780
GGGCCCCATG GTGCCATACA ACCAACAGCC AAGTGCGGTG GGAGTACTGT AAGATACCGT 840
CCTGTGACTC CTCCCCAGTA TCCACGGAAC AATTGGCTCC CACAGCACCA CCTGAGCTAA 900
CCCCTGTGGT CCAGGACTGC TACCACGGTG ATGGACAGAG CTACCGAGGC ACATCCTCCA 960
CCACCACCAC AGGAAAGAAG TGTCAGTCTT GGTCATCTAT GACACCACAC CGGCACCAGA 1020
AGACCCCAGA AAACTACCCA AATGCTGGCC TGACAATGAA CTACTGCAGG AATCCAGATG 1080
CCGATAAAGG CCCCTGGTGT TTTACCACAG ACCCCAGCGT CAGGTGGGAGTACTGCAACC 1140
TGAAAAAATG CTCAGGAACA GAAGCGAGTG TTGTAGCACC TCCGCCTGZT GTCCTGCTTC 1200
CAGATGTAGA GACTCCTTCC GAAGAAGACT GTATGTTTGG GAATGGGAAA GGATACCGAG 1260
GCAAGAGGGC GACCACTGTT ACTGGGACGC CATGCCAGGA CTGGGCTGCC CAGGAGCCCC 1320
ATAGACACAG CATTTTCACT CCAGAGACAA ATCCACGGGC GGGTCTGGAA AAAAATTACT 1380
GCCGTAACCC TGATGGTGAT GTAGGTGGTC CCTGGTGCTA CACGACAAAT CCAAGAAAAC 1440
TTTACGACTA CTGTGATGTC CCTCAGTGTG CGGCCCCTTC ATZTGATTGT GGGAAGCCTC 1500
AAGTGGAGCC GAAGAAATGT CCTGGAAGGG TTGTGGGGGG GTGTGTGGCC CACCCACATT 1560
CCTGGCCCTG GCAAGTCAGT CTTAGAACAA GGTTTGGAAT GCACTTCTGT GGAGGCACCT 1620
TGATATCCCC AGAGTGGGTG TTGACTGCTG CCCACTGCTI' GGAGAAGTCC CCAAGGCCTT 1680
CATCCTACAA GGTCATCCTG GGTGCACACC AAGAAGTGAA TCTCGAACCG CATGTI'CAGG 1740
AAATAGAAGT GTCTAGGCTG TTCTTGGAGC CCACACGAAA AGATATTGCC TTGCTAAAGC 1800
TAAGCAGTCC TGCCGTCATC ACTGACAAAG TAATCCCAGC ZTGTCTGCCA TCCCCAAATT 1860
ATGTGGTCGC TGACCGGACC GAATGTZTCA TCACTGGCTG GGGAGAAACC CAAGGTACTT 1920
TTGGAGCTGG CCTTCTCAAG GAAGCCCAGC TCCCTGTGAT TGAGAATAAA GTGTGCAATC 1980
GCTATGAGTT TCTGAATGGA AGAGTCCAAT CCACCGAACT CTGTGCTGGG CATTTGGCCG 2040
GAGGCACTGA CAGTTGCCAG GGTGACAGTG GAGGTCCTCT GGTTTGCTTC GAGAAGGACA 2100
AATACATTTT ACAAGGAGTC ACTI'CTTGGG GTCTTGGCTG TGCACGCCCC AATAAGCCTG 2160
GTGTCTATGT TCGTGTITCA AGGTTTGTTA CTTGGATTGA GGGAGTGATG AGAAATAATT 2220
AATTGGACGG GAGACAGAGT GACGCACGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGA 2280
AGGCCTCGGA CCGGGC 2296
-144-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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
(vii) IMMEDIATE SOURCE:
(B) CLONE: pN2gpt-gpl6O
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
TTTTTGGCAT ATAAATCGTT ATCCACCATG TAAGATAACG AATTCCATGG CCCGGG 56
(2) INFORMATION FOR SEQ ID NO: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 NO:20:
TIZTITITGG 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 NO: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) IMMDIATE SOURCE:
(B) CLONE: pEcoK-dhr
-145-
CA 02558864 1992-08-25
~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
ATTAGCGTCT CGTTTCAGAC GCGGCCGCGG TAATTAGATT CTCCCACATT 50
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1209 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: pdhr-gpt
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
AZTAGCGTCT CGTTTCAGAC GCGGCCGCTC TAGAACTAGT GGATCCCCCA ACTTAAGGGT 60
ACCGCCTCGA CATCTATATA CTATATAGTA ATACCAATAC TCAAGACTAC GAAACTGATA 120
CAATCTC'ITA TCATGTGGGT AATGTTCTCG ATGTCGAATA GCCATATGCC GGTAGTTGCG 180
ATATACATAA ACTGATCACT AATTCCAAAC CCACCCGCTT TTTATAGTAA GT'lTPI'CACC 240
CATAAATAAT A.AATACAATA ATTAATTTCT CGTAAAAGTA GAAAATATAT TCTAATTTAT 300
TGCACGGTAA GGAAGTAGAA TCATAAAGAA CAGTGACGGA TGATCCCCAA GCZTGGACAC 360
AAGACAGGCT TGCGAGATAT GITTGAGAAT ACCACTTTAT CCCGCGTCAG GGAGAGGCAG 420
TGCGTAAAAA GACGCGGACT CATGTGAAAT ACTGGTTTTT AGTGCGCCAG ATCTCTATAA 480
TCTCGCGCAA CCTATTTTCC CCTCGAACAC TTTTTAAGCC GTAGATAAAC AGGCTGGGAC 540
ACTI'CACATG AGCGAAAAAT ACATCGTCAC CTGGGACATG TTGCAGATCC ATGCACGTAA 600
ACTCGCAAGC CGACTGATGC CTTCTGAACA ATGGAAAGGC ATTATTGCCG TAAGCCGTGG 660
CGGTCTGGTA CCGGGTGCGT TACTGGCGCG TGAACTGGGT ATTCGTCATG TCGATACCGT 720
TTGTATTTCC AGCTACGATC ACGACAACCA GCGCGAGCTT AAAGTGCTGA AACGCGCAGA 780
AGGCGATGGC GAAGGCTTCA TCGTTATTGA TGACCTGGTG GATACCGGTG GTACTGCGGT 840
TGCGATTCGT GAAATGTATC CAAAAGCGCA CTTTGTCACC ATCTTCGCAA AACCGGCTGG 900
TCGTCCGCTG GTTGATGACT ATGTTGTI'GA TATCCCGCAA GATACCTGGA TTGAACAGCC 960
GTGGGATATG GGCGTCGTAT TCGTCCCGCC AATCTCCGGT CGCTAATCTT TTCAACGCCT 1020
GGCACTGCCG GGCGTTGTTC TTTI'rAACTT CAGGCGGGTT ACAATAGTTT CCAGTAAGTA 1080
TTCTGGAGGC TGCATCCATG ACACAGGCAA ACCTGAGCGA AACCCTGTTC AAACCCCGCT 1140
TTGGGCTGCA GGAAZTCGAT ATCAAGCTTA TCGATACCGT CGCGGCCGCG GTAATTAGAT 1200
TCTCCCACA 1209
-146-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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 NO:23:
GGCCGATATC TTAATTTAAA AGGCCT 26
(2) INFORMATION FOR SEQ ID NO: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
(vii) IMMEDIATE SOURCE:
(B) CLONE: odN3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CCAATGTTAC GTGGGTTACA TCAG -. 24
(2) INFORMATION FOR SEQ ID NO: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-Scel linker 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
TAGGGATAAC AGGGTAAT 18
-147-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: I-SceI linker 2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATTACCCTGT TATCCCTA 18
(2) INFORMATION FOR SEQ ID NO: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 NO:27:
GTATAAAGTC CGACTATTGT TCT 23
(2) INFORMATION FOR SEQ ID NO: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) IM[4EDIATE SOURCE:
(B) CLONE: odS3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
TCTGAGGCCT AATAGACCTC TGTACA 26
-148-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:29:
(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 (1)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GGCCGGCTAG GCC 13
(2) INFORMATION FOR SEQ ID NO: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 NO:30:
GGCCATATAG GCC 13
(2) INFORMATION FOR SEQ ID NO: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) IMMDIATE SOURCE:
(B) CLONE: odTKl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GAGTCGATGT AACACTTTCT ACAGGATCCG TTAACTCGCG AGAATTCCAT CACGTTAGAA 60
AATGCG 66
-149-
CA 02558864 1992-08-25
lm~
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: P-J(1)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
GATCCGGCCG GCTAGGCCGC GGCCGCCCGG GTTT'TTATCT CGAGACAAAA AGACGGACCG 60
GGCCCGGCCA TATAGGCCC 79
(2) INFORMATION FOR SEQ ID NO: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 NO:33:
AATTGGGCCT ATATGGCCGG GCCCGGTCCG TCTTTTTGTC TCGAGATAAA AACCCGGGCG 60
GCCGCGGCCT AGCCGGCCG 79
(2) INFORMATION FOR SEQ ID NO:34:
(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: odTK2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
AGAAGCCGTG GGTCATTG 18
-150-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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 NO:35:
TACCGTGTCG CTGTAACTTA C 21
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE 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 NO:36:
AGGCCGGCTA GGCCGCGGCC GCCCGGGTTT TTATCTCGAG ACAAAAAGAC GGACCGGGCC 60
CGGCCATATA GGCCA 75
(2) INFORMATION FOR SEQ ID NO: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 NO:37:
GTACTGGCCT ATATGGCCGG GCCCGGTCCG TCTTTTTGTC TCGAGATAAA AACCCGGGCG 60
GCCGCGGCCT AGCCGGCCTA GCT 83
-151-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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 NO:38:
GGCCACGTTT ZTATGGGAAG CTT=IT!' TTTPTZTTZT TGGCATATAA ATCGC 55
(2) INFORMATION FOR SEQ ID NO: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
(vi i ) IMMEDIATE SOURCE :
(B) CLONE: P-artP(12)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
GGCCGCGATT TATATGCCAA AAAAAAAAAA AAAAAAAAGC TTCCCATAAA AACGT 55
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid;
(A) DESCRIPTION: Synthetic DNA oligonucleotide
(vii) IMNMEDIATE SOURCE:
(B) CLONE: P-artP(8)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
CGCTGGCCTA TATGGCCGGG CCCGGTCCGA GGCCTTCCCG GGCCATGGAA TTCATTTATA 60
TGCCAAAAAA AAAAAAAAAA AAAAGCTTCT GCA 93
-152-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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 NO:41:
CGCTGGCCTA TATGGCCGGG CGTCCGAGGC CTTCCCGGGC CATGGAATTC GTTAACGATT 60
TATATGCCAA AAAAAAAAAA AAAAAAAAGC TTCTGCA 97
(2) INFORMATION FOR SEQ ID NO: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 NO:42:
ATTAGCGTCT CGTTTCAGAC GCGGCCGCGG TAATTAGATT CTCCCACATT 50
(2) INFORMATION FOR SEQ ID NO: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 NO:43:
CTAGCCCGGG 10
-153-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:44:
(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: P-P2 5' (1)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GTACGTACGG CTGCAGTTGT TAGAGCTTGG TATAGCGGAC AACTAAG 47
(2) INFORMATION FOR SEQ ID NO:45:
(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: P-P2 3'(1)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
TCTGACTGAC GTTAACGATT TATAGGCTAT AAAAAATAGT ATTTTCTACT 50
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 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-SM(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
GTCTTGAGTA ZTGGTATTAC 20
-154-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: P-SM(3)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
CGAAACTATC AAAACGCTTT ATG 23
(2) INFORMATION FOR SEQ ID NO:48:
(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: P-MN(1)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
AGCTAGCTGA ATTCAGGCCT CATGAGAGTG AAGGGGATCA GGAGGAATTA TCA 53
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: P-MN(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
CATCTGATGC ACAAAATAGA GTGGTGGTTG 30
-155-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: P-Seq(2)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
CTGTGGGTAC ACAGGCTTGT GTGGCCC 27
(2) INFORMATION FOR SEQ ID NO: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 NO:51:
CAATTITrCT GTAGCACTAC AGATC 25
(2) INFORMATION FOR SEQ ID NO: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) IMiMEDIATE SOURCE:
(B) CLONE: o-542
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
CGATTACGTA GTTAACGCGG CCGCGGCCTA GCCGGCCATA AAAAT 45
-156-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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: o-544
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
CTAGATI'ITT ATGGCCGGCT AGGCCGCGGC CGCGTTAACT ACGTAAT 47
(2) INFORMATION FOR SEQ ID NO: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: o-541
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
CZTIITCTGC GGCCGCGGAT ATGGCCCGGT CCGGTTAACT ACGTAGACGT 50
(2) INFORMATION FOR SEQ ID NO: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: o-543
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
CTACGTAGTT AACCGGACCG GGCCATATAG GCCGCGGCCG CAGAAAAAGC ATG 53
-157-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 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-selPI
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
CGATAAAAAT TGAAATTTTA TTTITITTTT TTGGAATATA AATAAGGCCT C 51
(2) INFORMATION FOR SEQ ID NO:57:
(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: o-selPII
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
CATGGAGGCC TTATTTATAT TCCAAAAAAA AAAAATAAAA TTTCAATTTT TAT 53
(2) INFORMATION FOR SEQ ID NO: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: o-830
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
TCGACTTTZT ATCA 14
-158-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 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-857
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
TATGATAAAA AC 12
(2) INFORMATION FOR SEQ ID NO: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 NO:60:
GAGCAGAAGA CAGTGGCCAT GGCCGTGAAG GGGATCAGGA 40
(2) INFORMATION FOR SEQ ID NO: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 NO:61:
CATAAACTGA TTATATCCTC ATGCATCTGT 30
-159-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4145 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: pS2gpt-84.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACA'IT 60
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 120
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTITT'I'I' GCGGCATT'I'I' 180
GCCTI'CCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 240
TGGGTGCACG AGTGGGZTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 300
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 420
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 480
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC ZTACZTCTGA 540
CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTI'ITGCA CAACATGGGG GATCATGTAA 600
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 660
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 720
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGZT GCAGGACCAC 780
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 840
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 900
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 960
TAGGTGCCTC ACTGATTAAG CATI'GGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 1020
AGATTGATTT AAAACTTCAT TTZTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 1080
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGZTCCA CTGAGCGTCA GACCCCGTAG 1140
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTI'ITCTGCG CGTAATCTGC TGCTTGCAAA 1200
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 1320
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 1380
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGZTCG TGCACACAGC 1500
-160-
CA 02558864 1992-08-25
Inw-
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 1560
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTZTAT AGTCCTGTCG 1680
GGTZTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTZTTGC TGGCCTTTTG 1800
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 2100
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGAZTACG 2160
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220
GCCGCTCTAG CCCGGGCTAG AACTAGTGGA TCCCCCAAAG CGGGGITTGA ACAGGGTTTC 2280
GCTCAGGTTT GCCTGTGTCA TGGATGCAGC CTCCAGAATA CTTACTGGAA ACTAZTGTAA 2340
CCCGCCTGAA GTTAAAAFIGA ACAACGCCCG GCAGTGCCAG GCGTTGAAAA GATTAGCGAC 2400
CGGAGATTGG CGGGACGAAT ACGACGCCCA TATCCCACGG CTGTTCAATC CAGGTATCTT 2460
GCGGGATATC AACAACATAG TCATCAACCA GCGGACGACC AGCCGGTTTT GCGAAGATGG 2520
TGACAAAGTG CGCTTTTGGA TACATTTCAC GAATCGCAAC CGCAGTACCA CCGGTATCCA 2580
CCAGGTCATC AATAACGATG AAGCCZTCGC CATCGCCTTC TGCGCGTTTC AGCACTTTAA 2640
GCTCGCGCTG GTTGTCGTGA TCGTAGCTGG AAATACAAAC GGTATCGACA TGACGAATAC 2700
CCAGZTCACG CGCCAGTAAC GCACCCGGTA CCAGACCGCC ACGGCTTACG GCAATAATGC 2760
CTTTCCATTG TTCAGAAGGC ATCAGTCGGC TTGCGAGTTT ACGTGCATGG ATCTGCAACA 2820
TGTCCCAGGT GACGATGTAT TTTTCGCTCA TGTGAAGTGT CCCAGCCTGT TTATCTACGG 2880
CTTAAAAAGT GTTCGAGGGG AAAATAGGTT GCGCGAGATT ATAGAGATCT GGCGCACTAA 2940
AAACCAGTAT TTCACATGAG TCCGCGTCTT TZTACGCACT GCCTCTCCCT GACGCGGGAT 3000
AAAGTGGTAT TCTCAAACAT ATCTCGCAAG CCTGTCTTGT GTCCAAGCTT GGGGATCATC 3060
CGTCACTGTT CTTTATGATT CTACTTCCTT ACCGTGCAAT AAATTAGAAT ATATTTTCTA 3120
CTTTTACGAG AAATTAATTA TTGTATTTAT TATTTATGGG TGAAAAACTT ACTATAAAAA 3180
GCGGGTGGGT TTGGAATTAG TGATCAGZTT ATGTATATCG CAACTACCGG CATATGGCTA 3240
TTCGACATCG AGAACAZTAC CCACATGATA AGAGATTGTA TCAGTTTCGT AGTCZTGAGT 3300
ATTGGTATTA CTATATAGTA TATAGATGTC GAGGCGGTAC CCTTAAGTTG GGCTGCAGAA 3360
GCTTTTTTIT TrI7'I'I'IT1T TTGGCATATA AATCGTTAAC GAATTCCATG GCCCGGGAAG 3420
GCCTCGGACC GGGCCCGGCC ATATAGGCCA GCGATACCGT CGCGGCCGCG ACCTCGAGGG 3480
GGGGCCCGGT ACCCAATTCG CCCTATAGTG AGTCGTATTA CGCGCGCTCA CTGGCCGTCG 3540
-161-
CA 02558864 1992-08-25
TTTT ACAACG TCGTGACTGG GAAAACCCTG GCGTTACCCA ACTTAATCGC CTTGCAGCAC 3600
ATCCCCCTIT CGCCAGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC 3660
AGTTGCGCAG CCTGAATGGC GAATGGAAAT TGTAAGCGTT AATATTTT GT TAAAATTCGC 3720
GTTAAATTTT TGZTAAATCA GCTCATIZTP TAACCAATAG GCCGAAATCG GCAAAATCCC 3780
TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG 3840
TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA 3900
TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTRITZTG GGGTCGAGGT GCCGTAAAGC 3960
ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTT AGAGCT TGACGGGGAA AGCCGGCGAA 4020
CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT 4080
AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC 4140
GTCAG 4145
(2) INFORMATION FOR SEQ ID NO: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: SEQ ID NO:63:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TZTATTTITC TAAATACAZT 60
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 120
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTITITT GCGGCATTTT 180
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 240
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGZT 300
TTCGCCCCGA AGAACGTTIT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 420
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 480
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAAC.AC TGCGGCCAAC TTACTTCTGA 540
CAACGATCGG AGGACCGAAG GAGCTAACCG CTITITTGCA CAACATGGGG GATCATGTAA 600
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 660
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 720
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 780
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 840
-162-
CA 02558864 1992-08-25
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 900
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 960
TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 1020
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 1080
ATCTCATGAC CAAAATCCCT TAACGTGAGT T'ITCGTTCCA CTGAGCGTCA GACCCCGTAG 1140
AAAAGATCAA AGGATCZTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CC.AACTCTTI' 1260
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 1320
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 1380
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 1560
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTZTAT AGTCCTGTCG 1680
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCC'ITITGC TGGCCTTTTG 1800
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 2100
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG 2160
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220
GCCGCTCTAG CCCGGGCTAG AACTAGTGGA TCCCCCAAAG CGGGGT7TGA ACAGGGTTTC 2280
GCTCAGGTTT GCCTGTGTCA TGGATGCAGC CTCCAGAATA CTTACTGGAA ACTATTGTAA 2340
CCCGCCTGAA GTTAAAAAGA ACAACGCCCG GCAGTGCCAG GCGTTGAAAA GATTAGCGAC 2400
CGGAGATTGG CGGGACGAAT ACGACGCCCA TATCCCACGG CTGTTCAATC CAGGTATCTT 2460
GCGGGATATC AACAACATAG TCATCAACCA GCGGACGACC AGCCGGTTTT GCGAAGATGG 2520
TGACAAAGTG CGCZZTTGGA TACATTTCAC GAATCGCAAC CGCAGTACCA CCGGTATCCA 2580
CCAGGTCATC AATAACGATG AAGCCTTCGC CATCGCCTTC TGCGCGTTTC AGCACTTTAA 2640
GCTCGCGCTG GTTGTCGTGA TCGTAGCTGG AAATACAAAC GGTATCGACA TGACGAATAC 2700
CCAGTTCACG CGCCAGTAAC GCACCCGGTA CCAGACCGCC ACGGCTTACG GCAATAATGC 2760
CTTTCCATTG TTCAGAAGGC ATCAGTCGGC TTGCGAGTTT ACGTGCATGG ATCTGCAACA 2820
TGTCCCAGGT GACGATGTAT TTTTCGCTCA TGTGAAGTGT CCCAGCCTGT TTATCTACGG 2880
-163-
CA 02558864 1992-08-25
CZTAAAAAGT GTTCGAGGGG AAAATAGGTT GCGCGAGATT ATAGAGATCT GGCGCACTAA 2940
AAACCAGTAT TTCACATGAG TCCGCGTCTT TTTACGCACT GCCTCTCCCT GACGCGGGAT 3000
AAAGTGGTAT TCTCAAACAT ATCTCGCAAG CCTGTCTTGT GTCCAAGCTT GGGGATCATC 3060
CGTCACTGTT CTZTATGATT CTACTTCCTT ACCGTGCAAT AAATTAGAAT ATATITTCTA 3120
CTT'I'TACGAG AAATTAATTA TTGTATTTAT TATTT ATGGG TGAAAAACTT ACTATAAAAA 3180
GCGGGTGGGT TTGGAATTAG TGATCAGTTT ATGTATATCG CAACTACCGG CATATGGCTA 3240
TTCGACATCG AGAACATTAC CCACATGATA AGAGATTGTA TCAGTTT CGT AGTCTTGAGT 3300
ATTGGTATTA CTATATAGTA TATAGATGTC GAGGCGGTAC CCTTAAGTTG GGCTGCAGTT 3360
GZTAGAGCTT GGTATAGCGG ACAACTAAGT AATTGTAAAG AAGAAAACGA AACTATCAAA 3420
ACCGTTTATG AAATGATAGA AAAAAGAATA TAAATAATCC TGTATTZTAG TTTAAGTAAC 3480
AGTAAAATAA TGAGTAGAAA ATACTATTTP TTATAGCCTA TAAATCGTTA ACGAATTCCA 3540
TGGCCCGGGA AGGCCTCGGA CCGGGCCCGG CCATATAGGC CAGCGATACC GTCGCGGCCG 3600
CGACCTCGAG GGGGGGCCCG GTACCCAATT CGCCCTATAG TGAGTCGTAT TACGCGCGCT 3660
CACTGGCCGT CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC CAACTTAATC 3720
GCCTTGCAGC ACATCCCCCT TTCGCCAGCT GGCGTAATAG CGAAGAGGCC CGCACCGATC 3780
GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGAA ATTGTAAGCG TTAATATTTT 3840
GTTAAAATTC GCGTTAAATT TTTGZTAAAT CAGCTCAT'IT TTTAACCAAT AGGCCGAAAT 3900
CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG TTGTTCCAGT 3960
TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC GTCAAAGGGC GAFIAAACCGT 4020
CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA TCAAGTTTIT TGGGGTCGAG 4080
GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG 4140
AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG GCGCTAGGGC 4200
GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA CCCGCCGCGC TTAATGCGCC 4260
GCTACAGGGC GCGTCAG 4277
(2) INFORMATION FOR SEQ ID NO: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
(vii) IMMEDIATE SOURCE:
(B) CLONE: pTZ-L2
-164-
CA 02558864 1992-08-25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTTTT 60
TCTGCGGCCG CGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCG AGGATTTCGC 120
GTGGGTCAAT GCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATAC CAGTGAGGGA 180
TGGTTTTACC ATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTA AAGCGGCGGA 240
CTAGCGTCGA GGTTTCAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTI'PCG CTCAGGTTTG 300
CCTGTGTCAT GGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAAC CCGCCTGAAG 360
TTAAAAAGAA CAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATPAGCGACC GGAGAZTGGC 420
GGGACGAATA CGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG CGGGATATCA 480
ACAACATAGT CATCAACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGT GACAAAGTGC 540
GCTT'ITGGAT ACATZTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC CAGGTCATCA 600
ATAACGATGA AGCCTTCGCC ATCGCC2TCT GCGCGTTTCA GCACTZTAAG CTCGCGCTGG 660
TTGTCGTGAT CGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC CAGTTCACGC 720
GCCAGTAACG CACCCGGTAC CAGACCGCCA CGGCTTACGG CAATAATGCC TTTCCATTGT 780
TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT GTCCCAGGTG 840
ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC TTAAAAAGTG 900
ZTCGAGGGGA AAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA AACCAGTAZT 960
TCACATGAGT CCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA AAGTGGTATT 1020
CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC GTCACTGTTC 1080
TTTATGAZTC TACTTCCZTA CCGTGCAATA AATTAGAATA TATTITCTAC TTTTACGAGA 1140
AATTAATTAT TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG CGGGTGGGTT 1200
TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGGCTAT TCGACATCGA 1260
GAACATTACC CACATGATAA GAGATTGTAT CAGTTTCGTA GTCTTGAGTA TTGGTATTAC 1320
TATATAGTAT ATNNNNNNGG TAACNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1380
NNNNNNNAGA TCTCGATCCG GATATAGTTC CTCCZTTCAG CAAAAAACCC CTCAAGACCC 1440
GTTI'AGAGGC CCCAAGGGGT TATGCTAGTT ATTGCTCANN NNNNNNNNGT CGACTTAATT 1500
AATTAGGCCT CTCGAGCTGC AGGGATCCAC TAGTGAGCTC CCCGGGGAAT TCCCATGGTA 1560
TTATCGTGTT TTTCAAAGGA AAAAAACGTC CCGTGGTTCG GGGGGCTCTN NNINNNNNNNN 1620
NNNNNNNNNN NN NNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1680
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1740
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1800
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1860
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1920
NNNNNNNNNN Nf~OJNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N2tl~1NNNNNNN 1980
-165-
CA 02558864 1992-08-25
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2040
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2100
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NCCGCTAGAG GGAAACCGTT GTGGTCTCCC 2160
TATAGTGAGT CGTATTAATT TCGCGGGATC GATCGATTAC GTAGTTAACG CGGCCGCGGC 2220
CTAGCCGGCC ATAAAAATCT AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT 2280
CCCAACAGTT GCGCAGCCTG AATGGCGAAT GGGAAATTGT AAACGTTAAT ATTT'TGTTAA 2340
AATTCGCGTT AAATTTTTGT TAAATCAGCT CAT'I"ITITAA CCAATAGGCC GAAATCGGCA 2400
AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGZT CCAGTTTGGA 2460
ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA ACCGTCTATC 2520
AGGGCGATGG CCCACTACGT GAACCATCAC CCTAATCAAG TPITI'I'GGGG TCGAGGTGCC 2580
GTAAAGCACT AAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA CGGGGAAAGC 2640
CGGCGAACGT GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG 2700
CAAGTGTAGC GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT GCGCCGCTAC 2760
AGGGCGCGTC AGGTGGCACT TITCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT 2820
TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT 2880
AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT AZTCCCTTTt' 2940
TTGCGGCATT TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG 3000
CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA 3060
TCCTTGAGAG TTTTCGCCCC GAAGAACGZT TTCCAATGAT GAGCACTTZT AAAGTTCTGC 3120
TATGTGGCGC GGTATTATCC CGTGTTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC 3180
ACTATTCTCA GAATGACTI'G GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG 3240
GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA 3300
ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTI'ITG CACAACATGG 3360
GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG 3420
ACGAGCGTGA CACCACGATG CCTGCAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG 3480
GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG 3540
TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATI'GCT GATAAATCTG 3600
GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT 3660
CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC 3720
AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT 3780
CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA 3840
TCCTITITGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 3900
CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC ZTITTTTCTG CGCGTAATCT 3960
GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC 4020
-166-
CA 02558864 1992-08-25
TACCAACTCT TTTT CCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC 4080
TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC 4140
TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 4200
GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT 4260
CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG 4320
AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 4380
GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTZT 4440
ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 4500
GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTT ACGGTTC CTGGCCTTTT 4560
GCTGGCCTTT TGCTCACATG TTCTTT CCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 4620
TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 4680
CAGTGAGCGA GGAAGCGGAA G 4701
(2) INFORMATION FOR SEQ ID NO: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
(vii) IMMEDIATE SOURCE:
(B) CLONE: pselP-gpt-L2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATI'CATTAA TGCAGCT'TiT 60
TCTGCGGCCG CGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCG AGGATTT CGC 120
GTGGGTCAAT GCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATAC CAGTGAGGGA 180
TGGT'ITTACC ATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTA AAGCGGCGGA 240
CTAGCGTCGA GGTTT CAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTTT CG CTCAGGTTTG 300
CCTGTGTCAT GGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAAC CCGCCTGAAG 360
TTAAAAAGAA CAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATTAGCGACC GGAGATTGGC 420
GGGACGAATA CGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG CGGGATATCA 480
ACAACATAGT CATCAACCAG CGGACGACCA GCCGGTTZTG CGAAGATGGT GACAAAGTGC 540
GCTTTTGGAT ACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC CAGGTCATCA 600
ATAACGATGA AGCCTTCGCC ATCGCCTTCT GCGCGTTT CA GCACTTT AAG CTCGCGCTGG 660
TTGTCGTGAT CGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC CAGTTCACGC 720
GCCAGTAACG CACCCGGTAC CAGACCGCCA CGGCTTACGG CAATAATGCC TTTCCATTGT 780
-167-
CA 02558864 1992-08-25
TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT GTCCCAGGTG 840
ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC TTAAAAAGTG 900
TTCGAGGGGA AAATAGGTTG CGCGAGAZTA TAGAGATCTG GCGCACTAAA AACCAGTATT 960
TCACATGAGT CCGCGTCTZT TTACGCACTG CCTCTCCCTG ACGCGGGATA AAGTGGTATT 1020
CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC GTCACTGTTC 1080
TTTATGATTC TACTTCCTTA CCGTGCAATA AATTAGAATA TATTZTCTAC TT'ITACGAGA 1140
AATTAATTAT TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG CGGGTGGGTT 1200
TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGATAAA AAGTCGACTT 1260
AATTAATTAG GCCTCTCGAG CTGCAGGGAT CCACTAGTGA GCTCCCCGGG GAATTCCCAT 1320
GGAGGCCTTA TTTATATTCC AAAAAAAl114A AATAAAATTT CAATT7TTAT CGATTACGTA 1380
GTTAACGCGG CCGCGGCCTA GCCGGCCATA AAAATCTAGC TGGCGTAATA GCGAAGAGGC 1440
CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT GGCGAATGGG AAATTGTAAA 1500
CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTPAA ATCAGCTCAT TTTTTAACCA 1560
ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA TAGACCGAGA TAGGGZTGAG 1620
TGTTGTTCCA GTZ'TGGAACA AGAGTCCACT ATTAAAGAAC GTGGACTCCA ACGTCAAAGG 1680
GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCT AATCAAGTTT 1740
TZTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCC CCCGATTTAG 1800
AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA GGGAAGAAAG CGAAAGGAGC 1860
GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT CACGCTGCGC GTAACCACCA CACCCGCCGC 1920
GCTTAATGCG CCGCTACAGG GCGCGTCAGG TGGCACTTTT CGGGGAAATG TGCGCGGAAC 1980
CCCTATTTGT TTAZZTITCT AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC 2040
CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC ATTTCCGTGT 2100
CGCCCTTATT CCCTTTPPTG CGGCATTIZ'G CCTTCCTGTT TTTGCTCACC CAGAAACGCT 2160
GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA TCGAACTGGA 2220
TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTZTC CAATGATGAG 2280
CACTTZTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT GTTGACGCCG GGCAAGAGCA 2340
ACTCGGTCGC CGCATACACT ATTCTC.AGAA TGACTTGGTT GAGTACTCAC CAGTCACAGA 2400
AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA TAACCATGAG 2460
TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG AGCTAACCGC 2520
TITITTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC CGGAGCTGAA 2580
TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GCAGCAATGG CAACAACGTT 2640
GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT TAATAGACTG 2700
GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG CTGGCTGGTT 2760
TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG CAGCACTGGG 2820
-168-
CA 02558864 1992-08-25
~
"NW
GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC AGGCAACTAT 2880
GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC ATTGGTAACT 2940
GTCAGACCAA GTTI'ACTCAT ATATACTTTA GATTGATZTA AAACTTCATT TTTAATTTAA 3000
AAGGATCTAG GTGAAGATCC T'ITITGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT 3060
TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTZT 3120
TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG 3180
TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA 3240
GATACCAIaAT ACTGTCCTTC TAGTGTAGCC GTAGZTAGGC CACCACTTCA AGAACTCTGT 3300
AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA 3360
TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC 3420
GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT 3480
GAGATACCTA CAGCGTGAGC ATTGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA 3540
CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC ZTCCAGGGGG 3600
AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTT CGCCAC CTCTGACTTG AGCGTCGATT 3660
TZTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT 3720
ACGGTI'CCTG GCCTTITGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA 3780
1TCTGTGGAT AACCGTATTA CCGCCTTT GA GTGAGCTGAT ACCGCTCGCC GCAGCCGAAC 3840
GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAG 3878
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6474 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: pse1P-gp160MN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTTTT 60
TCTGCGGCCG CGGCCTATAT GGCCCGGTCC GGTTAACTAC GTAGACGTCG AGGATTTCGC 120
GTGGGTCAAT GCCGCGCCAG ATCCACATCA GACGGTTAAT CATGCGATAC CAGTGAGGGA 180
TGGTI'ZTACC ATCAAGGGCC GACTGCACAG GCGGTTGTGC GCCGTGATTA AAGCGGCGGA 240
CTAGCGTCGA GGTTTCAGGA TGTTTAAAGC GGGGTTTGAA CAGGGTTTCG CTCAGGTZTG 300
CCTGTGTCAT GGATGCAGCC TCCAGAATAC TTACTGGAAA CTATTGTAAC CCGCCTGAAG 360
TTAAAAAGAA CAACGCCCGG CAGTGCCAGG CGTTGAAAAG ATTAGCGACC GGAGATTGGC 420
-169-
CA 02558864 1992-08-25
i
GGGACGAATA CGACGCCCAT ATCCCACGGC TGTTCAATCC AGGTATCTTG CGGGATATCA 480.
ACAACATAGT CATCAACCAG CGGACGACCA GCCGGTTTTG CGAAGATGGT GAC.AAAGTGC 540
GCTITTGGAT ACATTTCACG AATCGCAACC GCAGTACCAC CGGTATCCAC CAGGTCATCA 600
ATAACGATGA AGCCTTCGCC ATCGCCTTCT GCGCGTTTCA GCACTTTAAG CTCGCGCTGG 660
TTGTCGTGAT CGTAGCTGGA AATACAAACG GTATCGACAT GACGAATACC CAGTTCACGC 720
GCCAGTAACG CACCCGGTAC..;:CAGACCGCCA CGGCTTACGG CAATAATGCC TITCCATTGT 780
TCAGAAGGCA TCAGTCGGCT TGCGAGTTTA CGTGCATGGA TCTGCAACAT GTCCCAGGTG 840
ACGATGTATT TTTCGCTCAT GTGAAGTGTC CCAGCCTGTT TATCTACGGC TTAAAAAGTG 900
TTCGAGGGGA AAATAGGTTG CGCGAGATTA TAGAGATCTG GCGCACTAAA AACCAGTATT 960
TCACATGAGT CCGCGTCTTT TTACGCACTG CCTCTCCCTG ACGCGGGATA AAGTGGTATT 1020
CTCAAACATA TCTCGCAAGC CTGTCTTGTG TCCAAGCTTG GGGATCATCC GTCACTGTTC 1080
TZTATGATPC TACTTCCTTA CCGTGCAATA AAZTAGAATA TATTTTCTAC TTTTACGAGA 1140
AATTAATTAT TGTATZTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG CGGGTGGGTT 1200
TGGAATTAGT GATCAGTTTA TGTATATCGC AACTACCGGC ATATGATAAA AAGTCGACTT 1260
AATTAATTAG GCTGGTTCAG CTCGTCTCAT TCTTTCCCTT ACAGTAGGCC ATCCAGTCAC 1320
ACGTTTTGAC CATTTGCCAC CCATCTTATA GCAAAGCCCT TTCCAAGCCC TGTCTTATTC 1380
TTGTAGGTAT GTGGAGAATA GCTCTACCAG CTCTTTGCAG TACTTCTATA ACCCTATCTG 1440
TCCCCTCAGC TACTGCTATA GCTGTGGCAT TAAGCAAGCT AACAGCACTA CTCTTTAGTT 1500
CCTGACTCCA ATACTGTAGG AGATTCCACC AATATTTGAG GACTTCCCAC CCCCTGCGTC 1560
CCAGAAGTTC CACAATCCTC GCTGCAATCA AGAGTAAGTC TCTGTGGTGG TAGCTGAAGA 1620
GGAACAGGCT CCGCAGGTCG ACCCAGATAA TTGCTAAGAA TCCATGCACT AATCGACCGG 1680
ATGTGTCTCT GTCTCTCTCT CCACCTTCTT CTTCGATTCC TTCGGGCCTG TCGGGTCCCC 1740
TCGGAACTGG GGGGCGGGTC TGCAACGACA ATGGTGAGTA TCCCTGCCTA ACTCTATTCA 1800
CTATAGAAAG TACAGCAAAA ACTATTCTTA AACCTACCAA GCCTCCTACT ATCATTATGA 1860
ATATTTTTAT ATACCACAGC CAATTTGTTA TGTCAAACCA ATTCCACAAA CTTGCCCAZT 1920
TATCCAATTC CAATAATTCT TGTTCATTCT TZTCTTGTTG GGTTTGCGAT TTTTCTAGTA 1980
ATGAGTATAT TAAGCTTGTG TAATTGTCAA TTTCTCTTTC CCACTGCATC CAGGTCATGT 2040
TATTCCAAAT ATCATCCAGA GATTTATTAC TCCAACTAGC ATTCCAAGGC ACAGTAGTGG 2100
TGC.AAATGAG TTZTCCAGAG CAACCCCAAA ACCCCAGGAG CTGTTGATCC T'ITAGGTATC 2160
TZTCCACAGC CAGGACTCTT GCCTGGAGCT GCTTGATGCC CCAGACTGTG AGTTGCAACA 2220
TATGCTGTTG CGCCTCAATG GCCCTCAGCA AATTGTTCTG CTGTTGCACT ATACCAGACA 2280
ATAATAGTCT GGCCTGTACC GTCAGCGTCA CTGACGCTGC GCCCATAGTG CTTCCTGCTG 2340
CTCCTAAGAA CCCAAGGAAC AGAGCTCCTA TCGCTGCTCT TTTTTCTCTC TGCACCACTC 2400
TTCTCTTTGC CTTGGTGGGT GCTACTCCTA ATGGTTCAAT TGTTACTACT TTATATTTAT 2460
-170-
CA 02558864 1992-08-25
.~ _
ATAATTCACT TCTCCAATTG TCCCTCATAT CTCCTCCTCC AGGTCTGAAG ATCTCGGTGT 2520
CGTTCGTGTC CGTGTCCTTA CCACCATCTC TTGTTAATAG TAGCCCTGTA ATATTTGATG 2580
AACATCTAAT TTGTCCTTCA ATGGGAGGGG CATACATTGC TTTI'CCTACT TCCTGCCACA 2640
TGTTTATAAT TTGTTTTATT ZTGCATTGAA GTGTGATATT GZTAT'ITGAC CCTGTAGTAT 2700
TATTCCAAGT ATTATTACCA TTCC.AAGTAC TAZTAAACAG TGGTGATGTA TTACAGTAGA 2760
AAAATTCCCC TCCACAA'ITA AAACTGTGCA TTACAATTTC TGGGTCCCCT CCTGAGGATT 2820
GATTAAAGAC TATTGTTTTA TTCTTAAATT GTTCTTTTAA TTTGCTAACT ATCTGTCTTA 2880
AAGTGTCATT CCATTTTGCT CTACTAATGT TACAATGTGC TTGTCTTATA GTTCCTATTA 2940
TATTIZTTGT TGTATAAAAT GCTCTCCCTG GTCCTATATG TATCCTTTZT CTTITATTGT 3000
AGTTGGGTCT TGTACAATTA ATTTGTACAG ATTCATTCAG ATGTACTATG ATGGTTTTAG 3060
CATTATCAGT GAAATTCTCA GATCTAATTA CTACCTCTTC TTCTGCTAGA CTGCCATZTA 3120
ACAGCAGTTG AGTTGATACT ACTGGCCTAA TTCCATGTGT ACATTGTACT GTGCTGACAT 3180
TTTTACATGA TCCTTTTCCA CTGAACTTTT TATCGTTACA TTTTAGAATC GCAAAACCAG 3240
CCGGGGCACA ATAGTGTATG GGAATTGGCT CAAAGGATAT CTZ'TGGACAA GCTTGTGTAA 3300
TGACTGAGGT ATTACAACTT ATCAACCTAT AGCTGGTACT ATCATTATCT ATTGATACTA 3360
TATCAAGTTT ATAAAGAAGT GCATATTCIT TCTGCATCTT ATCTCTTATG CTTGTGGTGA 3420
TATTGAAAGA GCAGTTT'TTC ATTTCTCCTC CCTTTATTGT TCCCTCGCTA TTACTATTGT 3480
TATTAGCAGT ACTATTATTG GTATTAGTAG TATTCCTCAA ATCAGTGCAA TTTAAAGTAA 3540
CACAGAGTGG GGTTAATTTT ACACATGGCT TTAGGCTTTG ATCCCATAAA CTGATTATAT 3600
CCTCATGCAT CTGZTCTACC ATGTTATTIT TCCACATGTT AAAATTTTCT GTCACATTTA 3660
CCAATTCTAC TTCTTGTGGG TTGGGGTCTG TGGGTACACA GGCTTGTGTG GCCCAAACAT 3720
TATGTACCTC TGTATCATAT GCTTTAGCAT CTGATGCACA AAATAGAGTG GTGGTTGCTT 3780
CTTTCCACAC AGGTACCCCA TAATAGACTG TGACCCACAA ZZTITCTGTA GCACTACAGA 3840
TCATTAATAA CCCAAGGAGC ATCGTGCCCC ATCCCCACCA GTGCTGATAA TTCCTCCTGA 3900
TCCCCTTCAC GGCCATGGAG GCCTTATTTA TATTCCAAAA AAAAAAAATA AAATTTCAAT 3960
TTTTATCGAT TACGTAGTTA ACGCGGCCGC GGCCTAGCCG GCCATAAAAA TCTAGCTGGC 4020
GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG 4080
AATGGGAAAT TGTAAACGZT AATATTTTGT TAAAAZTCGC GTTAAATTTT TGTTAAATCA 4140
GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA 4200
CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG 4260
ACTCC.AACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT 4320
CACCCTAATC AAGZZTI'TTG GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG 4380
GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA 4440
AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA 4500
-171-
CA 02558864 1992-08-25
CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTCAGGTGGC ACTTTTCGGG 4560
GAAATGTGCG CGGAACCCCT A'ITTGTT'I'AT TTTTCTAAAT ACATTCAAAT ATGTATCCGC 4620
TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA 4680
TTCAACATTT CCGTGTCGCC CTTATTCCCT TZTITGCGGC ATTZTGCCTT CCTGTTTTTG 4740
CTCACCCAGA AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG 4800
GTTACATCGA ACTGGATCTCAACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC 4860
GTTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGZTG 4920
ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT 4980
ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG 5040
CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC . 5100
CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT 5160
GGGAACCGGA GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGCAG 5220
CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC 5280
AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC 5340
TTCCGGCTGG CTGGTZTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA 5400
TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG 5460
GGAGTCAGGC AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA 5520
TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATI' GATTTAAAAC 5580
TTCATTTZTA ATZTAAAAGG ATCTAGGTGA AGATCCTT'IT TGATAATCTC ATGACCAAAA 5640
TCCCTTAACG TGAGTT'TTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT 5700
CTTCTTGAGA TCCZTI'IT'IT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC 5760
TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG 5820
GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC 5880
ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG 5940
CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG 6000
ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA 6060
CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCATTG AGAAAGCGCC ACGCTTCCCG 6120
AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA 6180
GGGAGCTTCC AGGGGGAAAC GCC'TGGTATC TTTATAGTCC TGTCGGGTZT CGCCACCTCT 6240
GACTTGAGCG TCGATrIZTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA 6300
GCAACGCGGC CTTTZTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCT'I'TC 6360
CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG 6420
CTCGCCGCAG CCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAG 6474
-172-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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
(vi i ) IMMEDIATE SOURCE :
(B) CLONE: pN2-gpta ProtS
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 60
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 120
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCT'ITIIT GCGGCATTTT 180
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 240
TGGGTGC.ACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 300
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360
TATTATCCCG TA'PTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 420
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 480
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 540
CAACGATCGG AGGACCGAAG GAGCTAACCG CTPI"ITI'GCA CAACATGGGG GATCATGTAA 600
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 660
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 720
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 780
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 840
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 900
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 960
TAGGTGCCTC.ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 1020
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC C'ITI'T'I'GATA 1080
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140
AAAAGATCAA AGGATCTI'CT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 1320
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 1380
TCCTG'ITACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500
-173-
CA 02558864 1992-08-25
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 1560
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTZTGTGATG CTCGTCAGGG GGGCGGAGCC 1740
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG 1800
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATPCTGTGGA TAACCGTATT ACCGCCTTTG 1860
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980
GCAGCTGGCA CGACAGGTIT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040
TGAGTTAGCT CACTCAZTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 2100
TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG 2160
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220
GCCGCGTCGA CAGAAAAATT AATTAATTAT GGCCTCTCGA GCTGCAGCTG CCAAGAAGAA 2280
GATTCCTGTG CTGCTCTCAG GAAAATATGT CCCACTTGTT TTCTAATTCA ATAAAGATAC 2340
TGGTTTAAAT GTGAAGCCAC ACAAGAGAAA GATGAAGCCA AAGCTGGTCC CCCTGAGGAA 2400
TTGTTZTGAA ATAAGGCATT AGGACCCTCC AZTCAAZTCA TATTTAATAG ACCACCATCT 2460
CZTCTGCCTT CATCAGGAAA AAAACAAAAA CATAAACAAA ATAGTATCTG CCTATGATTA 2520
ATAGTATZTA ATTACACGCA CTTTTGTTTG AGTTTACTTC CTTGCTTTCT GAAAAAAACA 2580
TAGGTATTTA GACACTAGTT CATGATGATA AAATTAAAAA TTTAGTTTTA CAAACAAAAA 2640
TTGAAACTGT CATTTGTAGG AAAAAAAZTC AAATTTAAAA TTGTTATTZT TCACTATTCT 2700
TAGATAGCAA GAGAAGTAAG AATZTCTZTA CTGTGATZTA TATCACAACA GAATTITITT 2760
CCTTGACAAA GGACCTTTTA AAAATCCCAG GAAAGGACCA CAAAATAATC AAAGACTGCA 2820
CATTGTAAAT AAAACCCTTC AGCTGTTATT GAAACATAAG TATAATTACA CACAAGGAAA 2880
AGGTATTATA AGCAGAGAAA AGATGCCTTA AGAATTCTTT GTCTTTITCC AAACTGATGG 2940
ACATGAGTGA GCTCTAATAT CATTATGTZT AGAAATGGCT TCATCCAGAT CCAACTGTAC 3000
ACCATTAATA TTCACTTCCA TGCAGCCATT ATAAAAGGCA TTCACTGGTG TGGCACTGAA 3060
TGGAACATCT GGAAGGCCAC CCAGGTATGT GGCCACTTTT GCTTTCATTG CTTTGTCCAA 3120
GACGGCAAGT TGTCTTTGAA GGTCTTCATG GGAGATGGTT TCTATT'ITAA GTGGTGTCGA 3180
CAACTCCAGA TTGTZTCTGT TGACTCTAAA TTCCAGATGA GATTGTTGAT CGGAACATAG 3240
ACTTAGGGCC TGTATCCGAT ATATTACAGT ATTZTCAACA GATAACAGAA TATCCTGTGA 3300
TTTTI'CAGAG GTGGAGTCCA CCAAGGACAC AGCAAAGGGC ACTGTGTTGT TACCAGAAAC 3360
CAAGGCAAGC ATAACACCAG TGCCCGTGGA TGGACGAATA TTCAAGGTCA CATTTACATG 3420
CCAACCCTCA GCACTGGATA CATTATTATA ATCTATGTGA AATTGAGCAA TTCCAGAACC 3480
AGGATAGTAG GAGCCCTTCT CCACAGTAAC CAGGCAATGC TTATTZTGTT TTTCTTGAAT 3540
-174-
CA 02558864 1992-08-25
AATZTCCTTT ATTCCAGAAG CTCCTTGCTT CATCAAATTC CAGCZTCGTA TACATCCATC 3600
TAGACGAGGG TTAATCGGTT TAATGAGTTC ACTTTCCACT TTCCGAGGGA ATCCTGCAAA 3660
GTATACTT'TG GTTTCCAGCA ATCCATTTTC CGGCTTAAAA AGGGGTCCAG GTTTATTTAT 3720
ATCCATCACA GC'ITCTZTAG CTATTTI'AAT GCTAATACTA TGTTCTAATT CTTCCACAGA 3780
CACCATATTC CATAGACCAT TATTAATAAC ATCACCTCCA GTTGTGATTT TGGATGTATG 3840
TTCATTCTTA AGCTGAACTT CAATCTTTCC ACCACGAAGT GCAATCAGGA GCCACGCTGA 3900
GTGATCGATA GATTCTGCGT ACAGTATCAC GCCTTCTGAA TCATATGTCC GGAAATCAAA 3960
TTCTGCTGAA AATCTGCTGA TZTCTGGCAA ACGAAATTTT AAATATAAAA CAACCCCTGC 4020
AAACTGCTCC GCCAAGTAAA GTAATTCATA CTTTGTGTCA AGGTI'CAAGG GAAGGCACAC 4080
TGAAACAACC TCACAACTCT TCTGATCTTG GGCAAGTTTG AATCCTTTCT TCCCATCACA 4140
ATAGCAAGTG TAACCTCCAG GGTAATTGAC ACAAAGCTGA GCACACATGT TCTCAGAGCA 4200
TTCATCTATA TCTTCACAAG ACTTTGATTT GAGATTATAT CTGTAGCCTT CGGGGCATTC 4260
ACATTCAAAA TCTCCTGGGA TGTTCTTGCA CACAGCTGTG CCACAAATGC TTGGCTTCAA 4320
AGAGCATTCA TCCACATCTT TACAATCTTT CTTATTTGAA AGCATAACAA AACCATTTZT 4380
ACAGGAACAG TGGTAACTTC CAGGTGTATT ATCACAAA'IT TGACTGCAAC CTCCATTTAT 4440
ATTTGAGGGA TCZTPGCATT CATZTATGTC AAATTCACAC T'I'ITCTCCTT GCCAACCTGG 4500
TTTACAAGTG CAAGTAAAAG AAGCTTTTCC ATCTTTGCAG CTCATATATC CATCTTCATT 4560
GCATGGCAGA GGACTACACT GGTCTGGAAT GGCATTGACA CAGCTTCTTA GGTCAGGATA 4620
AGCATTAGTT GACTGACGTG CAGCAGTGAA TAACCCAGTT TGAAAAGAGC GAAGACAAAC 4680
TAAGTATTTT GGATAAAAAT AATCCGTTTC CGGGTCATTT TCAAAGACCT CCCTGGCTTC 4740
TTCTTTATTG CACAGTTCTT CGATGCAZTC TCTTTCAAGA ZTACCCTGTT TGGTTTCTTC 4800
AAGTAAAGAA TTTGCACGAC GCTTCCTAAC CAGGACTTGT GAAGCCTGTT GCTTTGACAA 4860
AAAGTTTGCC TCTGAGACGG GAAGCACTAG GAGGAGACAC GCCAGCAACG CCCCGCAGCG 4920
CCCACCCAGG ACCCCCATGG AGGCCTTATT TATATTCCAA AAAAAAAAAA TAAAATTTCA 4980
ATTTTTAGAT CCCCCAACTT AAGGGTACCG CCTCGACATC TATATACTAT ATAGTAATAC 5040
CAATACTCAA GACTACGAAA CTGATACAAT CTCTTATCAT GTGGGTAATG TTCTCGATGT 5100
CGAATAGCCA TATGCCGGTA GTTGCGATAT ACATAAACTG ATCACTAATT CCAAACCCAC 5160
CCGCTTTTTA TAGTAAGTTT TTCACCCATA AATAATAAAT ACAATAATTA ATTTCTCGTA 5220
AAAGTAGAAA ATATATTCTA ATTTATTGCA CGGTAAGGAA GTAGAATCAT AAAGAACAGT 5280
GACGGATGAT CCCCAAGCTT GGACACAAGA CAGGCTTGCG AGATATGTTT GAGAATACCA 5340
CTTTATCCCG CGTCAGGGAG AGGCAGTGCG TAAAAAGACG CGGACTCATG TGAAATACTG 5400
GTTTTTAGTG CGCCAGATCT CTATAATCTC GCGCAACCTA TTZTCCCCTC GAACACTTTI' 5460
TAAGCCGTAG ATAAACAGGC TGGGACACTT CACATGAGCG AAAAATACAT CGTCACCTGG 5520
GACATGTTGC AGATCCATGC ACGTAAACTC GCAAGCCGAC TGATGCCZTC TGAACAATGG 5580
-175-
CA 02558864 1992-08-25
AAAGGCATTA ZTGCCGTAAG CCGTGGCGGT CTGGTACCGG GTGCGTTACT GGCGCGTGAA 5640
CTGGGTATTC GTCATGTCGA TACCGTTTGT ATTT CCAGCT ACGATCACGA CAACCAGCGC 5700
GAGCTTAAAG TGCTGAAACG CGCAGAAGGC GATGGCGAAG GCTTCATCGT TATI'GATGAC 5760
CTGGTGGATA CCGGTGGTAC TGCGGTTGCG ATTCGTGAAA TGTATCCAAA AGCGCACTZT 5820
GTCACCATCT TCGCAAAACC GGCTGGTCGT CCGCTGGTTG ATGACTATGT TGTTGATATC 5880
CCGCAAGATA CCTGGATTGA ACAGCCGTGG GATATGGGCG TCGTATTCGT CCCGCCAATC 5940
TCCGGTCGCT AATCTTTTCA ACGCCTGGCA CTGCCGGGCG TTGTTCTTTT TAACTTCAGG 6000
CGGGTTACAA TAGTTTCCAG TAAGTATTCT GGAGGCTGCA TCCATGACAC AGGCAAACCT 6060
GAGCGAAACC CTGTTCAAAC CCCGCTTTGG GCTGCAGGAA TTCGATATCA AGCTTATCGA 6120
TACCGTCGCG GCCGCGACCT CGAGGGGGGG CCCGGTACCC AATTCGCCCT ATAGTGAGTC 6180
GTATTACGCG CGCTCACTGG CCGTCGTTTT ACAACGTCGT GACTGGGAAA ACCCTGGCGT 6240
TACCC.AACTT AATCGCCTTG CAGCACATCC CCCTTT CGCC AGCTGGCGTA ATAGCGAAGA 6300
GGCCCGCACC GATCGCCCTT CCCAACAGTT GCGCAGCCTG AATGGCGAAT GGAAATTGTA 6360
AGCGTTAATA TTTTGZTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTITAAC 6420
CAATAGGCCG AAATCGGCAA AATCCCZTAT AAATCAAAAG AATAGACCGA GATAGGGTTG 6480
AGTGTTGTTC CAGTTT GGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA 6540
GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC CTAATCAAGT 6600
TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT 6660
AGAGCZTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA 6720
GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC 6780
GCGCTTAATG CGCCGCTACA GGGCGCGTCA G 6811
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: oProtSl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
ACCCAGGACC GCCATGGCGA AGCGCGC 27
-176-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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
(vii) IMMEDIATE SOURCE:
(B) CLONE: pP2-gp160MN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 60
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 120
GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTITIT GCGGCATTTT 180
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 240
TGGGTGCACG AGTGGGZTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 300
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTITAA AGZTCTGCTA TGTGGCGCGG 360
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 420
ATGACZTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 480
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 540
CAACGATCGG AGGACCGAAG GAGCTAACCG CTT'ITPTGCA CAACATGGGG GATCATGTAA 600
CTCGCCZTGA TCGZTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 660
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 720
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 780
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 840
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 900
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 960
TAGGTGCCTC ACTGATPAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 1020
AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CT'ITITGATA 1080
ATCTCATGAC CAAAATCCCT TAACGTGAGT TZTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 1200
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 1320
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 1380
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500
-177-
CA 02558864 1992-08-25
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 1560
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620
CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740
TATGGAAAAA CGCCAGCAAC GCGGCCTTTI' TACGGTTCCT GGCCTZTI'GC TGGCCTTTTG 1800
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG 1860
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTZTACACZT TATGCTTCCG GCTCGTATGT 2100
TGTGTGGAAT TGTGAGCGGA TAACAAT'ITC ACACAGGAAA CAGCTATGAC CATGATTACG 2160
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220
GCCGCTCTAG CCCGGGCTAG AACTAGTGGA TCCCCCAAAG CGGGGTTTGA ACAGGGTTTC 2280
GCTCAGGTTT GCCTGTGTCA TGGATGCAGC CTCCAGAATA CTTACTGGAA ACTATTGTAA 2340
CCCGCCTGAA GTTAAAAAGA ACAACGCCCG GCAGTGCCAG GCGTTGAAAA GATTAGCGAC 2400
CGGAGATTGG CGGGACGAAT ACGACGCCCA TATCCCACGG CTGTTCAATC CAGGTATCTT 2460
GCGGGATATC AACAACATAG TCATCAACCA GCGGACGACC AGCCGGTTTi' GCGAAGATGG 2520
TGACAAAGTG CGCTTTTGGA TACATTTCAC GAATCGCAAC CGCAGTACCA CCGGTATCCA 2580
CCAGGTCATC AATAACGATG AAGCC'TTCGC CATCGCCTTC TGCGCGTTTC AGCACTTI'AA 2640
GCTCGCGCTG GTTGTCGTGA TCGTAGCTGG AAATACAAAC GGTATCGACA TGACGAATAC 2700
CCAGTTCACG CGCCAGTAAC GCACCCGGTA CCAGACCGCC ACGGCTTACG GCAATAATGC 2760
CTZTCCATTG TTCAGAAGGC ATCAGTCGGC TTGCGAGTTT ACGTGCATGG ATCTGCAACA 2820
TGTCCCAGGT GACGATGTAT TTTTCGCTCA TGTGAAGTGT CCCAGCCTGT ZTATCTACGG 2880
CTTAAAAAGT GTTCGAGGGG AAAATAGGTT GCGCGAGATT ATAGAGATCT GGCGCACTAA 2940
AAACCAGTAT TTCACATGAG TCCGCGTCTT TTTACGCACT GCCTCTCCCT GACGCGGGAT 3000
AAAGTGGTAT TCTCAAACAT ATCTCGCAAG CCTGTCTTGT GTCCAAGCTT GGGGATCATC 3060
CGTCACTGTT CTTTATGATT CTACTTCCTT ACCGTGCAAT AAATTAGAAT ATATTTTCTA 3120
CTTTTACGAG AAATTAATTA TTGTATTTAT TATTTATGGG TGAAAAACTT ACTATAAAAA 3180
GCGGGTGGGT TTGGAATTAG TGATCAGTTT ATGTATATCG CAACTACCGG CATATGGCTA 3240
TTCGACATCG AGAACATTAC CCACATGATA AGAGATTGTA TCAGTTTCGT AGTCTTGAGT 3300
ATTGGTATTA CTATATAGTA TATAGATGTC GAGGCGGTAC CCTTAAGTTG GGCTGCAGTT 3360
GTTAGAGCTT GGTATAGCGG ACAACTAAGT AATTGTAAAG AAGAAAACGA AACTATCAAA 3420
ACCGTTTATG AAATGATAGA AAAAAGAATA TAAATAATCC TGTATTTTAG TTTAAGTAAC 3480
AGTAAAATAA TGAGTAGAAA ATACTATTTT TTATAGCCTA TAAATCGTTC CTCATGAGAG 3540
-178-
CA 02558864 1992-08-25
TGAAGGGGAT CAGGAGGAAT TATCAGCACT GGTGGGGATG GGGCACGATG CTCCTTGGGT 3600
TATTAATGAT CTGTAGTGCT ACAGAAAAAT TGTGGGTCAC AGTCTATTAT GGGGTACCTG 3660
TGTGGAAAGA AGCAACCACC ACTCTATTTT GTGCATCAGA TGCTAAAGCA TATGATACAG 3720
AGGTACATAA TGTTTGGGCC ACACAAGCCT GTGTACCCAC AGACCCCAAC CCACAAGAAG 3780
TAGAATTGGT AAATGTGACA GAAAATTTTA ACATGTGGAA AAATAACATG GTAGAACAGA 3840
TGCATGAGGA TATAATCAGT TTATGGGATC AAAGCCTAAA GCCATGTGTA AAATTAACCC 3900
CACTCTGTGT TACTTTAAAT TGCACTGATT TGAGGAATAC TACTAATACC AATAATAGTA 3960
CTGCTAATAA CAATAGTAAT AGCGAGGGAA CAATAAAGGG AGGAGAAATG AAAAACTGCT 4020
CTTTCAATAT CACCACAAGC ATAAGAGATA AGATGCAGAA AGAATATGCA CTTCTZTATA 4080
AACTTGATAT AGTATCAATA GATAATGATA GTACCAGCTA TAGGTTGATA AGTTGTAATA ~4140
CCTCAGTCAT TACACAAGCT TGTCCAAAGA TATCCTTTGA GCCAATTCCC ATACACTAZT 4200
GTGCCCCGGC TGGTTZTGCG ATTCTAAAAT GTAACGATAA AAAGTTCAGT GGAAAAGGAT 4260
CATGTAAAEIA TGTCAGCACA GTACAATGTA CACATGGAAT TAGGCCAGTA GTATCAACTC 4320
AACTGCTGTT AAATGGCAGT CTAGCAGAAG AAGAGGTAGT AATTAGATCT GAGAATZTCA 4380
CTGATAATGC TAAAACCATC ATAGTACATC TGAATGAATC TGTAC,AAATT AATTGTACAA 4440
GACCCAACTA CAATAAAAGA AAAAGGATAC ATATAGGACC AGGGAGAGCA TTTTATACAA 4500
CAAAAAATAT AATAGGAACT ATAAGACAAG CACATTGTAA CATTAGTAGA GCAAAATGGA 4560
ATGACACTTT AAGACAGATA GTTAGCAAAT TAAAAGAACA ATTTAAGAAT AAAACAATAG 4620
TCZTTAATCA ATCCTCAGGA GGGGACCCAG AAATTGTAAT GCACAGTZTT AATTGTGGAG 4680
GGGAATTTTT CTACTGTAAT ACATCACCAC TGTTTAATAG TACTTGGAAT GGTAATAATA 4740
CTTGGAATAA TACTACAGGG TCAAATAACA ATATCACACT TCAATGCAAA ATAAAACAAA 4800
TTATAAACAT GTGGCAGGAA GTAGGAAAAG CAATGTATGC CCCTCCCATT GAAGGACAAA 4860
TTAGATGTTC ATCAAATATT ACAGGGCTAC TAZTAACAAG AGATGGTGGT AAGGACACGG 4920
ACACGAACGA CACCGAGATC TTCAGACCTG GAGGAGGAGA TATGAGGGAC AAZTGGAGAA 4980
GTGAATTATA TAAATATAAA GTAGTAACAA TTGAACCATT AGGAGTAGCA CCCACCAAGG 5040
CAAAGAGAAG AGTGGTGCAG AGAGAAAAAA GAGCAGCGAT AGGAGCTCTG TTCCTTGGGT 5100
TCTTAGGAGC AGCAGGAAGC ACTATGGGCG CAGCGTCAGT GACGCTGACG GTACAGGCCA 5160
GACTATTATT GTCTGGTATA GTGCAACAGC AGAACAATTT GCTGAGGGCC ATTGAGGCGC 5220
AACAGCATAT GTTGCAA.CTC ACAGTCTGGG GCATCAAGCA GCTCCAGGCA AGAGTCCTGG 5280
CTGTGGAAAG ATACCTAAAG GATCAACAGC TCCTGGGGTT TTGGGGTTGC TCTGGAAAAC 5340
TCATTTGCAC CACTACTGTG CCTTGGAATG CTAGTTGGAG TAATAAATCT CTGGATGATA 5400
TITGGAATAA CATGACCTGG ATGCAGTGGG AAAGAGAAAT TGACAATTAC ACAAGCTTAA 5460
TATACTCATT ACTAGAAAAA TCGCAAACCC AACAAGAAAA GAATGAACAA GAATTATTGG 5520
AAZTGGATAA ATGGGCAAGT TTGTGGAATT GGTTTGACAT AACAAATTGG CTGTGGTATA 5580
-179-
CA 02558864 1992-08-25
TAAAAATATT CATAATGATA GTAGGAGGCT TGGTAGGTTT AAGAATAGTT TTTGCTGTAC 5640
TI'TCTATAGT GAATAGAGTT AGGCAGGGAT ACTCACCATT GTCGTTGCAG ACCCGCCCCC 5700
CAGTTCCGAG GGGACCCGAC AGGCCCGAAG GAATCGAAGA AGAAGGTGGA GAGAGAGACA 5760
GAGACACATC CGGTCGATTA GTGCATGGAT TCTTAGCAAT TATCTGGGTC GACCTGCGGA 5820
GCCTGT"TCCT CTTCAGCTAC CACCACAGAG ACTTACTCTT GATTGCAGCG AGGATTGTGG 5880
AACTTCTGGG ACGCAGGGGG TGGGAAGTCC TCAAATATTG GTGGAATCTC CTACAGTATT 5940
GGAGTCAGGA ACTAAAGAGT AGTGCTGTTA GCTTGCTTAA TGCCACAGCT ATAGCAGTAG 6000
CTGAGGGGAC AGATAGGGTT ATAGAAGTAC TGCAAAGAGC TGGTAGAGCT ATTCTCCACA 6060
TACCTACAAG AATAAGACAG GGCTTGGAAA GGGCTTTGCT ATAAGATGGG TGGCAAATGG 6120
TCAAAACGTG TGACTGGATG GCCTACTGTA AGGGAAAGAA TGAGACGAGC TGAACCAGAA 6180
CGAATTCCAT GGCCCGGGAA GGCCTCGGAC CGGGCCCGGC CATATAGGCC AGCGATACCG 6240
TCGCGGCCGC GACCTCGAGG GGGGGCCCGG TACCCAATTC GCCCTATAGT GAGTCGTATT 6300
ACGCGCGCTC ACTGGCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCCT GGCGTTACCC 6360
AACTTAATCG CCTTGCAGCA CATCCCCCTT TCGCCAGCTG GCGTAATAGC GAAGAGGCCC 6420
GCACCGATCG CCCTTCCCAA CAGTTGCGCA GCCTGAATGG CGAATGGAAA TTGTAAGCGT 6480
TAATATTTTG TTAAAATTCG CGTTAAATTT TTGZTAAATC AGCTCATTZT TTAACCAATA 6540
GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT 6600
TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG 6660
AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTrITIT 6720
GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTT AGAGC 6780
TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG 6840
CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT 6900
TAATGCGCCG CTACAGGGCG CGTCAG 6926
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 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: selP promoter
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
TATGAGATCT AAAAATTGAA ATTTTATTTT TIZTiRTTGG AATATAAAT 49
-180-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: oFIX.l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
TCATGTTCAC GCGCTCCATG GCCGCGGCCG CACC 34
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5532 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: pN2gpta-FIX
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
GTGGCACTZT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 60
CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 120
GGAAGAGTAT GAGTAZTCAA CATTTCCGTG TCGCCCTTAT TCCCTITPIT GCGGCATTTT 180
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 240
TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 300
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 360
TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 420
ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 480
GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 540
CAACGATCGG AGGACCGAAG GAGCTAACCG CTTT'ITTGCA C.AACATGGGG GATCATGTAA 600
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 660
CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 720
CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 780
TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 840
GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 900
-181-
CA 02558864 1992-08-25
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 960
TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 1020
AGATTGATTT AAAACTTCAT TITTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 1080
ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG 1140
AAAAGATCAA AGGATCTTCT TGAGATCCTT TTITTCTGCG CGTAATCTGC TGCTTGCAAA 1200
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 1260
TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 1320
CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 1380
TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 1440
GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 1500
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 1560
GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 1620
CAGGAGAGCG CACGAGGGAG CZTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 1680
GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 1740
TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTI'TG 1800
CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTT'TG 1860
AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 1920
AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 1980
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 2040
TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 2100
TGTGTGGAAT TGTGAGCGGA TAACAATZTC ACACAGGAAA CAGCTATGAC CATGATTACG 2160
CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT GGAGCTCCAC CGCGGTGGCG 2220
GCCGCTTGZT AATTTTCAAT TCCAATGAAT TAACCTTGGA AATCCATCTT TCATTAAGTG 2280
AGCTTTGTTT TTTCCTTAAT CCAGZTGACA TACCGGGATA CCTTGGTATA TATTCCATAT 2340
TTGCCTTTCA TTGCACACTC TTCACCCCAG CTAATAATTC CAGTTAAGAA ACTGGTCCCT 2400
TCCACTTCAG TAACATGGGG TCCCCCACTA TCTCCTTGAC ATGAATCTCT ACCTCCTTCA 2460
TGGAAGCCAG CACAGAACAT GTTGTTATAG ATGGTGAACT TTGTAGATCG AAGACATGTG 2520
GCTCGGTCAA CAAGTGGAAC TCTAAGGTAC TGAAGAACTA AAGCTGATCT CCCTTTGTGG 2580
AAGACTCTTC CCCAGCCACT TACATAGCCA GATCC.AAATT TGAGGAAGAT GTTCGTGTAT 2640
TCCTTGTCAG CAATGCAAAT AGGTGTAACG TAGCTGTTTA GCACTAAGGG TTCGTCCAGT 2700
TCCAGAAGGG CAATGTCATG GTTGTACTTA TTAATAGCTG CATTGTAGTT GTGGTGAGGA 2760
ATAATTCGAA TCACATTTCG CTTT'I'GCTCT GTATGTTCTG TCTCCTCAAT ATTATGTTCA 2820
CCTGCGACAA CTGTAATTTT AACACCAGTT TCAACACAGT GGGCAGCAGT TACAATCCAT 2880
TTTTCATTAA CGATAGAGCC TCCACAGAAT GCATCAACTT TACCATTCAA AACAACCTGC 2940
-182-
CA 02558864 1992-08-25
CAAGGGAATT GACCTGGTZ'T GGCATCTTCT CCACCAAC.AA CCCGAGTGAA GTCATTAAAT 3000
GATI'GGGTGC TTTGAGTGAT GTTATCCAAA ATGGT'ITCAG CTTCAGTAGA ATTTACATAG 3060
TCCACATCAG GAAAAACAGT CTCAGCACGG GTGAGCTTAG AAGT'ITGTGA AACAGAAACT 3120
CTTCCACATG GAAATGGCAC TGCTGGTTCA CAGGACTTCT GGTTZTCTGC AAGTCGATAT 3180
CCCTCAGTAC AGGAGCAAAC CACCTTGTTA TCAGCACTAT TT1'TACAAAA CTGCTCGCAT 3240
CTGCCATTCT TAATGTTACA TGZTACATCT AATTCACAGT TCTTTCCTTC AAATCCAAAG 3300
GGACACCAAC ATTCATAGGA ATTAATGTCA TCCTTGCAAC TGCCGCCATT TAAACATGGA 3360
TTGGACTCAC ACTGATCTCC ATCAACATAC TGCTTCCAAA ATTCAGTTGT TCTTTCAGTG 3420
TTTTCAAAAA CTTCTCGTGC TTCTTCAAAA CTACACTTT'T CTTCCATACA ZTCTCTCTCA 3480
AGGTTCCCTT GAACAAACTC TTCCAATTTA CCTGAATTAT ACCTCTTTGG CCGATTCAGA 3540
ATTITGTTGG CGTTTTCATG ATCAAGAAAA ACTGTACATT CAGCACTGAG TAGATATCCT 3600
AAAAGGCAGA TGGTGATGAG GCCTGGTGAT TCTGCCATGA TCATGTTCAC GCGCTCCATG 3660
GAGGCCTTAT TTATATTCCA AAAAAAAAAA ATAAAATTTC AATT'I'TTAGA TCCCCCAACT 3720
TAAGGGTACC GCCTCGACAT CTATATACTA TATAGTAATA CCAATACTCA AGACTACGAA 3780
ACTGATACAA TCTCTTATCA TGTGGGTAAT GTTCTCGATG TCGAATAGCC ATATGCCGGT 3840
AGTTGCGATA TACATAAACT GATCACTAAT TCCAAACCCA CCCGC77 T1T ATAGTAAGTT 3900
TTTCACCCAT AAATAATAAA TACAATAATT AATTTCTCGT AA.AAGTAGAA AATATATTCT 3960
AATTTATTGC ACGGTAAGGA AGTAGAATCA TAAAGAACAG TGACGGATGA TCCCCAAGCT 4020
TGGACACAAG ACAGGCTTGC GAGATATGTT TGAGAATACC ACTTTATCCC GCGTCAGGGA 4080
GAGGCAGTGC GTAAAAAGAC GCGGACTCAT GTGAAATACT GGTTTITAGT GCGCCAGATC 4140
TCTATAATCT CGCGCAACCT ATTTTCCCCT CGAACACTTT TTAAGCCGTA GATAAACAGG 4200
CTGGGACACT TCACATGAGC GAAAAATACA TCGTCACCTG GGACATGTTG CAGATCCATG 4260
CACGTAAACT CGCAAGCCGA CTGATGCCTT CTGAACAATG GAAAGGCATT ATTGCCGTAA 4320
GCCGTGGCGG TCTGGTACCG GGTGCGTTAC TGGCGCGTGA ACTGGGTATT CGTCATGTCG 4380
ATACCGTTTG TATTTCCAGC TACGATCACG ACAACCAGCG CGAGCTTAAA. GTGCTGAAAC 4440
GCGCAGAAGG CGATGGCGAA GGCTTCATCG TTATTGATGA CCTGGTGGAT ACCGGTGGTA 4500
CTGCGGTTGC GATTCGTGAA ATGTATCCAA AAGCGCACTT TGTCACCATC TTCGCAAAAC 4560
CGGCTGGTCG TCCGCTGGTT GATGACTATG TTGTTGATAT CCCGCAAGAT ACCTGGATTG 4620
AACAGCCGTG GGATATGGGC GTCGTATTCG TCCCGCCAAT CTCCGGTCGC TAATCTTITC 4680
AACGCCTGGC ACTGCCGGGC GTTGTTCTTT TTAACTTCAG GCGGGZTACA ATAGTTTCCA 4740
GTAAGTATTC TGGAGGCTGC ATCCATGACA CAGGCAAACC TGAGCGAAAC CCTGTTCAAA 4800
CCCCGCTTTG GGCTGCAGGA ATTCGATATC AAGCTTATCG ATACCGTCGC GGCCGCGACC 4860
TCGAGGGGGG GCCCGGTACC CAATTCGCCC TATAGTGAGT CGTATTACGC GCGCTCACTG 4920
GCCGTCGTTT TACAACGTCG TGACTGGGAA AACCCTGGCG TTACCCAACT TAATCGCCTT 4980
-183-
CA 02558864 1992-08-25
GCAGCACATC CCCCTTi'CGC CAGCTGGCGT AATAGCGAAG AGGCCCGCAC CGATCGCCCT 5040
TCCCAACAGT TGCGCAGCCT GAATGGCGAA TGGAAATTGT AAGCGTTAAT ATTTTGTTAA 5100
AATTCGCGTT AAATTTTTGT TAAATCAGCT CATIZTITAA CCAATAGGCC GAAATCGGCA 5160
AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT CCAGTTT GGA 5220
ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA ACCGTCTATC 5280
AGGGCGATGG CCCACTACGT GAACCATCAC CCTAATCAAG TIITITGGGG 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 NO:73:
(i) SEQUENCE CHARACTERISTICS:
(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) IMMEDIATfi SOURCE:
(B) CLONE: wild-type gp160MN
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..14
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
CA ATG AGA GTG AAG 14
Met Arg Val Lys
1
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
Met Arg Val Lys
1
-184-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(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) IMMBDIATE SOURCE:
(B) CLONE: gp160 in vse1P-gp160 virus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..14
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
CC ATG GCC GTG AAG 14
Met Ala Val Lys
1
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
Met Ala Val Lys
1
(2) INFORMATION FOR SEQ ID NO: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
(vii) IMMEDIATE SOURCE:
(B) CLONE: wild-type Protein S
(ix) FEATURE :
(A) NAME/ICEY: CDS
(B) LOCATION: 4..18
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
GAA ATG AGG GTC CTG GGT 18
Met Arg Val Leu Gly
1 5
-185-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO:78:
(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:78:
Met Arg Val Leu Gly
1 5
(2) INFORMATION FOR SEQ ID NO:79:
(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
(vii) IMNMEDIATE SOURCE:
(B) CLONE: Protien S in the chimeras
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4..18
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
GCC ATG GCG GTC CTG GGT 18
Met Ala Val Leu Gly
1 5
(2) INFORMATION FOR SEQ ID NO: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
-186-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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 NO:81:
TT ATG CAG CGC GTG AAC 17
Met G1n Arg Val Asn
1 5
(2) INFORMATION FOR SEQ ID NO: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 NO:82:
Met Gln Arg Val Asn
1 5
(2) INFORMATION FOR SEQ ID NO: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) IMMEDIATE SOURCE:
(B) CLONE: factor IX vFIX#5
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..17
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
CC ATG GAG CGC GTG AAC 17
Met Glu Arg Val Asn
1 5
-187-
CA 02558864 1992-08-25
(2) INFORMATION FOR SEQ ID NO: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 NO:84:
Met Glu Arg Val Asn
1 5
-188-
