Note: Descriptions are shown in the official language in which they were submitted.
WO g5/16694 PCT/US94/14563
2 1 78965
ORIGIN OF REPLICATION COMPLEX GENES,
PROIEINS AND METHODS
INTRODUCTION
The research carried out in the subject application was ~uppolLed in part by
grants from the National Institutes of Health. The government may have rights inany patent issuing on this application.
Technical Field
The technical field of this invention concerns Origin of Replication
Complex genes which are invovled with DNA transcription and replication.
10 Background
The elements involved in the early events of eukaryotic DNA replication
have begun to emerge in the yeast Saccharomyces cerevisiae. A critical first step
was the identification of ARS elements derived from yeast chromosomes, a subset
of which were subsequently shown to act as chromosomal origins of DNA
15 replication (reviewed in 11). Sequence co..-pafison of a number of ARS elements
resulted in the identification of the ARS consellc~c sequence (ACS, 12). This
sequence is essential for the function of yeast origins of DNA replication (7, 12,
13). Three additional elements required for efficient ARSI function have been
identified. When mutated individually, these el~rnentC, referred to as Bl, B2, and
20 B3, result in a slight reduction of ARS1 activity. When two or three of the B elements are simultaneously mutated, however, ARS1 function is severely
co~ ol.,ised (14) .
wo 95/16694 2 1 7 8 9 6 5 PcrlUS94/14s63
Proteins that recognize two elements of ARS1 have been identified. The
yeast transcription factor ABFl binds to and me~i~tes the function of the B3
element (11, 14). More recently we have identified a multi-protein complex that
spe~ ific~lly recognizes the highly conserved ACS (15). This activity, referred to
as the origin recognition complex (ORC), has several propellies that make it an
attractive candidate to act as an initiator protein at yeast origins of replication.
Binding of this protein ~ uires the ACS, and the effect of mutations in the
con~n~-ls sequence on ARSl function parallels the effect of the same mutations on
ORC DNA binding. ORC binds to more than 10 yeast ARS elements, several of
which are known origins of DNA replication (15). Specific DNA binding by ORC
requires ATP, suggesting that ORC binds ATP, a plupelly of a number of known
initiator proteins (17). ORC also interacts with other sequences outside of the ACS
that are known to be important for ARS function (18, 19). Further support for
the hypothesis that ORC mYli~t~s the function of the ACS is provided by in situ
deoxyribonuclease I (DNase I) footprinting experiments that identify a protectedregion of ARSl relll~l~bly similar to that observed with ORC in vitro (20).
Relevant Literature
A multi-protein complex that recognizes cellular origins of DNA replication
was reported in Bell and Stillman (1992) Nature 357, 128-134. Much of the
present disclosure was published by Foss et al. (1993), Bell et al. (1993) and Li
and Herskowicz (1993), in Science 262, 1838, 1843 and 1870, respectively, issue
date December 17, 1993. Wang and Reed (1993) Nature 364, 121-126 report using
a single-hybrid screen as disclosed herein.
SUMMARY OF THE INVENTION
Origin of DNA Replication Complex (ORC) genes, recombinant ORC
peptides and methods of identifying DNA binding proteins and using the subject
co"~posilions are provided.
Provided are compositions comprising isolated nucleic acids encoding
unique ORC gene portions, especially portions encoding biologically active unique
portions of ORCl-ORC6 proteins. Vectors and cells comprising such DNA
molecules find use in the production of recombinant ORC peptides.
wo95/16694 ~ ~ 2 1 78 ~65 p~US94/14563
The subject compositions are used to isolate ORC genes from a wide
variety of species, including human. The subject ORC peptides also find particular
use in screening for ORC selective agents useful in the diagnosis, prognosis or
treatment of disease, particulary fungal infections and neoproliferative disease.
S Particularly useful are agents capable of distinguishing an ORC protein of an
infectious organism or transformed cell from the wild-type human homologue.
Also disclosed are methods for identifying a gene encoding a protein which
directly or indirectly ~csoCi~t~s with a selected DNA sequence. Generally, the
methods involve tran~ro.",ing an eA~lession library of hybrid proteins into a
10 re~,ler strain, wherein the library comprises protein-coding sequences fused to a
constitutively eApl~ssed transcription activation domain and the reporter straincomprises a reporter gene with at least one copy of a selected DNA sequence in its
promoter region. Clones e~-p-cssillg the transcription or translation product of the
c;po-lel gene are detected and recovered. A p-~ d method employs an
15 activation domain from GAL4 and a lacZ reporter gene.
BREIF DESCRIPTION OF SEQUENCE ID NUMBERS
SEQUENCE ID NO: 1. DNA Sequence of ORCl .
SEQUENCE ID NO:2. Amino Acid Sequence of ORCl.
20 SEQUENCE ID NO:3. DNA Sequence of ORC2.
SEQUENCE ID NO:4. Amino Acid Sequence of ORC2.
SEQUENCE ID NO:S. DNA Sequence of ORC3.
SEQUENCE ID NO:6. Amino Acid Sequence of ORC3.
SEQUENCE ID NO:7. DNA Sequence of ORC4.
25 SEQUENCE ID NO:8. Amino Acid Sequence of ORC4.
SEQUENCE ID NO:9. DNA Sequence of ORCS.
SEQUENCE ID NO:10. Amino Acid Sequence of ORCS.
SEQUENCE ID NO: 11. DNA Sequence of ORC6.
SEQUENCE ID NO: 12. Amino Acid Sequence of ORC6.
DESCRIPTION OF SPECIFIC EMBODIMENTS
The recombinant polypeptides of the invention comprise unique portions of
the ~lis~lose~ ORC proteins which retain an binding affinity specific to the subject
W0 95/16694 2 ~ ~ ~ 9 6 5 PCT/US94114563
full-length ORC protein. A "unique portion" has an amino acid sequence unique tcsubject ORC in that it is not found in previously known protein and has a length at
least long enough to define a peptide specific to that ORC. Unique portions are
found to vary from about 5 to about 25 residues, usually from 5 to 10 residues in
5 length, depending on the particular amino acid sequence and are readily idçntified
by co",p~ing the subject portion sequences with known peptide/protein sequence
data bases. Hence, the term polypeptide as used herein defines an amino acid
polymer with as few as five residues. ORCs used in the subject screening assays
are frequently smaller deletion mutants of full-length ORC proteins. Typically,
10 such deletion mutants are readily generated using conventional molecular
techniques and screened for an ORC-specific binding affinity using the various
assays described below, e.g. footprint analysis, coimmunoprecipitation, etc.
ORC-specific retained binding affinities include the ability to selectively
bind a nucleic acid of a defined sequence, an ORC protein or an compound such as15 an antibody which is capable of selectively binding an ORC protein. As such,
binding specificity may be provided by an ORC-specific immunological epitope,
lectin binding site, etc. Selective binding is conveniently shown by competition with labeled ligand using recombinant ORC peptide either in vitro or in cell based
systems as disclosed herein. Generally, seiective binding requires a binding
20 affinity of 10~M, preferably 10-8M, more preferably 10-'M, under in vitro
conditions as exemplified below.
The subject recombinant polypeptides may be free or covalently coupled to
other atoms or molecules. Frequently the polypeptides are present as a portion of
a larger polypeptide comprising the subject polypeptide where the remainder of the
25 larger polypeptide need not be ORC-derived. The subject polypeptides are
typically "isolated", m~ning unaccompanied by at least some of the m~teri~l withwhich they are ~soci~t~ in their natural state. Generally, an isolated polypeptide
constitutes at least about 1%, preferably at least about 10%, and more preferably at
least about 50% by weight of the total poly/peptide in a given sample. By pure
30 peptidepolypeptide is intended at least about 60%, preferably at least 80%, and
more preferably at least about 90% by weight of total polypeptide. Included in the
subject polypeptide weight are any atoms, molecules, groups, etc. covalently
WO95/16694 2 1 7 8 ~ 6 5 Pcrluss4ll4563
coupled to the subject polypeptides, such as detectable labels, glycosylations,
phosphorylations, etc.
The subject polypeptides may be isolated or purified in a variety of ways
known to those skilled in the art depending on what other colllponents are present
5 in the sample and to what, if anything, the polypeptide is covalently linked.
Purification methods include electrophoretic, molecular, immunological and
- chromatog,dphic techniques, especi~lly affinity chr~--latog,dphy and RP-HPLC in
the case of peptides. For general guidance in suitable purification techniques, see
Scopes, R., Protein Purification, Springer-Verlag, NY (1982).
The polypeptides may be modified or joined to other compounds using
physical, chemical, and molecular techniques disclosed or cited herein or otherwise
known to those skilled in the relevant art to affect their ORC/receptor binding
specificity or other prope,lies such as solubility, membrane transportability,
stability, toxicity, bioavailability, loc~li7~tion, detect~bility, in vivo half-life, etc.
15 as assayed by methods disclosed herein or otherwise known to those of ordinary
skill in the art. Other modifications to further modulate binding specificity/affinity
include chemical/enzymatic inteNention (e.g. fatty acid-acylation, proteolysis,
glycosylation) and especially where the poly/peptide is integrated into a largerpolypeptide, se~ection of a particular eAl)~ession host, etc. Amino and/or carboxyl
20 termini may be functionalized e.g., for the amino group, acylation or alkylation,
and for the carboxyl group, esterification or amidification, or the like.
Many of the disclosed poly/peptides contain glycosylation sites and patterns
which may be disrupted or modified, e.g. by enzymes like glycosidases. For
instance, N or O-linked glycosylation sites of the disclosed poly/peptides may be
25 deleted or substituted for by another basic amino acid such as Lys or His for N-
linked glycosylation alterations, or deletions or polar substitutions are introduced at
Ser and Thr residues for modul~ing O-linked glycosylation. Glycosylation
variants are also produced by selecting approp,iate host cells, e.g. yeast, insect, or
various m~mm~ n ~lls, or by in vitro methods such as neuraminidase digestion.
30 Other covalent modifications of the disclosed poly/peptides may be introduced by
reacting the targeted amino acid residues with an organic derivatizing (e.g. methyl-
3-[(p azido-phenyl)dithio] propioimidate) or crosclinking agent (e.g. 1,1-
bis(di~70~retyl)-2-phenylethane) capable of reacting with selected side chains or
W095116694 . ~ 1 78965 PCT/US94/14563
termini. For theld~ulic and diagnostic loc~li7~tion, the subject poly/peptides
thereof may be labeled directly (radioisotopes, fluorescers, etc.) or indirectly with
an agent capable of providing a dete~t~hle signal, for example, a heart muscle
kinase labeling site.
ORC poypeptides with ORC binding specificity are identified by a variety
of ways including crosclinking, or prefeMbly, by screening such polypeptides forbinding to or disruption of ORC-ORC complexes. Additional ORC-specific agents
include specific antibodies that can be modified to a monovalent form, such as Fab,
Fab', or Fv, specifically binding oligopeptides or oligonucleotides and most
preferably, small molecular weight organic compounds. For example, the
disclosed ORC peptides are used as immunogens to generate specific polyclonal ormonoclonal antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory, for geneMl methods.
Other pros~ecli~e ORC specific agents are screened from large libMries of
synthetic or natural co.,-pounds. Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant and animal extracts are available or readily
producible. Additionally, natural and synthetically produced libraries and
co"lpounds are readily modified through conventional chemical, physical, and
biochPmi~l means. See, e.g. Houghten et al. and Lam et al (1991) Nature 354,
84 and 81, respectively and Blake and Litzi-Davis ~1992), Bioconjugate Chem 3,
510.
Useful agents are identified with assays employing a compound comprising
the subject polypeptides or encoding nucleic acids. A wide variety of in vitro,
cell-free binding assays, espe~i~lly assays for specific binding to immobilized
col--pounds comprising ORC polypeptide find convenient use. For example,
immobilized ORC-ORC or ORC-nucleic acid complexes provide convenient targets
for disruption, e.g. as measured by the ~lic~csoci~tion of a labelled component of
the complex. Such assays are amenable to scale-up, high throughput usage suitable
for volume drug screening. While less plefelled, cell-based assays may be used to
determine specific effects of prospective agents.
~efelled agents are ORC- and species-specific. Useful agents may be
found within numerous chemical classes, though typically they are organic
co",pounds; prefeMbly small organic compounds. Small organic compounds have
WOgS/16694 ~; ~ ~ 2 1 /8965 PCT/USs4/14563
a molecular weight of more than 150 yet less than about 4,500, preferably less
than about 1500, more preferably, less than about 500. Exemplary classes includesteroids, heterocyclics, polycyclics, substituted aromatic compounds, and the like.
Sele~t~d agents may be modified to enhance efficacy, stability,
S pharm~ceutical colllpalibility, and the like. Structural identification of an agent
may be used to identify, generate, or screen additional agents. For example,
where peptide agents are identified, they may be modified in a variety of ways as
described above, e.g. to enh~nce their proteolytic stability. Other methods of
stabilization may include encapsulation, for example, in liposo-lles, etc. The
10 subject binding agents are ~repar~d in any convenient way known to those in the
art.
For the~ eulic uses, the compositions and agents disclosed herein may be
~rlministçred by any convenient way. Small organics are preferably ~iminictered
orally; other compositions and agents are preferably administered parenterally,
15 conveniently in a pharmaceutically or physiologically acceptable carrier, e.g.,
phosphate buffered saline, or the like. Typically, the compositions are added to a
retained phyciological.fluid. As examples, many of the disclosed thel~peulics are
~m~n~hle to direct injection or infusion, topical, intratracheal/nasal ~rlminictration
e.g. through aerosal, intraocularly, or within/on implants e.g. collagen, osmotic
20 pumps, grafts comprising applupliately transformed cells, etc. Generally, theamount ~dminict~red will be empirically determined, typically in the range of about
10 to 1000 ~lg/kg of the recipient. For peptide agents, the concentration will
generally be in the range of about 50 to 500 ~gtml in the dose adminict~red.
Other additives may be included, such as stabilizers, bactericides, etc. These
25 additives will be present in conventional amounts.
The invention provides icol~ted nucleic acids encoding ORC genes, their
transcriptional regulatory regions and the disclosed unique ORC polypeptides which
retain ORC-specific function. As used herein: an "isolated" nucleic acid is present
as other than a naturally occurring chromosome or transcript in its natural state and
30 is typically joined in sequence to at least one nucleotide with which it is not
normally associated on a natural chromosome; nucleic acids with substantial
sequence simil~rity hybridize under low stringency conditions, for example, at
50C and SSC (0.9 M saline/0.09 M sodium citrate) and remain bound when
WO 95/16694 2 1 7 8 9 6 5 PCT/US94/14563
subject to washing at 55C with SSC, wherein regions of non-identity of
subst~nti~lly similar nucleic acid sequences preferably encode redundant codons; a
partially pure nucleotide sequence con~tituteC at least about 5%, preferably at least
about 30%, and more preferably at least about 90% by weight of total nucleic acid
5 present in a given fraction; unique portions of the disclosed nucleic acids are of
length sufficient to distinguish previously known nucleic acids, hence a unique
portion has a nucleotide sequence at least long enough to define a novel
oligonucleotide, usually at least about 18 bp in length, preferably at least about 36
nucleotides in length.
Typically, the invention's ORC polypeptide encoding polynucleotides are
associated with heterologous sequences. Examples of such heterologous sequences
include regulatory sequences such as promoters, enhancers, response elements,
signal sequences, polyadenylation sequences, etc., introns, 5' and 3' noncoding
regions, etc. According to a particular embodiment of the invention, portions of15 the coding sequence are spliced with heterologous sequences to produce soluble,
secreted fusion proteins, using appropliate signal sequences and optionally, a
fusion partner such as ~-Gal. For antisense applications where the inhibition ofeAples~ion is indicated, especially useful oligonucleotides are between about 10 and
30 nucleotides in length and include sequences surrounding the disclosed ATG start
20 site, espe~i~lly the oligonucleotides defined by the disclosed sequence beginnin~
about 5 nucleotides before the start site and ending about 10 nucleotides after the
disclosed start site. The ORC encoding nucleic acids can be subject to alternative
purification, synthesis, modification, sequencing, expression, transfection,
~ministration or other use by methods disclosed in standard manuals such as
25 Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More,
Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, NY,
1992) or that are otherwise hnown in the art.
The invention also provides vectors comprising the described ORC nucleic
acids. A large number of vectors, including plasmid and viral vectors, have been30 described for eApr~ssion in a variety of euharyotic and prokaryotic hosts.
Advantageously, vectors will often include a promotor operably linked to an ORC
polypeptide-encoding portion, one or more replication systems for cloning or
t;~ression, one or more l--alhel~ for selection in the host, e.g. antibiotic
wo 95/16694 2 1 7 8 9 6 5 Pcr,Us94/l4563
recict~nce. The inserted coding sequences may be synthPsi7PA, isolated from
natural sources, p~ aled as hybrids, etc. Suitable host cells may be
transformed/transfected/infected by any suitable method including electroporation,
CaCl2 mPAi~tPA DNA uptake, viral infection, microinjection, microprojectile, or
5 other methods.
Appropriate host cells include bacteria, archeb~teria, fungi, espe~i~lly
yeast, and plant and animal cells, espe~i~lly m~mm~ n cells. Of particular
interest are E. coli, B. subtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells,
293 cells, Neurospora, and CHO, COS, HeLa cells, immortalized m~mm~ n
10 myeloid and lymphoid cell lines, and pluripotent cells, esperi~lly m~mm~ n EScells and zygotes. Preferred eA~l~ession systems include COS-7, 293, BHK, CHO,
TM4, CVl, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT 060562,
TRI cells, and baculovirus systems. Preferred replication systems include M13,
ColEl, SV40, baculovirus, lambda, adenovirus, AAV, BPV, etc. A large number
15 of transcription initiation and termination regulatory regions have been isolated and
shown to be effective in the transcription and translation of heterologous proteins in
the various hosts. Examples of these regions, methods of isolation, manner of
manipulation, etc. are known in the art.
For the production of stably transformed cells and transgenic ~nim~l.c, the
- 20 subject nucleic acids may be integrated into a host genome by recombination
events. For example, such a nucleic acid can be electroporated into a cell, and
thereby effect homologous recombination at the site of an endogenous gene, an
analog or pseudogene thereof, or a sequence with substantial identity to an ORC-encoding gene. Other recombination-based methods such as nonhomologous
25 recombinations, deletion of endogenous gene by homologous recombination,
especi~lly in plu~i~lent cells, etc., provide additional applications. Preferredtransgenics and stable transrol,--ants over-express or under-express (e.g. knock-out
cells and ~nim~l.c) a disclosed ORC gene and find use in drug development and asa disease model. Methods for making transgenic ~nim~lc, usually rodents, from
30 ES cells or zygotes are known to those skilled in the art.
The compositions and methods disclosed herein may be used to effect ~erle
therapy. See, e.g. Zhu et al. (1993) Science 261, 209-211; Gutierrez et al. (.992)
Lancet 339, 715-721. For example, cells are transfected with ORC-encoding
WO95tl6694 2 1 7 8 ~ 6 5 PCT/USg4/l4s63
sequences operably linked to gene regulatory sequences capable of effecting altered
ORC expression or regulation. To modulate ORC translation, target cells may be
transfected with complementary antisense polynucleotides. For gene therapy
involving the grafting/implanting/transfusion of transfected cells, a~lmini~tration
5 will depend on a number of variables that are ascertained empirically. For
example, the number of cells will vary depending on the stability of the transfered
cells. Transfer media is typically a buffered saline solution or other
pharmacologically acceptable solution. Similarly the amount of other ~mini~ttored
compositions, e.g. transfected nucleic acid, protein, etc., will depend on the
10 manner of administration, purpose of the therapy, and the like.
The genes encoding six ORC subunits from S. cerevisiae are used to obtain
the functional homologues of the ORC proteins from other species. For example,
we have demonstrated that the ORCI gene is conserved in a related fungi
klyuermyces lactis. The ORCl gene in both S. cerevisie and k lactis contain
lS conserved primary protein sequence that are utliized to obtain the ORCI gene from
other species including other fungi and from human. Using oligonucleotide
- primers based on the conserved sequences between S. cerevisiae and k lactis, PCR
is used to identify the ORCl protein in any eukaryotic species. The cloned gene
- encoding ORCI polypeptide from any fungi or from human cells is used to express
20 the protein in a bacterial eAI)ression system to make antibodies against the
polypeptide. These antibodies are used to immunoprecipi~te the ORC complex
from the relevant species. Using the disclosed techniques for protein sequencing,
the sequence the ORC polypeptides is obtained. Using the protein sequencing
methodologies disclosed herein for cloning the S. cerevisiae protein, other genes or
25 cDNAS encoding the ORC polypeptides from other fungi species and from human
cells are obtained. As we demonstrate herein how to reconstitute the ORC
complex by eApres~ing each of the S. cerevisiae genes in a baculovirus e~pr~ssion
vector and infecting Sf 9 insect cells with viruses expressing. each of the ORC
subunits, these genes are used to express the ORC polypeptides and reconstitute
30 activity. In this way, large amounts of ORC protein from any fungi or
m~mm~ n species, including human cells, are obtained.
Inhibitors of ORC protein in fungi provide valuable reagents to selectively
inhibit proliferation of fungal cell division by inhibiting the initiation of DNA
- -
wo 95tl6694 ~ ~ 2 1 7 8 9 6 5 Pcrluss4ll4563
replication. This offers a powerful, selective target for antifungal agents valuable
in controlling fungal infections in human and other species. For example, as
disclosed herein, inhibiting the ORC function by mutation in S. cerevisiae can
actually cause the death of the mutant cells.
S In human proliferative disorders such as cancer, cells of the dice~ced tissue
undergo uncontrolled cell proliferation. A key event in this cell proliferation is the
initiation of DNA replication. Inhibiting the initiation of DNA replication through
inhibition of ORC function provides a valuable target for inhibitors of cell growth.
By e,~p,essing each of the cDNAS encoding the ORC proteins, either individually
or together in an c;Apre~ion system, ORC function is reconstituted in vitro. Using
this recombinant, expressed protein, inhibitors of ORC function are obtained that
block the initiation of DNA replication in cell cycle. As described above, smallmolecular inhibitors of ORC DNA binding or other activities provide valuable
reagents as anti-cancer and anti-proliferation drugs.
The following examples are offered by way of illustration and not by way
of limitation.
EXAMPLES
Fy~rnple 1.
Transcriptional silencing and ORC.
The binding of purified ORC to the ARS consenc~ls sequence (ACS) at each
of the mating type silencers was tested using a DNase I protection assay (22).
ORC p,ulecled the match to the ACS at each of the four silencers in an ATP
dependent manner. In addition, at each silencer characteristic hypersensitive sites
of DNAse I cleavage were observed initiating 12-13 bp from the ACS and
extending away from the conc~ncuc sequence at approximately 10 bp intervals.
This pattern of DNase I l,iol~;Lion and enhanced cleavage is nearly identical to that
observed at non-silencer sequences and indicates that ORC binding to these
elements is not fund~mentally different from its binding at other ARS elements.
At HML-E, HML-I, and HMR-E the only protection observed included the
ACS. At HMR-I, however, we observed a second unexpected footprint that did
not overlap a strong match to the ACS. Moreover, unlike all previous sites boundby ORC, this p~oleclion showed little dependence upon the addition of ATP to the
11
WOgS/16694 2 1 7896~ PcT/uS94/14563
binding reaction. Although there are two partial matches to the ACS in this
region, similar sequences in other ARS elements and silencers were not recognized
by ORC, suggesting that these sequences did not direct this unusual ATP-
independent binding of ORC to DNA. In combination with the proteclion observed
5 at the ACS, the boundaries of the ORC footprint at HMR-I were very similar to
the boundaries of HMR-I defined by deletion mutagenesis (23). These experiments
demonstrate that ORC binds all four of the mating-type silencers, that ORC can
bind sequences other than the ACS and that it plays an important role at HML andHMR.
A clear link between ORC function and transcriptional ~ilen~in~ was
provided by the finding that a mutation in a gene encoding a subunit of ORC was
defective for lepression at HMR (below). To clone the genes encoding the variousORC subunits, peptides derived from each of the ORC subunits were sequenced
(24). A candidate gene, referred to as ORC2, was isolated by complementation of
15 a ~"")el~ture sensitive mutation that showed silencing defects at the permissive
le-"~ lu~e. Genetic experiments suggested that ORC2 me li~ted the silencing
function of the ACS at HMR-E, making it a good candidate to encode a subunit of
ORC (below). Comparison of the predicted amino acid sequence of ORC2 showed
that all of the peptides derived from the 72 kd subunit of ORC were within the
20 open reading frame of the ORC2 gene indicating that it encoded the second largest
subunit of ORC.
ORC2 mutations alter ORC function in vitro.
To address the effect of ORC2 mutations on ORC function in vitro, extracts
were p~epaled from both orc2-1 and ORC2 strains (25). Fractions derived from
25 wild-type cells showed strong ORC DNAse I protection over the ACS and B1
elementc of ARSl in DNAse I footprinting. In contrast, fractions derived from
orc2-1 cells showed a dramatic reduction in ORC DNA binding activity. The ACS
and the B1 element were no longer protected from DNase I cleavage. Only the
char~t~ri~tic enhanced DNase I cleavages in the B domain of ARS1 rem~in~d.
30 Mutations that disrupt ORC DNA binding at ARS1 prevented the residual DNA
binding observed with the mutant fractions, indicating that this binding required the
ACS. The DNA binding defects were also not due to a general inhibition of DNA
binding as mixing of mutant and wild type fractions did not reduce binding of the
12
wo gsl16694 2 1 7 8 9 6 5 P~ ,5 ~rl4s63
wild type protein. Incubation of the mutant cells at the non-permissive te"~peldlure
was not necesc~ry to observe defects in ORC DNA binding, which explains the
defect observed in mating-type regulation at the permissive te",pt;ldture (below).
To investig~te the polypeptide composition of ORC derived from orc2-1
5 and ORC2 cells, immuno-blots of these fractions were probed with polyclonal
antibodies raised against ORC. 30 ~g of partially purified ORC derived from
either JRY3688 (ORC2) or JRY3687 (orc2-1) was separated on a 10% SDS-
polyacrylamide gel and transferred to nitrocellulose. The resllltin~ protein blot was
incub~ted with polyclonal mouse sera raised against the entire ORC complex. This10 sera detects all but the 50 kd subunit of ORC. Antibody-antigen complexes were
detected with horseradish peroxidase conjugated secondary antibodies followed byincubation with a chemiluminescent substrate.
Wild type fractions contained the 120, 72, 62, 56, and 53 kd subunits of
ORC in roughly equal quantity. The mutant fractions, however, showed a
15 distinctly different subunit composition. While the amount of the 120 and 56 kd
subunits was only slightly reduced relative to the wild type fraction, the amount of
the 72, 62, and 53 kd subunits was reduced dramatically. In UV cross-linking
experiments the same three subunits are specifically cross-linked to DNA in an
ACS and ATP dependent manner, suggesting an important role for one or more of
20 these ~ubunils in ORC DNA binding (15). Thus, the absence of these subunits
explains the defects in DNA binding observed in vitro and indicates that the orc2-1
mutation results in a reduction of ORC stability or a defect in Orc2p also results in
reduced DNA binding of an intact ORC complex.
orc2-1 cells are defective for entry into S-phase.
The point in the cell cycle the essential function of ORC2 is pelrolllled in
vivo was investig~t~d using alpha factor and hydroxyurea (HU) as cell cycle
l~n-lm~rkc (26). Our results were consistent with the execution of the essentialfunction of Orc2p between late Gl and the initiation of DNA synthesis. Arrest
with HU followed by release into the non-permissive lelllpeldlul~ resulted in 89%
of the cells completing an addition~.' cell cycle, indicating that the essential function
for Orc2p was executed before the J arrest point in the cell cycle. In contrast,blocking the cell cycle with alpha-factor followed by release at the non-permissive
le",peldture resulted in the only 41 % of the cells completing an additional cell
13
WO 95/16694 2 1 7 8 '~ 6 5 PCT/US94/14563
cycle. This phenotype indicates that the Orc2p function was ~l~ll,led at or nearthe Gl-S phase boundary.
To address the role of ORC in yeast DNA replication more directly, the
DNA content of asynchronous cultures of either orc2-1 or isogenic wild type cells
5 was measured at various times after shifting from the permissive to the non-
permissive tellll)e,dture by fluorescent cytometric analysis (27). JRY3687 (orc2-1)
or JRY3688 (ORC2) cells grown at 24C (0 minute time point) or at various times
after shifting to the non-permissive tel-.pel~ re (37C) were fixed, stained with
propidium iodide, and analyzed for DNA content using a Coulter Model Epics-C
10 Flow Cytometer. In addition, a small number of cells (app~o~imately 1000) from
each time point were returned to the permissive le-..peldture to determine the
pe~ent~ge of cells that rem~inecl viable at a given time point. Initially, the DNA
content of both wild type and mutant cells was equally divided between lC and 2Cwith approximately 10% of the cells in S phase. At early time points after the
15 telllpeldlure shift (15-70 minutes) there was a dramatic loss of orc2-1 cells in S-
phase suggesting that entry into S-phase had been halted. Consistent with this
hypothesis, as the time course continued the orc2-1 mutant showed a rapid
accumulation of cells with a lC DNA content and a commencurate decrease in
cells with a 2C DNA content (50-100 minutes). Between l00 and 120 minutes, a
20 new population of orc2-1 cells was observed that appeared to enter into a delayed S
phase. By 150 minutes the bulk of the mutant cells were in this population and
after 180 minutes Qnly a few cells rem~ine~ with a lC DNA content.
Interestingly, we observed a strong correlation between entry into the new
round of DNA synthesis and a loss of orc2-1 cell viability. Similar experiments
25 with isogenic ORC2 cells showed that these effects were specific to the orc2-1
mutation. These findings indicate that at the non-permissive temperature the orc2-
1 cells were initially unable to enter S phase, but later entered into an abortive
round of DNA replication. Entry into this type of replication appears to be a lethal
event. Overall, the analysis of the orc2-1 mutation provides in vivo evidence
30 showing that ORC acts early in S-phase in general, and as the initiator protein at
yeast origins of replication in particular.
Identification of the ORC6 gene.
14
wo 95/16694 2 1 7 8 9 6 5 PCTtUS94/14563
A second gene that represen~ed a strong candidate to encode one of the
subunits of ORC was the AAPI gene. This gene was cloned using a novel screen
for proteins that bound to the ACS in vivo (below). When col"pa~t;d to the
predicted amino acid sequence of this gene, we found that all of the peptides
5 derived from the 50 kd subunit of ORC were encoded by the open reading frame
of the AAPI gene (28). For this reason we now refer to AAPI as ORC6, as it
encodes the sm~ st of the six ORC subunits. The identification of this gene as asubunit of ORC provides direct evidence that ORC is bound to the ACS in vivo.
Numbered Citations for Introduction and Example 1
1. Callan, Cold Spring Harbor Symp. Quant. Biol. 38, 195-203 (1973).
2. F~gm~n and Brewer, Cell 71, 363-366 (1992).
3. P. Laurenson and J. Rine, Micro. Rev. 56, 543-560 (1992).
4. D. D. Dubey, et al., Mol. Cell. Biol. 11, 5346-5355 (1991).
5. D. H. Rivier and J. Rine, Science 256, 659-663 (1992).
6. A. M. Miller and K. A. Nasmyth, Nature 312, 247-251 (1984).
7. L. Pillus and J. Rine, Cell 59, 637-647 (1989).
8. A. Axlerod and J. Rine, Mol. Cell. Biol. 11, 1080-1091 (1991).
9. J. L. Campbell and C. S. Newlon, in The Molecular and Cel1ul(7r Biology
- of the Yeast Saccharomyces J. R. Broach,~J. R. Pringle, E. W. Jones, Eds. (Cold
20 Spring Harbor Labol~toly Press, Gold Spring Harbor, NY, 1991) pp. 41-146.
10. J. Broach, et al., CSH Symp. Quant. Biol. 47, 1165-1173 (1983).
11. Deshpande and Newlon, Mol. & Cell. Bio 12, 4305-4313 (1992).
12. Y. Marahrens and B. Stillman, Science 255, 817-823 (1992)
13. S. P. Bell and B. Stillman, Nature 357, 128-134 (1992).
14. Kornberg & Baker, DNA Replicat'n (Freeman & Co, NY,1991) v2.
15. C. S. Newlon, Microbiol. Rev. 52, 568-601 (1988).
16. Newlon and Theis, Current opinion in gen. and dev. 3, (1993).
17. J. F. X. Diffley and J. H. Cocker, Nature 357, 169 172 (1992).
18. Jacob, et al., CSH Symp. Quant. Biol., 28, 329-348 (1963).
19. DNAse I footprinting was pelro""ed as previously described (15).
20. J. B. Feldman,et al., J. Mol. Biol. 178, 815-834 (1984).
21. To obtain sufficient protein for peptide sequencing, a revised pllnfic~tionprocedure for ORC was devised. Whole cell extract was p~epal~d from 400g of
wo gS/16694 2 1 7 8 q 6 5 PCT/US94/14563
frozen BJ926 cells using a bead beater (Biospec Products) until greater than 90%breakage was achieved. One twelfth volume of a saturated (at 4C) solution of
ammonium sulfate was added to the broken cells and stirred for 30 minutes. This
solution was then spun at 13,000 x g for 20 minutes. The resulting supernatant
5 was spun in a 45Ti rotor (Reckm~n) at 44,000 RPM for 1.5 hrs. 0.27g/ml of
ammonium sulfate was added to the resulting supernatant. and the resulting
precipitate was collected by spinning in the 45 Ti rotor at 40,000 RPM for 30
minutes. The res~-lting pellet was resuspended in buffer H/0.0 (15) and dialyzedversus H/0.lSM KCI (H with 0.15 M KCl added). Preparation of ORC from this
10 extract was similar to (15) with the following changes. The dsDNA cellulose
column was omitted from the prepaMtion and only a single glycerol gradient was
pelrol---ed. Sequencing of peptides derived from ORC subunits was performed
using a modification of an "in gel" protocol described previously (40, 41).
Purified ORC (~ 10 ~g per subunit) was separated by SDS-PAGE and stained with
15 0.1 % Coomassie Brilliant Blue G (Aldrich). After dest~ining the gel was soaked
in water for one hour. The protein bands were excised, transferred to a
microcentrifuge tube and treated with 200 ng of Achromobacter protease I
(Lysylendopeptidase: Wako). The resulting peptides were separated by reverse-
phase chromatography and sequenced by automated Edman degradation (Applied
20 Biosystems model 470).
22. To isolate and assay ORC from ORC2 and orc2-1 cells four liters of
JRY3687 (orc2-1, MATa, hmrDA::TRPI ade2 his3 leu2 trpl ura3) or the isogenic
wild-type strain JRY3688 (ORC2 MATa, hmrDA::TRPI ade2 his3 leu2 trpl ura3)
were grown to a density of 2 x 107 cells per ml. Extracts were prepared as
25 described (24) and fractionated over the first two columns in the preparation of
ORC. The peak fraction of ORC DNA binding activity eluted from the Q-
Sepharose (Pharmacia) column of each preparation was used for subsequent
analysis. Antibodies were raised against the entire ORC complex using a single
mouse. The resulting sera was able to recognize all but the 50 kd subunit of ORC.
30 Proteins were transferred to nitrocellulose and antigen-antibody complexes were
detected with horse radish peroxidase conjugated secondary anitbodies and a
chemiluminescent substrate.
16
wo 95/16694 2 1 7 8 9 6 5 PCT/US94114563
23. Yeast cells were grown to a density of 1-4 x 107 cells per ml at 24C then
diluted to a density of 2-4 x 106 cells per ml into YPD containing 6 ~M alpha-
factor and incub~ted for 2-2.5 hours at 24C (> 90% unbudded cells). For the
hydroxyurea arrest experiments alpha factor was washed away and the cells were
S res~-~pen-iecl in YPD conl;~ining 100 mM hydroxyurea and incubated an additional
2.5 hours (> 90% large budded cells). After incubation with the growth
inhibitor, cells were briefly sonicated and plated on YPD plates pre-incubated at
either 24C or 37C and observed at 0, 3, and 6 hours after plating.
24. Yeast cells were grown to a density of 1-4 x 107 cells per ml at 24C and
10 diluted into fresh YPD at either 37C or 24C and a density of 2-4 x 106 cells per
ml. At times after dilution, 3 x 106cells were processed as described (42).
25. The position of the five peptides derived from the 50 kd subunit of ORC in
the ORC6 gene were residues: 51-65; 91-102; 110-105; 207-226; 424-430.
26. K. M. Hennessy, et al, Genes Dev. 4, 2252-2263 (1990).
27. H. Renauld, et al., Genes Dev. 7, 1133-1145 (1993).
28. A. H. Brand, G. Micklem, and K. Nasmyth, Cell 51, 709-719 (1987).
29. McNally and Rine, Mol. an~ Cell. Biol. 11, 5648-5659 (1991).
30. D. D. Brown, Cell 37, 359-365 (1984).
31. A. P. Wolffe, J. Cell Sci. 99, 201-206 (1991).
32. D. Kitsberg, et al., Nature 364, 459-463 (1993).
33. K. S. Hatton, et al., Mol. Cell. Biol. 8, 2149-2158 (1988).
34. V. Dhar, et al., Mol. Cell. Biol. 9, 3524-3532 (1989).
35. L. G. Edgar and J. D. McGhee, Cell 53, 589-599 (1988).
36. L. P. Villarreal, Micro. Rev. 55, 512-542 (1991).
37. H. Kawasaki, et al., Anal. Biochem. 191, 332-336 (1990).
38. H. Kawasaki and K. Suzuki, Anal. Biochem. 186, 264-268 (1990).
39. R. Nash, et al., EMBL Journal 7, 4335-4346 (1988).
40. J. Abraham, et al., J. Mol. Biol. 176, 307-331 (1984).
41. D. T. Stinchcomb, et al., Nature 282, 39-43 (1979).
Example 2.
ORC2, a gene required for viability and silencing
wo 95/16694 2 1 ~ 8 9 6 5 p~US94114563
In a mutant screen, a ~I~peldture-sensitive mutation called orc2-1 was
isolated that, at the permissive temperature, resulted in derepression of HMRa
flanked by the synthetic ~il.oncer and did not cause dercples~ion of HMRa flanked
by the wild-type silencer (20). Rer~usP the orc2-1 mutant was temperature-
S sensitive and silencing defective, it mPrit~d further analysis. The telll~ldturere~ict~nce of a heterozygus orc2-1/ORC2 diploid (JRY2640) established that themutation was recessive. The diploid was transformed with a plasmid cont~ining
HMRa flanked by a mutant silencer (pJR1212), to provide MATal function
required for sporulation. The telll~ldture-sensitive growth phenotype segregated 2
10 ts: 2 wild type in each of 23 tetrads, indicating that it was caused by a single
nuclear mutation. An HMLo~ matal HMRo~ orc2-1 segregant aRY3683) was
obtained from the diploid following sporulation.
Genetic crosses were used to determine which features in the wild-type
silencer distinguished it from the synthetic silencer with respect to derepression by
15 orc2-1. A matal HMRo~ strain (JRY3683) containing the orc2-1 mutation was
mated to a MATo~ strain cont~ining a mutation in the RAPl binding site of HMR-E
fl~nking HMRa (the HMRa-e-rapl-10 allele; 5401-la) to determine whether orc2-1
could dclcl~lcss HMRa in the absence of a functional RAPl binding site. All 29 of
the 96 MATo~ segregallts that had little or no mating ability were ~elllpeldture-
20 sensitive for growth. Nineteen of the MATo~ lel"~ldture-sensitive seg~cgant~ were
mating colllpetent, indicating that the orc2-1 mutation per se was in~ufficient to
disrupt mating ability, and suggesting that the HMRa-e-rapl-10 allele was required
in combination with orc2-1 to block mating ability of ~ strains. A MATo~
telllpeld~ure-sensitive segregant from this cross, which mated weakly as an cY
25 (JRY4133), was confirmed to have the genotype MATo~ HMRa-e-rapl-10 orc2-1.
As further evidence that orc2-1 in combination with HMRa-e-rapl-10
blocked the mating ability of MATa strains, a somewhat unusual cross was used tosimplify the previous cross by having orc2-1 as the only relevant heterozygous
marker. Two MATo~ HMRa-e-rapl-10 strains (JRY4133 and JRY4132) had
30 complementary auxotrophic markers, allowing for the selection of the rare
MAT~IMATo~ diploid formed by a mating event between these two strains. This
diploid was able to sporulate due to the low level of e~l)lession of HMRa in thediploid caused by the RAPl-site mutation in the HMR-E silencer (21). One of
18
wo 95/16694 2 1 7 8 9 6 5 PCT~ s94~l4s63
these strains had the orc2-1 mutation (JRY4133) and the other did not. As
expected, the le~ dture sensitivity segregated 2:2 in each of 34 tetrads. All ofthe ~ll~ tule-resistant segle~ts (two per tetrad) exhibited the ~ mating
phenotype, and all of the t~ ture-sensitive segregants were either very weak
5 ~-maters or were unable to mate at all. The absence of any recombinants between
the l~"~ldture sensitivity and mating phenotype placed the gene(s) responsible for
the lelll~ldlu~ sensitivity and the mating defect less than 1.5 centimorgans apart,
providing strong evidence that a lesion in a single gene was responsible for both
phenotypes. This result was in agreement with the co-reversion of the ts and
10 mating phenotypes described herein.
Isolation of multiple alleles of ORC2
Using the information from this analysis of orc2-1, a second screen was
o-llled to identify additional mutations in essential genes with a role in silencer
function. This second screen produced 50 mutants that were temperature sensitive15 for growth, and in which HMRo~ (flanked by a mutation in the RAP1-binding site)
was derepressed at a semi-permissive temperature. Complementation tests for bothgrowth at 37C and for mating phenotype were performed between orc2-1 and the
collection of temperature-sensitive mutants from the second screen. The collection
of te"-~lature sensitive mutants had the matal stel4 genotype, but were able to
20 mate as ~'s due to the derepl~;ssion of HMRo~. These mutants were mated to a
matal orc2-1 strain (JRY3683) and the diploids were tested for growth at 37C.
All but three diploids were able to grow at the restrictive te...~ldture. The three
temperature-sensitive diploids were each presumed to be orc2/orc2 homozygotes
due to the inability of the two mutations to complement one another. The mating
type of the diploids was che~k~l to determine whether the defect in repression of
HMR was complemented. All three diploids mated as ~'s. Thus, the three
mutants were unable to complement either the ten.~ldture sensitivity or the mating
phenotype of the original orc2-1 mutation. The new mutations (in strains
JRY4136, 4137 and 4138) were decign~t~d orc2-2, orc2-3, and orc2-4.
To investigate the possibility that the new mutations were in a gene other
than ORC2 ~e still failed to complement orc2-1, the allelism between orc2-1 and
orc2-3 was tested. The original matal orc2-3 stel4 mutant was cured of its
HMRo~ plasmid, creating JRY 4137, and mated with a MATo~ HMRa-e-rapl-10
19
wo 95/16694 - ~ l 7 ~ q 6 5 Pcr~US94/14563
orc2-1 strain aRY3685). In 24 tetrads from this diploid, all segr~ants were
le",peldture sensitive for growth, intli~.~ting strong linkage between orc2-1 and
orc2-3 (~2 centimorgans). All further studies were pelrol,--ed using the orc2-1
allele, which provided the stronger mutant phenotypes.
Map position of ORC2
Linkage between ORC2 and LYS2, on chromosome II, was evident in
crosses between two Iys2 strains (JRY2640 and PSY152) and the original orc2-1
isolate aRY2903) that placed ORC2 approximately 24 centimorgans from LYS2.
A third cross (JRY4130 x JRY4134) tested the linkage between secl8, which is
centromere proximal to LYS2, and ORC2. Re~ se both orc2-1 and secl8 are
~e..,peldture sensitive, an ORC2 allele marked by URA3 (from pJR1423) was used
to determine that SEC18 and ORC2 were separated by 6.6 centimorgans (Table 1).
No previously-mapped genes involved in silencing map near SEC18.
Table 1. Linkage of ORC2 to LYS2 and ORC2 to SEC18
Tetrad types Map
distadnce
Cross PD T NPD (cM
ORC2 vs LYS2 10 14 0 29
ORC2 vsLYS2 20 14 0 21
ORC2 vs LYS2 TOTAL 30 28 0 24
ORC2 vs SEC18 46 7 0 6.6
The ORC2 mutants arrested with a cell cycle terminal phenotype.
The effect of the orc2-1 mutation on the cell division cycle was explored:
25 mutant orc2-1 strains were grown in liquid medium at 23C, the permissive
t~lllpelature~ and then shifted to 37C to test whether the cells arrested with a
single lel",inal morphology. Specifically, orc2-1 cells (JRY3683) were grown to
log phase at the permissive lelllpela~re (23C) and the culture was split. Half of
the culture was grown an additional five hours at the permissive l~--l~;ldture and
30 the other half was shifted to the nonpell,lissive telllpeldture (37C) and grown for
an additional five hours. At that time, both cultures were fixed and stained with
DAPI to allow visll~li7~tion of the nucleus. In the culture m~int~ined at the
permissive lelllpeldture, cells at all phases of the cell cycle were observed. Cells
wo 95/16694 2 ~ 7 ~ 9 6 5 PCT/US94/14563
later in the cell cycle, as evidenced by the presence of large buds, frequently
exhibited nuclei in both the mother and the daughter cell. In contrast, in the
culture shifted to the restrictive ~e-,-peldture, approximately 90% of the cellsarrested as large budded cells. Nuclei were only present in the mother cell and not
- 5 in the daughter cells. In addition, the cells were larger than those grown at the
permissive le"~peldture, indicating that protein synthesis and cell wall synthesis
continued in the absence of ORC2 function. Similar results were obtained with two
additional orc2-1 strains aRY3685 and JRY36~7).
ORC2 cells harvested either after continuous growth at the permissive
l~;-.-~:ldtule or after a shift to the nonpelmissive len.pe~dlule were fixed andstained with DAPI allowing vi~ li7~tion of DNA with fluorescence microscopy.
The cells grown permissively displayed a range of morphologies from small
unbudded cells to cells with single buds of various sizes. The cells shifted to the
nonpel--,issive ~e"~peldture looked very different: the majority arrested as large
budded cells, and for the most part, each mother-daughter pair contained only a
single brightly-staining region, often at or near the neck. These data in-iic~tP~ that
orc2-1 mutants displayed cell cycle defects characteristic of mutants defective in
DNA replication.
Cloning of the ORC2 gene:
The ORC2 gene was cloned by complementation of the orc2-1 temperature
sensitivity (22). One complementing clone (pJR1416) was chosen for further
analysis. Subclones mi~cing various fragments from the insert were retransformedinto an orc2 strain to assay whether the deletion affected the clone's ability to
complement orc2-l 's te"~ ture sensitivity. The key observations were that the
deletion of a 2.8-kb SstI-SstI fragment destroyed complementation activity, whereas
the deletions of fl~nking sequences (XbaI, and the largerSstI fragment) had no
effect. The 2.8-kb fragment was subcloned (pJR1263), and shown to possess
complemP-nting activity.
To determine whether the gene on the clone was indeed allelic to the ORC2
mutation, a fragment of the original clone was subcloned into a yeast integrating
vector. This plasmid (pJR1423) was cleaved within the insert to direct homologous
inlegl~lion and transformed into a wild-type strain (W303-lA). As a result, the
21
wo 95/16694 ~ 2 1 7 8 9 6 5 P,~lus94ll4s63
site of integration was marked by the plasmid's URA3 gene. The resulting strain
(JRY4134) was crossed to an orc2-1 strain (JRY3685). In each of 59 tetrads,
URA3 seg~cgated opposile to the te,."~ldt~lre sensitivity caused by orc2-1,
indicating that ORC2 had indeed been cloned.
ORC2 was essential for cell viability.
ORC2 was disrupted by URA3, (23), and integrated into a diploid
homozygous for ura3 and ORC2, (JRY3444). Of the 41 tetrads ~i~cect~, 40
tetrads had two live and two dead segr~ants, and one tetrad had only one live
segregant. The colonies that grew were, without exception, Ura-. By inference,
10 the dead segregants contained the URA3 gene, and thus the ORC2 disruption,
indicating that ORC2 function was essential for cell viability at all temperatures.
The dead se~l~ants were .o~mined under a microscope to gain some insight into
the true null phenotype. Most of the spores germinated into cells that were
elongated or otherwise deformed and had not divided. In no case did the cell
15 divide more than two times. Thus in many spores, the absence of ORC2 blocked
cell division but not growth.
Role of ORC2 in Plasmid Replication
To test the role of ORC2 in plasmid stability, an isogenic pair of strains,
one wild type (W303-lB) and one orc2-1 (JRY4125), were transformed with a
2.0 plasmid cont~ining a centromere, a supp~essor tRNA (SUPII-I), URA3, and
ARS1, a chromosomal origin of replication (YRP14/CEN4/ARS1/ARS1; (24, 25),
s~l~ting for uracil pr~totlophy. Transrol-,-ants were grown on selective medium
at 23C, the permissive te~peldture for orc2-1. The colonies were picked from
the selective plate, serially diluted, plated onto solid rich medium and grown to
25 colonies at 23C. The wild-type tran~ro~l"ants grew into colonies most of which
were white with a few exhibiting red sectors. The small fraction of red colonieswere from cells in the selectively grown colony that had lost the plasmid. In
contrast, the majority of colonies from the orc2-1 mutant were red, reflecting ahigh degree of plasmid loss among the cells in the selectively grown colony.
30 Moreover, in the orc2-1 strain, red sectors were present in the majority of white
colonies with some white colonies displaying multiple red sectors.
It is possible to quanlilate the number of cell cycles in which a plasmid is
lost from the number of colonies that are half red and half white. Only those
WO 95/16694 2 1 7 8 9 6 5 PCT/US94114563
colonies that lose the plasmid in the first cell division form half red, half white
colonies. In the case of the wild-type strain, 0.9 % (10 / 1168) of the colonieswere half red and half white, indicating that the plasmid was lost in 0.9 % of cell
cycles. In contrast, the frequency of half red and half white colonies in the orc2-1
- S strain grown at the permissive le~,dture was 11% (58 / 512), in~ic~ting that the
same plasmid was lost appro~cimately 12 times as often in the strain with partially
defective Orc2p. These data indicated a profound defect in plasmid stability
specific to the orc2-1 strain, and in combination with the cell-cycle phenotype of
orc2-1, suggested that orc2-1 strains were defective in DNA replication. These
results were consistent with the flow cytometry studies of orc2-1 strains herein.
Sequence of ORC2
The sequence of the 2.8-kb SstI-SstI orc2-complementing fragment was
determined and deposited in Gçnb~nk (Accession #L23924). The only open
reading frame of significant length was deduced to be ORC2, and predicted a 620
residue protein of approximately 68 kD. The SstI fragment included 806-bp of
upstream sequence and 140-bp of downstream sequence.
The deduced Orc2p protein was 15% basic residues and 16%
serine/threonines. Fully 50% of the N-terminal residues (residues 15 280) were
lysine, arginine, proline, serine, or threonine. The KeyBank motif program
- 20 revealed several matches to peptide motifs within Orc2p. Orc2p contained many
potential phosphorylation sites: 3 for cAMP- and cGMP-dependent protein kinase
(starting at residues 57, 433 and 546), 12 for protein kinase C (24, 41, 42, 89,101, 102, 176, 321, 335, 431, 521, and 549) and 14 for caseine kinase II (60, 148,
149, 182, 238, 270, 389, 481, 486, 491, 505, 552, 595, and 605), and match to
25 the nuclear ~r~,~ling sequence (residues 103-107). A perfect match to the RAP1
binding site concenC~s (starting at nucleotide 595), and two near ."~t~hPs (12/15) to
the ABF1-binding concensus sequence (starting at 12 and 609). It was determined
by sequence homology that a lysyl tRNA synthet~ce gene is located to the left ofthe Sstl fragment shown here (MiMnde and Waller, 1988), and a kinase homolog
30 to the right.
Another homolgy is with the region near the catalytic domain of human
topoisomeMse I proteins which has diverged among topoisomeMse I proteins from
other species except for the region surrounding the invariant active-site tyrosine.
23
WO 95/16694 2 1 1 8 9 6 5 PcTlus94ll4563
This region includes a conC~-nCl~c sequence consicting of a serine and lysine residue
near the tyrosine (25). The Orc2p protein also contained such a con~Pn~us
sequence near its C-terminus. However, mutation of this putative active-site
tyrosine to phenylalanine had no detectable effect on the ability of ORC2 to
5 complement the te~lpe,dture-sensitivity or mating defect of an orc2-1 strain.
Table 2. Strain list.
Strain Genotype ( ~
DBY1034 MATa his4-539 Iys2-801 ura3-52 SUC
W303-lA MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-l
ura3-1
W303-lB MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-l
ura3-1
PSY152 MATa his3D200 leu2-3,112 Iys2-801 ura3-52
JRY4130 MATa his4 ura3 secl8
JRY438 MATa Gal+ his4519 leu2-3,112 SVC2 ura3-52
JRY543 MATalMATa ade2-101/ade2-101 his3~200/his3~200
Iys2-801/lys2-801 met2/MET2 TYRI/tyrl
ura3-52/ura3-52
- JRY2640 matal ade2 leu2-3,112 Iys2-801 ura3
JRY2698 MATa HMRa ade2-101 his3 leu2 trpl ura3-52
JRY2699 MATa HMRa ade2-101 his3 leu2 trpl ura3-52
sir4DN::H153
JRY2700 MATa HMRa ade2-101 his3 leu2 trpl ura3-52
+ pJR924
JRY2903 MATa HMRa ade2-101 his3 leu2 orc2-1 trpl ura3-52
JRY2904 MATa HMRa ade2-101 his3 leu2 orc2-1 trpl ura3-52
+ pJR924
JRY3444 MATalMATa ade2-101/ade2-101 his3D200mis3D200
Iys2-801/lys2-801met2/MET2 TYRI/tyrl
ura3-52/ura3-52 orc2::Tnl OLUK/ORC2
JRY3683 matal {HMRa} ade2 his3 leu2 orc2-lura3
JRY3685 MATa HMRa-e-rapl -10 ade2 leu2 trpl orc2-1 ura3
JRY3687 MATa hmrDA::TRPI ade2 his3 leu2 trpl ura3 orc2-1
24
wo 9S/16694 2 ~ 7 8 9 6 5 Pcrluss4ll4563
JRY3690 MATa HMRa-e-rapl-10 ade2 his3-11,15 leu2 orc2-1
trpl ura3
JRY4125 MATa ade2-1 canl-100 his3-11,15 leu2-3,112 orc2-1
trpl-1 ura3-1
JRY4132 MATol HMRa-e-rapl-10 ade2 his3 ura3
JRY4133 MATo~ HMRa-e-rapl-10 ade2 leu2 orc2-ltrpl ura3
JRY4134 MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-1
ura3-1 ORC2:.~JR1423
JRY4135 matal ade2 leu2-3,112 Iys2-801 ura3 stel4
JRY4136 matal ade2 leu2-3,112 Iys2-801 orc2-2 ura3 stel4
JRY4137 matal ade2 leu2-3,112 Iys2-801 orc2-3 ura3 stel4
JRY4138 matal ade2 leu2-3,112 Iys2-801 orc2-4 ura3 stel4
(a) Unless otherwise noted, all strains were HMLo~ and HMRa. HMRa-e-
15 rapl-10 refers to the allele of HMR-E, originally described as PAS1-1, that
contains a mutatlon in the RAPl binding site (21).
Numbered Citations for Example 2.
1. . I. Herskowitz, et al Cold Spring Harbor Laboratory Press 583 (1992).
20 2. J. Abraham, J. Feldman, K.A. Nasmyth, J.N. Strathern, J.R. Broach, and
J. Hicks, C.S.H. Symp. Quant. Biol. 47, 989 (1982). J.B. Feldman, J.B. Hicks,
and J.R. Broach, J. Mol. Biol. 178, 815 (1984).
3. J. Rine, and I. Herskowitz, Genetics 116, 9 (1987).
4. Kurtz et al, Genes Dev. 5, 616 (1991); Sussel et al, PNAS 88, 7749 (1991).25 5. J.R. Mullen, et al, PMBO J. 8, 2067 (1989).
6. P.S. Kayne, et al, Cell 55, 27 (1988). L.M. Johnson, et al, Proc. Natl.
Acad. Sci. USA 87, 6286 (1990). P.D. Megee, et al, Science 247, 841 (1990). E.
Park, and J. .Szost~k Mol. Cell. Biol. 10, 4932 (1990).
7. P. Laurenson, and J. Rine, Microbiol. Rev. 56, 543 (1992).
30 8. Brand, et al., Cell 41, 41 (1985); Kimmerly, et al., EMBO J. 7, 2241
(1988).
9. D. Shore, and K. Nasmyth, Cell 51, 721 (1987).
21 7~i~65
WO 95/16694 : PCTtUS94/14563
10. M.S. Longtine, et al., Curr. Genet. 16, 225 (1989).
11. A.R. Buchman, et al, Mol. Cell. Biol. 8, 5086 (1988).
12. J.F.X. Diffley, and J.H. Cocker, Science 357, 169 (1992).
13. A.S. Buc~m~n, and R.D. Kornberg, Mol. Cell. Biol. 10, 887 (1990).
14. J.A. Huberman, et al, Nucleic Acids Res. 16, 6373 (1988). B.J. Brewer,
and W.L. F~ngm~n, Cell 51, 463 (1987).
15. S.P. Bell and B. Stillman, Nature 357, 128 (1992).
16. F.J. McNally, and J. Rine, Mol. Cell. Biol. 11, 5648 (1991).
17. A.M. Miller, and K.A. Nasmyth, Nature 312, 247 (1984).
18. D.H. Rivier, and J. Rine, J. Science 256, 659 (1992).
19. Two genetic screens were devised to identify temperature sensitive
mutations in essential genes involved in silencing. The screen that led to isolation
of orc2-1 started with JRY2698 (HMLo~, MATo~, HMRo~, ade2, his3, leu2, trpl,
ura3-52), which had a mating-type c~settes at all three chromosomal mating-type
15 loci and was transformed with a plasmid (pJR924) containing the a mating-typec~ccette at HMR (JRY2700). The plasmid-borne HMRa locus had two synthetic
silencers substituted for the E silencer, and also had a deletion of the I element.
The use of two silencers rather than one minimi7ed the risk of being distracted by
site mutations in the silencer. One hundred and sixty two thousand colonies of
20 EMS-mutagenized colonies were grown on supplemented minimal media (without
uracil) at 25C and screened for del~pres~ion of the plasmid-borne a c~sette at
HMR. Mutagenized colonies were replica-plated onto lawns of the mating tester
strain DBY1034 (MATa, his4539, Iys2-801, ura3-52) on minimal media either
- with or without uracil supplementation. Replicas were incubated at 25C for one
25 hour, then overnight at 30C. Only plasmid-containing JRY2700 cells were able to
mate with the tester strain to yield diploids capable of growing on the
unsupplemented plates because the only functional URA3 gene was on the plasmid.
Cells bearing mutations causing de~epr~ssion of the plasmid-borne a
c~sette could be distinguished from the other classes of mutations by exploiting a
30 feature of yeast pl~cmi-ls. Approximately 10% of the cells in these colonies lacked
the plasmid and thus could, in principle, mate with the tester strain and form Ura~
diploids capable of growth on the plates supplemented with uracil. If a colony had
a mutation in the mating response pathway, the cells would be unable to mate even
26
WO95/16694 2 1 78965 PCT~US94/14563
in the absence of the plasmid, and thus would be unable to form diploids capableof growth on medium supplemented with uracil. Twenty eight strains were
identified that were te",~ld~ule-sensitive for growth and that mated with the tester
strain only on plates supplemented with uracil. Plasmid-free isolates of each strain
S were then retransformed with the plasmid bearing the synthetic ~ nc~r at the
HMRa locus (pJR924) and with the plasmid bearing the wild-type HMRa locus
(pJR919; McNally and Rine, 1991). Three strains were able to mate when
carrying the wild-type HMR locus (pJR919) but not when carrying the synthetic
silencer-con~ining HMR locus (pJR924). In order to determine if the ts growth
10 phenotype and the mating phenotype were due to the same mutation, spontaneousrevertants of the ts phenotype were selected. A spontaneous revertant of the ts
growth of one strain, JRY2904, mated as well as the wild-type JRY2700,
suggesting that the mating phenotype and temperature-sensitive growth were due to
the same mutation which was named orc2-1.
15 20. Y. Kassir, et al, Genet. 109, 481 (1985). Foss and Rine, Genetics. (1993)
21. The ORC2 gene was cloned by complementation of the tempelature
sensitivity of orc2-1. An orc2-1 strain (JRY3683) was transformed with a CEN
LEU2-based Saccharomyces cerevisiae genomic library (32) Approximately 1000
to 1500 tran~rorlllanls formed colonies at 23C. Replica prints of these colonies
2.0 were incub~t~ at 37C to screen for the ability to grow at elevated te---~l~tures.
Plasmids were isolated from ~e",pel~ture-resistant strains and retested. Those
plasmids that complemented the defect a second time were analyzed by restrictiondigestion. One plasmid from the CEN-LEU2 library (pJR1416) was chosen for
further analysis.
25 22. ORC2 was disrupted with the TnlO LUK transposon (33), which inserted
within the ORC2 coding sequence on the plasmid (pJR1146) carrying the SstI orc2-I complern~nting fragment. Plasmid pJR1147 had the TnlOLUK insertion within
the ORC2 coding region. The ORC2-cont~ining SstI fragment, disrupted by the
transposon, was removed from pJR1147 by partial digestion with SstI. The
30 fragment was transformed into the wild-type diploid JRY543. The integration of
this disruption allele at the ORC2 locus was confirmed by DNA blot hybridizationanalysis (Southern, 1975), and the diploid was named JRY3444.
23. P. Hieter, C. Mann, M. Snyder, and R.W. Davis, Cell 40, 381 (1985).
27
WO g5/16694 2 1 7 8 9 6 5 Pcr,l,S94,l4563
24. D. Koshl~nd, J.C. Kent, and L.H. Hartwell, Cell 40, 393 (1985). R.M.
Lynn, et al, Proc. Natl. Acad. Sci. USA 86, 3559 (1989). W.-K. Eng,S.D.
Pandit, and R. Sterngl~n7, J. Biol. Chem. 264, 13373 (1989).
25, 26. A.H. Brand, G. Micklem, and K. Nasmyth, Cell 51, 709 (1987).
5 27. S. Shuman, et al, Proc. Natl. Acad. Sci. USA 86, 9793 (1989).
28, 29. J. Singh, and A.J.S. Klar, A. J. S. Genes and Dev. 6, 186 (1992).
30. D.D. Dubey, et al, Mol. Cell. Biol. 11, 5346 (1991).
31. C.A. Hrycyna, et al, EMBO J. 10, 1699 (I991).
32. A mutation was introduced into the RAPl binding site at HMR-E ~dj~cçnt
10 to the HMRol locus by oligonucleotide-directed mutagenesis (35), and the change
confirmed by sequencing. The RAPl site mutation was identical to the PASI-l
mutation of HMR-E characterized previously that blocks RAPl protein binding in
vitro (21), and is described here as HMRo~-e-rapl-10. The plasmid con~i~ting of
the HMRo~-e-rapl-10 HindIII fragment in pRS316 was named pJR1425. The wild-
15 type HMRa version of the same plasmid was named pJR1426. Approximately100,000 mutagenized cells from 12 independent cultures of the HMLo~ matal
HMRa stel4 strain with the HMRo~ plasmid (pJR1425) were grown into colonies at
23C and replica-plated to a MATa ura3 mating-type tester lawn (PSY152) to
identify mutants exhibiting the a mating phenotype. The mating plates were
20 incubatçd at 30C in order to identify mutants defective enough to be derepressed
at HMR yet not so defective as to be inviable. Of nine hundred haploid mating
proficient colonies that were picked, fifty mutants were ~elllpeldture sensitive for
growth at 37F to some degree. These mutants were subjected to further study andthe remainder were discarded. All 50 mutants were recessive to wild-type. Only
25 the subset of mutants relevant to ORC2 are presented here; the remainder will be
~i~cucse~ elsewhere.
33. The ORC2 gene was defined by the orc2-1 mutation. An orc2-
complerne~ting plasmid (pJR1416) was obtained by complementation of the
te-"l)eldtulc sensitivity of orc2-1. In order to map the approximate position of the
30 orc2 -complementing gene in the plasmid, six derivatives of pJR1416 were madeand tested for complementation. The SalI-SalI fragment was removed from the
insert to yield pJR1418. Three adjacent XbaI-XbaI fragments were removed to
28
wo 95/16694 2 1 7 8 ~ 6 5 PcTrUSs4/14563
yield pJR1422. SphI cleaved once in the insert and once just inside the vector.
Deleting this SphI-SphI fragment produced pJR1417. Cleavage by SstI released
two fr~gmPnt~ from the insert. Deletion of both fragments created pJR1419.
Isolates in which only the larger SstI fragment (pJR1421) or only the smaller SstI
5 fragment (pJR1420) was deleted were also recovered. The 2.8-kb SstI-SstI orc2-compl~nting fragment was cloned into the SstI site of the CEN URA3 vector
pRS316 (36), to yield pJR1263. Two plasmids were made which allowed the
ch,o",oso",al integration of part or all of ORC2. The first, pJR1423, contained an
XhoIlKpnI insert (from pJR1416) which extended from a few kb upstream of the
10 ORC2 start codon to about 60-bp upstream of the stop codon inserted into X71oI-
KpnI-cut pRS306 (36), a yeast integrating vector marked by URA3. The second
plasmid, pJR1424, contained the SstI orc2-complementing fragment inserted into
the SstI site of pRS306.
34. F. Spencer, et al Genetics 124, 237 (1990).
35. O. Huisman, et al, Genetics 116, 191 (1987).
36. E.M. Southern, J. Mol. Biol. 98, 503 (1975).
37. T.A; Kunkel, et al, Methods Enymol. 154, 367 (1987).
38. R.S. Sikorski, and P. Hieter, Genetics 122, 19 (1989).
20 Example 3.
In order to identify potential yeast initiators, we developed a genetic
strategy, the one-hybrid system, to find proteins that recognize a target sequence of
interest. The one-hybrid system has two basic components: (i) a hybrid eA~ression
library, constructed by fusing a transcriptional activation domain to random protein
25 segments, and (ii) a lc~x~ltel gene conti~ining a binding site of interest in its
promoter region. Hybrid proteins that recognize this site are expected to induce,ression of the repoller gene, because of their dual ability to bind the promoter
region and activate transcription (8). This association may be indirect, since
hybrids that interact with endogenous proteins already occupying the binding site
30 will also activate transcription (7). Nevertheless, as long as the ~Csoci~tion is
sequence-specific the protein incorporated in the hybrid should be functionally
relevant.
29
wo 95/16694 2 1 7 8 q 6 ~ pCT/US94/14563
We have used this method to look for proteins from the yeast
Saccharomyces cerevisiae that recognize the ARS conCPn.cuc sequence (ACS) of
yeast origins of DNA replication. The protein co-,-ponent of this screen was
provided by a set of three complementary yeast hybrid eAplession libraries, YLl-3,
containing random yeast protein segmentc fused to the GAL4 transcriptional
activation domain (GAL4AD) (9). The ,e~,ler gene for our screen contained four
direct repeats of the ACS in its promoter region and was integrated into the yeast
strain GGYl to form JLY363(ACSW') (10). To determine the dependence of lacZ
induction on the ACS, we constructed in parallel JLY365(ACSMUrA~), which
harbors a l~ller gene carrying four copies of a nonfunctional multiply-mutated
ACS (Fig. 4) (10).
We isolated nine plasmids that induced greater lacZ activity in
JLY363(ACSWr) than JLY365(ACSMUr^NT) from a screen of 1.2 million YLl-3
transformants (11). Many of the plasmids that induced lacZ activity on initial
screening of the library in JLY363(ACSWT) failed to exhibit a dependence on the
ACS when introduced into JLY365(ACSMUrA~). Restriction analysis of these
pl~cmi~c sho~wed that the nine isolates repl~;sented five genomic clones, which we
initially labeled AAPI-5 for ACS associated protein. AAPI was isolated four times,
AAP5 twice, and the others only once.
To eY~mine the sequence specificity of lacZ induction with finer resolution,
repo"er constructs containing direct repeats of four ACS point mutants were eachinteg,~ted into GGYl to generate the set of reporter strains(10). The five AAP
clones were individually ex~min~d in these strains for the ability to induce lacZ
eA~,ession. AAPI displayed a collespondence between the induction of this set of~ r genes and the ARS function (12) of their ACS. The AAPS hybrid
exhibited a slightly weaker correlation, and the rem~ining clones showed poor
correlation. These findings suggest that AAPI, and possibly AAP5, encodes a
protein that recognizes the ACS in a sequence-specific manner. Constructs with
deletions in the AAPl coding sequence (14) were unable to induce lacZ expression,
indicating that recognition of the ACS resided in the protein segment fused to
GAL4.
The genomic segments fused to the GAL4AD in AAPI-S were sequenced (15)
to determine the extent of the hybrid proteins that were made. AAPl and AAPS
WO 95/16694 2 1 7 8 9 6 5 PCT/US94114563
had sizable protein coding sequences of 301 and 123 amino acids, respectively,
fused in frame with the GAL4AD. In principle, these segments are large enough todirect the hybrid protein to the promoter of the ,~ er gene. AAP2-4 encoded
hybrid proteins with only short peptide extensions (10, 22, and 38 amino acids
respectively) fused to the GAL4^D, suggesting that these hybrids were not
responsible for the transcriptional induction attributed to these clones. Rec~use of
this finding and the lack of proper sequence specificity for the ACS element,
AAP2-4 were not studied further.
The full-length gene for AAPI was cloned from a yeast genomic library
and sequenced (15) (Genb~nk accession no. L23323). AAPI contains an open
reading frame for a protein 435 amino acids long with a predicted molecular
weight of 50,302 daltons. The hybrid GAL4^D-AAPl protein obtained from the
screen was a fusion of the GAL4^D to the C-terminal two-thirds of the predicted
full-length protein (residues 135-435), indicating that this portion of the molecule
is sufficient for ~Csoci~tion with the ACS. Comparison of peptide sequences fromthe 50kd subunit of ORC with the predicted protein sequence from MPI
demonstrated that our gene encodes this subunit and confirmed the association
between the AAPl protein and the ACS. Rec~llse of this identity, we have
rPn~mPd our gene ORC6.
An overlapping ORF capable of encoding a protein 250 amino acids long
exists on the complementary strand. The positions of the predicted start and stop
codons for this ORF are at nt 1615-7 and nt 865-7, respectively. In pJL766 the Cresidue at 1471 was mutated to a T, preserving the amino acid sequence of ORC6
- but introducing a stop codon in this overlapping ORF. The sequence of ORC6
indic~tes a connection with the regulatory machinery goveming cell cycle
~rogression. Orc6p contains four phosphorylation sites, (S/T)PXK, for cyclin-
dependent protein kinases (20) clustered in the first half of the molecule. Using
the more relaxed conC~ncus site (S/T)P adds two more sites to this cluster. We
have observed Orc6p phosphorylated in vivo on serine and threonine residues.
30 However, since the initiation of yeast DNA replication commences pro~llplly in
response to the activatio!l of this protein kinase in Gl, we believe that Orc6p and
possibly other ORC subunits are regulated substrates of this kinase. Finally, asexpected for a protein participating in nuclear events, Orc6p contains a potential
2 1 7 ~ q 6~
WO 95/16694 PCTIUS94114563
nuclear loc~li7~tion signal (NLS) within the (S/T)PXK cluster and one in the C-
terminal domain (amino acld residues 117-122 and 263-279). Orc6p can be seen
in the nucleus by immunofluoresence.
A marked deletion of the ORC6 gene (pJL731) (21), removing all but 13
5 codons from its open reading frame, was introduced into diploids from three
different strain backgrounds. The resulting heterozygous ORC6 deletion strains,
JLY481, JLY475, and JLY469 were induced to undergo meiosis, and 20 tetrads of
each strain were di~ected (21). In all backgrounds the ORC6 disruption
coseglegated with inviability, demonstrating that ORC6 is essential for cell growth.
10 Microscopic ex~min~tion revealed that mutant spores from JLY481 and JLY475
g~l",in~t~d, completed 1-2 rounds of cell division, and then arrested with a
uniform large bud morphology rernini~cent of cell division cycle mutants defective
in DNA replication or nuclear division (22). The position of cell cycle arrest could
not be pinpointed, however, since the DNA content of these cells could not be
15 readily measured. Mutant spores derived from JLY469 germin~t~ poorly.
The inle,~"t;~tion of these ORC6 deletion experiments was complicated by
the presence of a second open reading frame (ORF2) of 250 amino acids on the
antisense strand of the ORC6 gene. ORF2 spans nucleotides 1617 to 868 of the
Genb~nk sequence and overlaps the C-terminal two-thirds of the ORC6 coding
- 20 sequence. A marked deletion that removed the N-terminal third of the ORC6coding sequence without affecting ORF2 (pJL733) was introduced into diploids
(21). Tetrad analysis again showed the ORC6 deletion cosegregating with cell
death. Finally, an ORC6 gene was constructed that contains a silent codon changefor the ORC6 ORF but introduces a UGA stop codon in ORF2 (22). This gene
25 was able to rescue a haploid strain containing a full deletion of the ORC6 ORF.
We conclude that ORC6 is essential for cell viability.
Our results validate the one-hybrid system screen as a method to identify
and clone genes for proteins that recognize a DNA sequence of interest. This
screen has also been successful in identifying DNA-binding proteins (23), and a
30 variation of this screen has been used to identify a binding site for a suspected
DNA-binding protein (24). The one-hybrid approach is particularly useful for
proteins that are difficult to detect biochemically or for which starting material in a
purification is difficult to obtain.
32
wo 95/16694 2 1 7 8 9 6 5 PCT/US94/14563
We identified genes that interact gçneti~lly with ORC6 using established
cdc mutants because gel..~in~ g spores bearing an ORC6 deletion al)peared to
exhibit a cell division cycle phenotype. pJL749 (28), a plasmid that overe~,esses
Orc6p several hundred-fold, was introduced into a virtually isogenic set of
5 te.l-pc~dture-sensitive cdc mutants arresting at various points in the cell cycle (29).
Ove.eAI,lc;ssion of ORC6 selectively affected cdc6 and cdc46 mutants, lowering
their restrictive telllpeldtu~e by 5-7 C; there was no significant effect on the other
mutants e~mine~ or on the wild-type strain (Table l).
viability with
Strain cdc mutantovereA~ression of ORC6
RDY488 wild-type +
RDY501 cdc28-1 +
RDY510 cdc4-1 +
RDY664 cdc34-2 +
RDY543 cdc7-4 +
JLY310 cdc6-1 -
JLY179 cdc46-1
JLY338 cdc2- 1 +
JLY353 cdc17-1 +
RDY619 cdc15-2 +
Table 1. Viability of cdc Mutants in the Presence of High Levels of ORC6
Expression. JL749 (GALp-HA-ORC6), JL772 (GALp-HA), and RS425 were
introduced into each cdc mutant, and eY~mine~ for growth at various telll~;~dtu~es
25 under conditions that induce ~A~,ession of ORC6 (28, 29). + in~ tes mutants
whose restrictive tell~ldture remains unchanged in the presence of JL749 relative
to JL772 and RS425. - indicates ll-ul~ls whose restrictive tel~lpeldtul~ is lowered
5-7 C when JL749 is present.
33
wo 95/16694 2 1 7 ~ 9 6 5 pCT/US94/14563
Numbered Citations for Fx~mple 3
1. Kelly, J. Biol. Chem. 263, 17889 (1988); Marians, Annu. Kev. Biochem.
61, 673 (1992); Kornberg, Baker, DNA Replication. (Freeman and Company,
New York, 1992); B. Stillman, Annu. Rev. Cell Biol. 5, 197 (1989).
5 2. M. L. DePamphilis, Annu. Rev. Biochem. 62, 29 (1993).
3. Campbell and Newlon, in The Molecular an~l Cellular Biology of the Yeast
Sa~charomyces Broach, et al, Eds. (CSHL Press, 1991), vol. 1, pp. 41-146.
4. F~ngm~n and Brewer, Annu. Rev. Cell Biol. 7, 375 (1991).
5. J.R. Broach et al., Cold Spring Harbor Symp. Quant. Biol. 47, 1165
10 (1983); Van Houton and C. S. Newlon, Mol. Cell. Biol. 10, 3917 (1990).
6. Y. Marahrens and B. Stillman, Science 255, 817 (1992).
7. S. Fields and O.-K. Song, Nature 340, 245 (1989); C.-T. Chien, P.T.
Bartel, R. Stemglanz, S. Fields, Proc. Natl. Acad. Sci. USA 88, 9578 (1991).
8. R. Brent and M. Ptdshne, Cell 43, 729 (1985).
15 9. The N-terminal portions of the hybrids from hree related hybrid expressionlibraries, YLl-3 (7), consist of the SV40 nuclear localization signal and amino
acids 768-881 of the GAL4 activation domain (GAL4AD). The C-terminal portions
were derived from random yeast protein segments which have been fused to the
end of the GAL4AD. These segments are encoded by short (1-3kb) fragments from
20 a Sau3a partial digest of yeast genomic DNA. Together, YLl-3 ensure that all
three reading frames of these fr~gmentc can be c~p,cssed.
10. pLRlDl is described in R.W. West Jr., R.R. Rogers, M. Ptashne, Mol.
Cell. Biol. 4, 2467 (1984). We generated pBgl-lacZ from pLRlDl by (i)
subsl;4~ g dn XhoI-BglII-XhoI polylinker for the XhoI linker and (ii) precisely
25 excising a Hind III fragment cont~ining 2m sequences. The resulting vector has a
unique Bgl II site appro~Limately 100 bp upstream of the TATA box for insertion of
DNA sequences in the promoter region and a unique Stul site for targeted
integld~ion of the plasmid at the URA3 locus. Multiple direct repeats of ARSl
domain A and several of its mutant derivatives were inserted into the Bgl II site of
30 pBgl-lacZ to generate all the reporter genes used in this work. The inserted repeat
elernP-ntc, derived from complementary oligonucleotides, were oriented with the
TATA box to their right. Each ~epo.ler gene construct was integrated into the
wo 95/16694 ' ~ 2 ! 7 8 ~ 6 5 PCT/US94/14563
URA3 locus of GGYl (MATa Dgal4 Dgal80 ura3 leu2 his3 ade2 tyr) [G. Gill and
M. Ptashne, Cell 51, 121 (1987)] to create a reporter strain. Integration of pBgl-
lacZ into GGYl generated JLY387.
11. YEPD (rich complete) and SD (synthetic dropout) media are as described
5 tJ.B. Hicks and I. Herskowitz, Genetics 83, 245 (1976)]. Standard methods wereused for manipulation of yeast cells ~C. Guthrie and G.R. Fink, Ed., Guide to
Yeast Genetics and Moleculat Biology (Academic Press, San Diego 1991)] and
DNA [F.M. Ausubel et al., Ed., Current Protocols in Molecular Biology (Wiley,
New York 1989)]. Libraries YLl-3 were transformed [R.H. Schiestl and R.D.
10 Geitz, Current Genetics 16, 339 (1989)] into JLY363 (10) and plated on SD-Leu at
a density of 2-5000 colonies/lOcm plate. 500,000 tran~ro~ ants were obtained forYLl and YL2, and 200,000 for YL3. Transformants were assayed on filters for
production of b-galactositl~ce [L. Breeden and K. Nasmyth, Cold Spring Harbor
Symp. Quant. Biol. 47, 643 (1985)]. 49 isolates remained positive after colony
15 purification (15 from YL-l; 22 from YL-2, 12 form YL-3), and library plasmidswere extracted from them . These plasmids were each transformed into both
JLY363 and its mutant counterpart JLY365 (10). Nine plasmids induced greater
b-g~ tocidase activity in the wild type reporter strain than the control. These
pl~cmi~is were cl~csified into five clones, AAPI-5, based on their Hind III
20 restriction pattern. Each clone was then retested in JLY360, JLY361, JLY387,
JLY429, JLY431, JLY433, JLY435. The AAPI hybrid clone was called pJL720.
The AAPI gene was later ren~med ORC6.2
12. The ARS function of the mutant sequences was analyzed in the context of
ARS1 domain B (BglII-Hinf~ fragment, nt 853-734) in the following CEN-based
25 URA3-containing plasmids: pJL347 (wt), pJL243 (multiple), pJL326 (A863T),
pJL338 (T869A), pJL330 (T862C), and pJL316 (T867G). These plasmids were
transformed into JLY106 (MATa ura3 leu2 his3 trpl Iys2 ade2) and its
homozygous diploid counte~ JLY162. pJL243, pJL326, and pJL338 did not
yield a high frequency of transformation and could not be assayed quantitatively30 for ARS function. pJL347, pJL330, and pJL316 transformed cells with high
efficiency and were assayed for mitotic stability [Stinchcomb, et al. Nature 282, 39
(1979)]-
wo 95/16694 2 1 7 8 9 6 5 PCT/USs4/14563
13. pJL720, the ORC6 hybrid construct originally isolated from the YL3library, has two BamHI sites. The 5' site created by the hybrid junction
collesponds to Sau3a site at nt. 843. Excision of the segment between the two
sites generated pJL721, leaving amino acids 339-435 in frame with the GAL4^D.
5 pGAD3R (11) the parent vector for the YL3 library, contains no ORC6 sequence.
pRS425, Christi~n~on, et al., Gene 110, 119 (1992), contains no co...ponents of the
fusion protein.
14. All sequçnçing was pe,l~ led with Sequenase (USB) on collapsed double-
stranded templates. The protein coding segments of the AAPI-S hybrid clones were10 sequenced from their junction with the GAL4^D to their stop codon. Two of theORC6 sequencing primers were used as colony hybridization probes to screen a
high copy number yeast genomic library [M. Carlson and D. Botstein, Cell 28,
145 (1982)] for a clone of the full-length ORC6 gene (pJL724). The full-length
gene was sequenced on both strands using oligonuclotide primers positioned
15 a~plo~imately 200 nt apart.
15. S. P. Bell and B. Stillman, Nature 357, 128 (1992).
16. Hodgman, Nature 333, 22 (1988);Walker et al., EMBO J. 1, 945 (1982). 17. P. Linder, et al., Nature 337, 121 (1989).
18. E. A. Nigg, Seminars in Cell Biology 2, 261 (1991).
lg. ORC6 deletions were constructed by replacing nuc!eotides 458-1721
(pJI,731) or nucleotides 458-846 (pJL733) of the Genbank sequence with the URA3
HindIII fragment oriented in the opposite direction to that of the ORC6 sequence.
Each construct was used to generate heterozygous deletions of ORC6 in diploid
strains by one-step gene repl~cçrn~nt. ORC6 deletion analysis was pel~l"led in
25 JLY461 (MATa/MATa ura3/ura3 leu2/leu2 his3/his3 trpl/trpl ade2/ade2 lcirl),
JLY462 (MATa/MATa ura3/ura3 leu2/leu2 trpl/trpl his4/his4 canl/canl), and
JLY463 (MATa/MATa ura3/ura3 leu2/leu2 trpl/trpl his3/HI53); their respective
genetic backgrounds are S288c, EG123, and A364a. Disruption of JLY461,
JLY462, and JLY463 by pJL731 (full deletion) created JLY481, JLY475, and
30 JLY469, l~eclively. Disruption of JLY461, JLY462, and JLY463 by pJL733
(N-terrninal deletion) created JLY485, JLY479, JLY473, respectively. These
36
WO 95/16694 . 2 1 7 8 9 6 5 PCT/US94/14563
helefozygous marked deletion strains were sporulated, and twenty tetrads of eachwere tli~c~te l and grown on YEPD to assess viability.
20. Pringle and Hartwell, in The Molecular Biology of the Yeast Saccharomyces
Strathern, et al, Eds. (CSHL Press, CSH, 1981), vol. 1, pp. 97-142.
21. A point mutant (pJL766) was made by replacing the BamHI-SphI fragment
of the full-length clone with a R~mH~/SphI fragment generated by PCR from
pJL720 using primers. One mutation changes nucleotide 1471 of the C~çnb~nk
sequence from C to T and was confirmed by sequence analysis.
22. M. M. Wang and R. R. Reed, Nature 364, 121 (1993).
23. T. E. Wilson, et alt, Science 252, 1296 (1991).
24. J. F. X. Diffley and J. H. Cocker, Nature 357, 169 (1992).
25. pJL749 contains the GALI promoter (nt 146-816) driving the t;A~ression of
ORC6 (nt 443-2298) in the high-copy yeast shuttle vector RS425 [T. W.
Christianson, et al., Gene 110, 119 (1992)] .
26. The cdc mutant strains have been backcrossed 4-5 times against two
congenic strains derived from A364a, RDY487 (MATa leu2 ura3 trpl) and
RDY488 (MATa leu2 ura3 trpl). All are ura3 leu2 trpl. RDY510, RDY664,
JLY310, and JLY179 are MATa; the rest are MATa. Additional markers can be
found in JLY310(ade2), RDY543(his3), and RDY619 (pep4D::TRPI his3 ade2).
20 pJL749, pJL772, and RS425 (28) were transformed into these strains and plated on
SD-LEU at 22 C. Four colony-purified isolates from each transformation were
p~tc~ed onto SD-LEU plates and replica-plated to SGAL-LEU plates, all at 22 C.
The patches on SGAL-LEU were replica-plated to a series of pre-warmed SGAL-
LEU plates at 22, 25, 27, 30, 32.5, 35, 37, and 38 C. The viability of cdc
lllu~nts con~ining pJL749 was col,lpal~d to those con~ining pJL772 and pRS425.
27. Hartwell, JMB 104, 803 (1976); Hennessy, et al G&D 4, 2252(1990).
28. Chen, et al., PNAS 89, 10459 (1992); Hogan, et al, ibid. 89, 3098.
29. B.J. Andrews and S.W. Mason, Science. 261, 1543 (1993).
30 Example 4. Orc protein purification and gene cloning
Protein Purification: To obtain sufficient protein for peptide
sequençing~ a revised purification procedure for ORC was devised, based on the
procedure reported previously (Bell and Stillman, 1992). Whole cell extract was
37
wo 95/16694 2 1 7 8 9 6 5 PcrlUSs4/l4s63
t d from 400g of frozen BJ926 cells (frozen immediately after harvesting a
300 liter logarithmically growing culture, total of 1.6 kg per 300 liters). All
buffers contained 0.5 mM PMSF, 1 mM ben7~mi~ine, 2 mM pep~Lin A, 0.1
mg/ml bacitracin and 2mM DTT. 400 mls of 2X buffer H/0.1-NP~ (100 mM
5 Hepes-KOH, pH 7.5, 0.2 M KCl, 2 mM EDTA, 2 mM EGTA, 10 mM Mg
Acetate, and 20% glycerol) was added to the cells and after thawing the cells were
broken using a bead beater (Biospec Products) until greater than 90% cell breakage
was achieved (twenty 30 second pulses separated by 90 second pauses). After
breakage is complete, the volume of the broken cells was measured and one twelfth
10 volume of a saturated (at 4C) solution of ammonium sulfate was added and stirred
for 30 minutes. This solution was then spun at 13,000 x g for 20 minutes. The
res--lting supernatant was transferred to 45Ti bottle assemblies (Re~m~n) and spun
in a 45Ti rotor at 44,000 RPM for 1.5 hrs. The volume of the resulting
su~",atant was measured and 0.27g/ml of ammonium sulfate was added. After
15 stirring for 30 minutes, the precipitate was collected by spinning in the 45 Ti rotor
at 40,000 RPM or 30 minutes. The resulting pellet was resuspended using a B-
pestle dounce in buffer H/0.0 (50 mM Hepes-KOH, pH 7.5, 1 mM EDTA, 1 mM
EGTA, 5 mM Mg Acetate, 0.02% NP-40, 10% glycerol) and dialyzed versus
- H/0.lSM KCl (Buffer H with 0.15 M KCl added). This preparation typically
20 yielded 12-16 g soluble protein (determined by Bradford assay with a bovine serum
albumin standard). Preparation of ORC from this extract was essentially as
described (Bell and Stillman, 1992) with the following changes (column sizes used
for preparation of ORC from 400g of cells are indicated in parenthesis). The S-
Sel)har~se column was loaded at 20 mg protein per ml of resin ( ~ 300 ml). The
Q-Sepharose (50 ml) and sequence specific affinity column (Sml) was run as
described but the dsDNA cellulose column was omitted from the preparation.
Only a single glycerol gradient was pelro",led in an SW-41 rotor spun at 41,000
RPM for 20 hrs. We estim~t~ a yield of 130 ~g of ORC complex (all subunits
combined) per 400 g of yeast cells.
Protein Sequencing: Digestion of ORC subunits was pelrol."ed using an
"in gel" protocol described by Kawasaki and Suzuki with some modification.
Briefly, purified ORC (~ 10 ~g per subunit) was first separated by 10% SDS-
PAGE and stained with 0.1 % Coomassie Brilliant Blue G (Aldrich) for 15 min.
38
wo 95/16694 ;~ 1 7 8 9 6 5 P~/US94/~4s63
After dest~ining (10% meth~nol, 10% acetic acid), the gel was soaked in water
for one hour, then the protein bands were excised, transferred to a microcentrifuge
tube and cut into 3-5 pieces to fit snugly into the bottom of the tube. A minimum
volume of 0. lM Tris-HCl (pH=9.0) containing 0.1 % SDS was added to
completely cover the gel pieces. Then 200 ng of Achromobacter protease I
(Lysylendopeptidase: Wako) was added and incub~ted at 30C for 24 hrs. After
digestion the samples were centrifuged and the su~lna~ was passed through an
Ultrafree-MC filter (Millipore, 0.22~m). The gel slices were then washed twice
in 0.1 % TFA for one hour and the washes were recovered and filtered as above.
All filtrates were combined and reduced to a volume suitable for injection on the
HPLC using a speed-vac. The digests were separated by reverse-phase HPLC
(Hewlett-Packard 1090 system) using a Vydac C18 column (2.1x 250 mm, S~m,
300 angstroms) with an ion exchange pre-column (Brownlee GAX-013, 3.2x
15mm). The peptides were eluted from the C-18 column by increasing acetonitrile
concentration and monitored by their absorbance at 214, 280, 295, and 550 nm.
Amino acid sequencing of the purified peptides was pelrolnled on an automated
sequencer (Applied Biosystems model 470) with on-line HPLC (Applied
Biosystems model 1020A) analysis of PTH-amino acids.
ORC SUBUNIT CLONING:
ORCI: To clone the gene for the largest (120 kd) subunit of ORC, the
following degenerate oligonucleoide primers 1201 and 1202 were syntheci7çd basedon the sequence of the first ORCl peptide. These oligos were used to ~~
PCR reactions using total yeast genomic DNA from the strain W303 a as target.
A 48 base pair fragment was specifically amplified. This fragment was subcloned
and sequencecl. The resulting sequence encoded the predicted peptide indicating
that it was the correct amplification product. A radioactively labeled form of the
PCR product was then used to probe a genomic library of yeast DNA sequences
r~s~-lting in the identification of two overlapping clones. Sequencing of these
clones resulted in the identification of a large open reading frame that encoded a
30 protein with a predicted molecular weight of 120 kd and that encoded all four of
the ORCl peptide sequences.
ORC3: To clone the gene for the 62 kd subunit of ORC, the following
degen~.dte oligonucleoide primers 621 and 624 were synthe~i7~A based on the
39
WOg5/16694 2 1 78965 PCTIUS94/14563
sequence of the third peptide. These oligos were used to pe~ PCR reactions
using total yeast genomic DNA from the strain W303 a as target. A 53 base pair
fragment was specifically amplified. This fragment was subcloned and sequenced.
The resulting sequence encoded the predicted peptide in~ic~ting that it was the
5 correct amplification product. A radioactively labeled form of the PCR productwas then used to probe a genomic library of yeast DNA sequences reslllting in the
i~lPntific~tion of two overlapping clones. Sequencing of these clones resulted in
the identification of a large open reading frame that encoded a protein with a
predicted molecular weight of 71 kd and encoded all three of the ORC3 peptide
10 sequences. The inconsistency of the molecular weight is presumably due to
anomalous migration of this protein during SDS-PAGE.
ORC4: By co",l)aling the sequnce of the ORC4 peptides to that of the
known potentially protein encoding sequnces in the genbank ~l~t~h~ce we found that
a portion of the ORC4 coding sequence had been previously cloned in the process
15 of cloning the adjacent gene. Using the information from the l~t~h~e we wereable to design a perfect match oligo and use this to immediately screen a yeast
library. Using this oligo as a probe of the same yeast genomic DNA library a
lambda clone was isolated that contained the entire ORC4 gene. This gene
encoded a protein of predicted molecular weight 56 kd and also all of the peptides
20 derived from the peptide sequencing of the 56 kd subunit.
ORCS: To clone the gene for the 53 kd subunit of ORC, the following
degenerate oligonucleoide primers 535 and 536 were synthesized based on the
sequence of the first ORC5 peptide. These oligos were used to perform PCR
reactions using total yeast genomic DNA from the strain W303 a as target. A 47
25 base pair fragment was specifically amplified. This fragment was subcloned and
sequenced. The reslllting sequence encoded the predicted peptide indicating that it
was the correct amplification product. A radioactively labeled form of the PCR
product was then used to probe a genomic library of yeast DNA sequences
resl-lting in the identification of a single lambda clone. Sequencing of this clones
30 resulted in the identification of a large open reading frame that encoded a several
of the peptide sequences derived from the 53 kd subunit of ORC indicating that
this was the correct gene. However the sequence of the 5' end of the gene wasno
present in this lambda clone. Fortuitoulsy, the mutations in the same gene had also
wo 95/16694 2 1 7 8 9 6 5 PCTIUS94tl4563
been picked up in the same sreen that resulted in the identification of the ORC2gene. A complementing clone to this mutation was found to overlap with the
l~mb(l~ clone and contain the entire 5' end of the gene. Sequencing of this
complementing DNA fragment resulted in the identification of the entire sequence5 of the ORCS gene.
All publications and patent applications cited in this specification are herein
inc~ ed by reference as if each individual publication or patent application
were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
10 illustration and example for purposes of clarity of understanding, it will be readily
a~alent to those of ordinary skill in the art in light of the teachings of this
invention that certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sr;Q~N~r; LISTING
(1) GENERAL INFORMATION:
(i~ APPLICANT: COLD SPRING HARBOR LABORATORY
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
(ii) TITLE OF INVENTION: ORC GENES, RECOMBINANT ORC PEPTIDES AND
METHODS OF IDENTIFYING DNA BINDING PROTEINS
(iii) NUMBER OF SEQUENCES: 12
` (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FLEHR, HOHBACH, TEST, ALBRITTON & HERBERT
(B) STREET: 4 Embarcadero Center, Suite 3400
(C) CITY: San Francisco
(D~ STATE: California
(E) COuh-~: USA
(F) ZIP: 94111-4187
(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) ~u~R~ APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Osman, Richard A
(B) REGISTRATION NUMBER: 36,677
(C) *~rr;k~N~r;/DOCRET NUMBER: FP-59032-PC/RAO
(ix) TFT-FCOMMUNICATION INFORMATION: ..
(A) TELEPHONE: (415) 781-1989
21 7896~
W O95/16694 - PCT~us94/14563
~B) TELEFAX: (415) 398-3249
(C) TELEX: 910 277299
5 ( 2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4940 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATAArATGcT CGCC~.-~-A TATTATGACA GAAAGAATAT ATATATTCAT ATATAArA-TG 60
CTTCTATTTA TTAGTTTTAT CTTTTAATTG ATGATGTGTC CATAGAATTT AAGTAAGTGC 120
ATGGTATGGA GTGTATAATG GTTTATAATT lCCC~lAAGA TGACACAAAA AAA1~Ll~lC 180
CCAAAAATTT ACCAAGAAAA AAAATTAAGA ATACTACACA ATTGATGCTT GGGTTATTTT 240
25 AAATATCCGG TACATTCTAT TACAAATATG TTTGTACAAT GTAAGCCCCT TCATAATGGT 300
CAGTATTAAG ATAAGGACTG CTATGGGGCA l~ l~l~i TACTGGGTAT CACAGGATAA 360
TAACTTGGCG CCAAATTAGA APArATATAA ACCTCAAATA TTTGAAATTC TTTGGTGACC 420
,~, ~ATCG TTATATCAAC AAATATTGCA CCAACGAACA CCACTACATA TGTAACTACT 480
~ C~CG ACTTATTTTT TATTAACGTT GACACGGCCA GATCGAAAAT CATAGAAAAA 540
35 rAACAACATT GAGAAGAGAT GAAGTTGCGC AAAGGGAAAG AAAACTGCAT AGGCGGCAAA 600
TTCAGCCTAA AAG~C~AG AAGCAGGAAC TCATTCCCTA TTGATTAATA CTCATTACAA 660
ApA-ccAcAAT AGAGTAGATA AGATGGCAAA AACGTTGAAG GATTTACAGG GTTGGGAGAT 720
pATAArAACT GATGAGCAGG GAAATATAAT CGATGGAGGT rArAAGAGAT TACGCCGAAG 780
AGGTGCAAAA ACTGAACATT ACTTAAAGAG AAGTTCTGAT GGAATTAAAC TAGGTCGTGG 840
45 TGATAGTGTA GTCATGCACA ACGAAGCCGC TGGGACTTAC ~CCG1~ATA TGATCCAGGA 900
GTTGAGACTT AATACATTAA ATAATGTTGT CGAACTCTGG GCTCTCACCT ATTTACGATG 960
GTTTGAAGTC AA~C~AG CTCATTATAG GCAGTTTAAT CCTGACGCTA ACATTTTGAA 1020
~CG~C~lA AATTATTACA ATAAACTGTT TTCTGAAACT GCAAATAAAA ATGAACTGTA 1080
TCTCACTGCA GAATTAGCCG AATTGCAGCT ATTTAACTTT ATCAGGGTTG CCAACGTAAT 1140
55 GGATGGAAGC AAATGGGAAG TATTGAAAGG AAATGTCGAT CrAr-AAAr7AG ACTTTACAGT 1200
TCGTTATATT TGTGAGCCGA CTGGGGAGAA ATTTGTGGAC ATTAATATTG AGGATGTCAA 1260
AGCTTACATA AAGAAAGTGG AGCCAAGGGA AGCCCAGGAA TATTTGAAAG ATTTAACACT 1320
TCCATCAAAG AAGAAAGAGA TCAAAAGAGG TCCTCAAAAG AAAGATAAGG CTACTCAAAC 1380
GGCACAAATT TCAGACGCAG AAACAAGAGC TArAGATATA ACGGATAATG AGGACGGTAA 1440
65 TGAAGATGAA TCATCTGATT ATGAAAGTCC GTCAGATATC GACGTTAGCG AGGATATGGA 1500
CAGCGGTGAA ATATCCGCAG ATGAGCTTGA GGAAGAAGAA GACGAAGAAr, AArA~r,AAr.A 1560
42
WO 95/16694 2 ~ 7 ~ 9 6 5 PCT/US94/14563
CrAACAAr~G AAAGAAGCTA GGCATACAAA TTrAC~AGG AAAAGAGGCC GTAA~-ATAAA 1620
ACTAGGTAAA GATGATATTG ACG~l-~-~ ACAACCTCCC CCCAAAAAAA GAGGTCGTAA 1680
S ACCTAAA~-AT CCTAGTAAAC CGCGTCAGAT GCTATTGATA TCTTCATGCC GTGrPAATAA 1740
TA~.C~.~.G ATTAGGAAAT TTArAAAAPA GAATGTTGCT AGGGCGAAAA AGAAATATAC 1800
CCCG.~..CG AAAAGATTTA AATCTATAGC TGCAATACCA GATTTAACTT CATTACCTGA 1860
ATTTTACGGA AA-~..CGG AATTGATGGC ATCAAGGTTT GAAAACAAAT TAAAAACAAC 1920
CCAPAAGCAT CAGATTGTAG AAArAATTTT TTCTAAAGTC AAAAAACAGT TGAACTCTTC 1980
lS GTATGTCAAA GPAGAAATAT TGAAGTCTGC AAATTTCCAA GATTATTTAC CGGCTAGGGA 2040
GAATGAATTC GCCTCAATTT ATTTAAGTGC ATATAGTGCC ATTGAGTCCG ACTCCGCTAC 2100
TACTATATAC GTGGCTGGTA CGCCTGGTGT AGGGAAAACT TTAACCGTAA GGGAAGTCGT 2160
AAAGGAACTA CTA~C~.~.. CTGr~CAACG A~AAATACCA GA~ ATGTGGAAAT 2220
AAATGGATTG AAAATGGTAA AACCCACAGA CTGTTACGAA ACTTTATGGA ACAAAGTGTC 2280
25 AG~-A~-AAAGG TTAACATGGG CAGCTTCAAT GGAGTCACTA GAGTTTTACT TTAAAAGAGT 2340
TCCAAAAAAT AAGAAGAAAA CCATTGTAGT ~.l~.~GGAC GAACTCGATG CCATGGTAAC 2400
GAAATCTCAA GATATTATGT ACAATTTTTT CAATTGGACT ACTTACGAAA ATGCCAAACT 2460
TATTGTCATT GCAGTAGCCA ATA~AATGGA cTTAcrA~AA CGTCAGCTAG GCAATAAGAT 2520
TACTTCAAGA ATTGGGTTTA CCAGAATTAT GTTCACTGGG TATACGCACG AAGAGCTAAA 2580
35 AAATATCATT GATTTAAGAC TGAAGGGGTT GAACGACTCA ..~..~.ATG TTGATACAAA 2640
AACTGGCAAT GCTATTTTGA TTGATGCGGC TGGAAACGAC ACTACAGTTA AGCAAACGTT 2700
GCCTGAAGAC GT~-AGr-AAAG TTCGCTTAAG AAT-GAGTGCT GATGCCATTG AAATAGCTTC 2760
~ AGPAAAGTA GCAAGTGTTA GTGGTGATGC AAGAA~AGCA TTGAAGGTTT GTAAAAGAGC 2820
AGCTGAAATT GCTGAAAAAC ACTATATGGC TAAGCATGGT TATGGATATG ATGGAAAGAC 2880
GGTTATTGAA GATGAAAATG AGGAGCAAAT ATACGATGAT GAAGACAAGG ATCTTATTGA 2940
AAGTAA~AA GCCAAAGACG ATAATGATGA CGATGATGAC AATGATGGGG TAcAAAcAGT 3000
TCACATCACG CACGTTATGA AAGCCTTAAA CGAAACTTTA AATTCTCATG TAATTACGTT 3060
TATGACGCGA CTTTCATTTA CAGCAAAACT GTTTATTTAT GCATTATTAA ACTTGATGAA 3120
pAAGAACGGA TCTCAAGAGC AAGAACTGGG CGATATTGTC GATGAAATCA AGTTACTTAT 3180
SS TGAAGTAAAT GGCAGTAATA AG~..~.~AT GGAGATAGCC AAAACATTGT TCCAACAGGG 3240
AAGTGATAAT ATTTCTGAAC AATTGAGAAT TATATCATGG GA...CGl.C TCAATCAGTT 3300
ACTTGACGCG GGAATATTGT TTAAA~AAAC TATGAAGAAC GATA~-AATAT G--~.~.~AA 3360
GCTAAATATA TCAGTAGAAG AAGCCAAAAG AGCCATGAAT GAGGATGAGA CATTGAGAAA 3420
TTTPTArATT CG~..~..AT TATTCATGAC CTAGCATACA CATACATATA CCTACATAGT 3480
65 AGCGCATTTA TCCAAAACAT ACGATATTGT GGATGTACAT AC~ ATA ~-C~.lAAA 3540
GCTATTGTGT AGCTTGATTT AAAATATGCT AACGCCAACT CTCACATGGT AGCAGGCGGG 3600
43
WO95/16694 2 1 7 8 9 6 5 PCT/US94/14563
TATAGTTGTT TTCATGTATT AACGCCCGGC GATGGTGCCT TAGATGAGGG CGACGAGGAG 366
GG~l.C~.GA TATTATGGCT ~l~ ATCC TGAC~ TATGATGTCG ATGTTGCTGG 3720
CCACCTAGGT GCTTATATAT CAAAAGAGGA TCGCCGATTT CATTGATTTC TGGGATGGTT 3780
AATGTCAAAT TAAAGATCTT TGCCAGTGCA ATTTTGAAAA l~ lGAAT GTTTATAGAT 3840
TTGGCAGTAG AG~AGAATAT AAGAGGAGCA TTCATGACCT GTGCATACTT CATACTCGTT 3900
CTCGAGATTT G-~C~GATA TTCCGGGTCT AAGTCTATTA GTAAATCGTA ~ GCCC 3960
A~APAATAG GAATTGCCGA ATCATTTAGC CCGTACGCCT GCCTATACrA ~C~ATT 4020
15 GAACTCAACG ~ GGACG TGTCAGGTCA AACAGAAATA TGATCACTGA A-r~AcccTAcc 4080
GTCGCAATTG GGAGCATGTT GATGAATTCT ~..~G~CCGC CTAAATCCAT TATAGAAAAT 4140
ATAATATCCG TGGAGCGTAT GCTTACTTTT CTTTTCAAAA AGTTCACTCC CAGCC~Gl 4200
GTGTATTCCT TATCGTATAT Gl~G~ACG TACTTCACCA TCAGCGATGT l~cc~AcT 4260
TGTGCATCCC CTACTAATCC AACCTGAACT TCAACCTGAT TTCGTACCGC AGGTATAGAA 4320
25 ,~ ,GCTC CCGTGCTTGG TGTAGCCATC TTAGCTTAAC TCAATTTAAT TTCTACAGCA 4380
AAATCCAAAC GTAATATCTA TA,,,ll~lC GAAAAACTGA GGACAAGAGC CAATCAATCA 4440
TCTATAATCC AATTTATATT A..~...CCC TTCTGGGTTC l,ll~llC~l l~llG1~ 4500
AC~...l~lG ~l~ ~ATA AAATAATTTC TCTAGATTTG AAGACAGCAT llll~lACAT 4560
CrATAr~CCA TACACCATAC ACCATAGCAC CAGTACACTA TATTTTTATG AATTTTACTA 4620
35 AGAATTATTC CTGCAGGAGC TCCACTGAAA AAAAAAGAGc AGCATGGATG TCATGTCGGT 4680
AGAGTGCTAC TGAGTAAATG GGAGGACGCG GTAGATCCAG TGTGGAATCA AGGTGGTGCC 4740
GGTGTGAAGC CGCCTCGGCC GGCTGGACTC TCCAGGCCGG AGTGATGATT GCCACGCTGA 4800
AGCTAACACA GTTTCACAAT ACCAGTGTCC TCATTAGTGA GTTCCAATGT ATAGTTAGTA 4860
GTGGTATTTT ~ATATATGTG AGTGGTAGCA GATTTGAACT TAGTTAGTTG TATTCGCCTT 4920
45 TGAGGAAACC AAGCCAAAAA 4940
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 914 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2
Met Ala Lys Thr Leu Lys Asp Leu Gln Gly Trp Glu Ile Ile Thr Thr
1 5 10 15
Asp Glu Gln Gly Asn Ile Ile Asp Gly Gly Gln Lys Arg Leu Arg Arg
20 25 30
Arg Gly Ala Lys Thr Glu His Tyr Leu Lys Arg Ser Ser Asp Gly Ile
44
W 095/16694 ~ ! 7 ~ 9 6 5 PCTrUS94/14563
Ly6 Leu Gly Arg Gly Asp Ser Val Val Met His Asn Glu Ala Ala Gly
50 55 60
Thr Tyr Ser Val Tyr Met Ile Gln Glu Leu Arg Leu Asn Thr Leu Asn
65 70 75 80
Asn Val Val Glu Leu Trp Ala Leu Thr Tyr Leu Arg Trp Phe Glu Val
0 Asn Pro Leu Ala Hi8 Tyr Arg Gln Phe Asn Pro Asp Ala Asn Ile Leu
100 105 110
Asn Arg Pro Leu Asn Tyr Tyr Asn Lys Leu Phe Ser Glu Thr Ala Asn
115 120 125
Lys Asn Glu Leu Tyr Leu Thr Ala Glu Leu Ala Glu Leu Gln Leu Phe
130 135 140
Asn Phe Ile Arg Val Ala Asn Val Met A~p Gly Ser Lys Trp Glu Val
145 150 155 160
Leu Lys Gly Asn Val Asp Pro Glu Arg Asp Phe Thr Val Arg Tyr Ile
165 170 175
cys Glu Pro Thr Gly Glu Lys Phe Val Asp Ile Asn Ile Glu Asp Val
180 185 190
Lys Ala Tyr Ile Lys Lys Val Glu Pro Arg Glu Ala Gln Glu Tyr Leu
195 200 205
Lys Asp Leu Thr Leu Pro Ser Lys Lys Lys Glu Ile Lys Arg Gly Pro
210 215 220
Gln Lys Lys Asp Lys Ala Thr Gln Thr Ala Gln Ile Ser Asp Ala Glu
225 230 235 240
Thr Arg Ala Thr Asp Ile Thr Asp Asn Glu Asp Gly Asn Glu Asp Glu
245 250 255
- 40 Ser Ser Asp Tyr Glu Ser Pro Ser Asp Ile Asp Val Ser Glu Asp Met
260 265 270
Asp Ser Gly Glu Ile Ser Ala Asp Glu Leu Glu Glu Glu Glu Asp Glu
275 280 285
Glu Glu Asp Glu Asp Glu Glu Glu Lys Glu Ala Arg His Thr Asn Ser
290 295 300
Pro Arg Lys Arg Gly Arg Lys Ile Lys Leu Gly Lys Asp Asp Ile Asp
305 310 315 320
Ala Ser Val Gln Pro Pro Pro Lys Lys Arg Gly Arg Lys Pro Lys Asp
325 330 335
Pro Ser Lys Pro Arg Gln Met Leu Leu Ile Ser Ser Cys Arg Ala Asn
340 345 350
Asn Thr Pro Val Ile Arg Lys Phe Thr Lys Lys Asn Val Ala Arg Ala
355 360 365
Lys Lys Lys Tyr Thr Pro Phe Ser Lys Arg Phe Lys Ser Ile Ala Ala
370 375 380
Ile Pro Asp Leu Thr Ser Leu Pro Glu Phe Tyr Gly Asn Ser Ser Glu
385 390 395 400
Leu Met Ala Ser Arg Phe Glu Asn Lys Leu Lys Thr Thr Gln Lys His
405 410 415
WO 95/16694 2 1 7 8 9 6 5 PCT/US94114563
Gln Ile Val Glu Thr Ile Phe Ser Lys Val Lys Lys Gln Leu Asn Ser
420 425 430
Ser Tyr Val Lys Glu Glu Ile Leu Lys Ser Ala Asn Phe Gln Asp Tyr
435 440 445
Leu Pro Ala Arg Glu Asn Glu Phe Ala Ser Ile Tyr Leu Ser Ala Tyr
450 455 460
0 Ser Ala I le Glu Ser Asp Ser Ala Thr Thr I le Tyr Val Ala Gly Thr
465 470 475 480
Pro Gly Val Gly Lys Thr Leu Thr Val Arg Glu Val Val Lys Glu Leu
485 490 495
Leu Ser Ser Ser Ala Gln Arg Glu I le Pro Asp Phe Leu Tyr Val Glu
500 505 510
Ile Asn Gly Leu Lys Met Val Lys Pro Thr Asp Cys Tyr Glu Thr Leu
515 520 525
Trp Asn Lys Val Ser Gly Glu Arg Leu Thr Trp Ala Ala Ser Met Glu
530 535 540
Ser Leu Glu Phe Tyr Phe Lys Arg Val Pro Lys Asn Lys Lys Lys Thr
545 550 555 560
Ile Val Val Leu Leu Asp Glu Leu Asp Ala Met Val Thr Lys Ser Gln
565 570 575
Asp Ile Met Tyr Asn Phe Phe Asn Trp Thr Thr Tyr Glu Asn Ala Lys
580 585 590
Leu Ile Val Ile Ala Val Ala Asn Thr Met Asp Leu Pro Glu Arg Gln
595 600 605
Leu Gly Asn Lys Ile Thr Ser Arg . Ile Gly Phe Thr Arg Ile Met Phe
610 615 620
Thr Gly Tyr Thr His Glu Glu Leu Lys Asn Ile Ile Asp Leu Arg Leu
625 630 635 640
Lys Gly Leu Asn Asp Ser Phe Phe Tyr Val Asp Thr Lys Thr Gly Asn
645 650 655
Ala Ile Leu Ile Asp Ala Ala Gly Asn Asp Thr Thr Val Lys Gln Thr
660 665 670
Leu Pro Glu Asp Val Arg Lys Val Arg Leu Arg Met Ser Ala Asp Ala
675 680 685
Ile Glu Ile Ala Ser Arg Lys Val Ala Ser Val Ser Gly Asp Ala Arg
690 695 700
Arg Ala Leu Lys Val Cys Lys Arg Ala Ala Glu Ile Ala Glu Lys His
705 710 715 720
Tyr Met Ala Lys His Gly Tyr Gly Tyr Asp Gly Lys Thr Val Ile Glu
725 730 735
Asp Glu Asn Glu Glu Gln Ile Tyr Asp Asp Glu Asp Lys Asp Leu Ile
740 745 750
Glu Ser Asn Lys Ala Lys Asp Asp Asn Asp Asp Asp Asp Asp Asn Asp
755 760 765
Gly Val Gln Thr Val His Ile Thr His Val Met Lys Ala Leu Asn Glu
770 775 780
46
WO 95/16694 2 1 7 8 9 6 5 PCT/US94/14563
Thr Leu Asn Ser HiR Val Ile Thr Phe Met Thr Arg Leu Ser Phe Thr
785 790 795 800
Ala Lys Leu Phe Ile Tyr Ala Leu Leu Asn Leu Met Lys Lys Asn Gly
805 810 815
Ser Gln Glu Gln Glu Leu Gly Asp Ile Val Asp Glu Ile Lys Leu Leu
820 825 830
Ile Glu Val Asn Gly Ser Asn Lys Phe Val Met Glu Ile Ala Lys Thr
835 840 845
Leu Phe Gln Gln Gly Ser Asp Asn Ile Ser Glu Gln Leu Arg Ile Ile
850 855 860
Ser Trp Asp Phe Val Leu Asn Gln Leu Leu Asp Ala Gly Ile Leu Phe
865 870 875 880
Lys Gln Thr Met Lys Asn Asp Arg Ile Cys Cy8 Val Lys Leu Asn Ile
885 890 895
Ser Val Glu Glu Ala Lys Arg Ala Met Asn Glu Asp Glu Thr Leu Arg
900 905 910
Asn Leu
(2) INFORMATION FOR SEQ ID NO:3
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2809 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 807 2666
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAGCTCAACA CCACCATTGA GAACGTAGAA TTTCAATTTT TAAGCTGATT ~.C~ GC 60
ATGAACTCTC CTAGCAATGT GAAACTTCTC TTAAGGGAAA TTTTCGCCTT TTTGAATGGG 120
CATACTTGGC rAAAAATTCA GGATTGAATA TATATAATCG GAACTTGTAT GrATAAAAAT 180
TTATATCAAG AGl~.~.-C TTAATTGGAT TTGCTGTGAT CTAGTATTGA GATGACTATA 240
AACCGGCCAG GAAATTAGTC TTTTCGAAGC TGGTTTTGGT TTCGCAAGAG l~lll.~GAC 300
AG~.l..lGG CCTCAATTTG TAl.CCC.lA ATACGCTTCT TCAACTCTGT CTTAGAr-ACC 360
A..~.CCAG TGGCCTCATC TAGGTGTAAA CTAGCAATAG CGTCACTAGC TGCCGTGACA 420
TTAACTTGCT GTGGCACCTT TATATGTAAT ATr-AAr,r~TC TTTCAATGGA Tr~TAAG~AT 480
60 AA~..,.C~,.A AAAGGCCAAA TATCCATGCA TAAATATCGA CTTATTCGCG TAAATGTGAT 540
ATGGATCAGC TAGTACr~AT TTCTAGTCTA GCA~AATCGG GAAAATTTTT CAGAACACCC 600
ACTCACCGCA TCATTGAGGT GGAAATGACA ATAGTAAGCA GAATTGTTAT TCTTCACAAT 660
GTGTAAAAGT TATAAAGAAA TAGGAACCAC CTTTAAATTA AGACAAAGTA GAATATATTA 720
GCTGAAATTG TATTTGATAA TTGATCATTG ATCTTATTTG CTATATCTTT AAAAcAArGTT 780
47
2 1 78965
WO 95/16694 PCT/US94/14563
TTTGTAGTAC TGCGAATTGC CATAAC ATG CTA AAT GGG GAA GAC TTT GTA GAG 8:
Met Leu Asn Gly Glu Asp Phe Val Glu
l 5
CAT AAT GAT ATC CTA TCG TCT CCG GCA AAA AGC AGG AAT GTA ACC CCA 881
His Asn Asp Ile Leu Ser Ser Pro Ala Lys Ser Arg Asn Val Thr Pro
AAA AGG GTT GAC CCA CAT GGA GAA AGA CAA CTG AGA AGA ATT CAT TCA 929
0 Lys Arg Val Asp Pro His Gly Glu Arg Gln Leu Arg Arg Ile His Ser
30 35 40
TCA AAG AAG AAT TTG TTG GAA AGA ATC TCG CTT GTA GGC AAC GAA AGG 977
Ser Ly~ Lys Asn Leu Leu Glu Arg Ile Ser Leu Val Gly Asn Glu Arg
45 50 55
AAA AAT ACA TCT CCA GAT CCG GCA CTC AAA CCT AAA ACG CCA AGT AAA 1025
Lys Asn Thr Ser Pro Asp Pro Ala Leu Lys Pro Lys Thr Pro Ser Lys
GCT CCC CGT AAA CGT GGA AGA CCA AGA AAG ATA CAG GAA GAA TTA ACT 1073
Ala Pro Arg Lys Arg Gly Arg Pro Arg Lys Ile Gln Glu Glu Leu Thr
25 GAT AGG ATC AAG AAG GAT GAG AAA GAT ACA ATT TCC TCT AAG AAA AAG 1121
Asp Arg Ile Lys Lys Asp Glu Lys Asp Thr Ile Ser Ser Lys Lys Lys
100 105
AGG AAA TTG GAC AAA GAT ACA TCA GGT AAT GTC AAT GAG GAA AGC AAG 1169
30 Arg Lys Leu Asp Lys Asp Thr Ser Gly Asn Val Asn Glu Glu Ser Lys
110 115 120
ACT TCT AAC AAC AAG CAG GTG ATG GAA AAG ACG GGG ATA AAA GAG AAA 1217
Thr Ser Asn Asn Lys Gln Val Met Glu Lys Thr Gly Ile Lys Glu Lys
125 130. 135
AGA GAA CGC GAA AAA ATA CAG GTA GCG ACC ACA ACA TAT GAA GAT AAT 1265
Arg Glu Arg Glu Lys Ile Gln Val Ala Thr Thr Thr Tyr Glu Asp Asn
140 145 150
GTG ACT CCA CAA ACT GAT GAT AAT TTT GTA TCA AAT TCA CCC GAG CCA 1313
Val Thr Pro Gln Thr Asp Asp Asn Phe Val Ser Asn Ser Pro Glu Pro
155 160 165
45 CCA GAA CCT GCA ACA CCA TCT AAG AAG TCT TTA ACC ACT AAT CAT GAT 1361
Pro Glu Pro Ala Thr Pro Ser Lys Lys Ser Leu Thr Thr Asn His Asp
170 175 180 185
TTT ACT TCG CCC CTA AAG CAA ATT ATA ATG AAT AAT TTA AAA GAA TAT 1409
Phe Thr Ser Pro Leu Lys Gln Ile Ile Met Asn Asn Leu Lys Glu Tyr
l90 195 200
AAA GAC TCA ACC TCC CCA GGT AAA TTA ACC TTG AGT AGA AAT TTT ACT 1457
Lys Asp Ser Thr Ser Pro Gly Lys Leu Thr Leu Ser Arg Asn Phe Thr
205 210 215
CCA ACC CCT GTA CCG AAA AAT AAA AAG CTC TAC CAA ACT TCG GAA ACC 1505
Pro Thr Pro Val Pro Lys Asn Lys Lys Leu Tyr Gln Thr Ser Glu Thr
220 225 230
AAG TCA GCA AGC TCG TTT TTG GAT ACT TTT GAA GGA TAT TTC GAC CAA 1553
Lys Ser Ala Ser ser Phe Leu Asp Thr Phe Glu Gly Tyr Phe Asp Gln
235 240 245
65 AGA AAA ATT GTC AGA ACT AAT GCG AAG TCA AGG CAC ACC ATG TCA ATG 1601
Arg Lys Ile Val Arg Thr Asn Ala Lys Ser Arg His Thr Met Ser Met
250 255 260 265
48
W 095/16694 2 1 7 8 9 65 pcTnus94/14563
GCA CCT GAC GTT ACC AGA GAA GAG TTT TCC CTA GTA TCA AAC TTT TTC 1649
Ala Pro Asp Val Thr Arg Glu Glu Phe Ser Leu Val Ser Asn Phe Phe
270 275 280
AAC GAA AAT TTT CAA AAA CGT CCC AGG CAA AAG TTA TTT GAA ATT CAG 1697
Asn Glu Asn Phe Gln Lys Arg Pro Arg Gln Lys Leu Phe Glu Ile Gln
285 290 295
AAA AAA ATG TTT CCC CAG TAT TGG TTT GAA TTG ACT CAA GGA TTC TCC 1745
0 Lys Lys Met Phe Pro Gln Tyr Trp Phe Glu Leu Thr Gln Gly Phe Ser
300 305 310
TTA TTA TTT TAT GGT GTA GGT TCG AAA CGT AAT TTT TTG GAA GAG TTT 1793
Leu Leu Phe Tyr Gly Val Gly Ser Lys Arg Asn Phe Leu Glu Glu Phe
315 320 325
GCC ATT GAC TAC TTG TCT CCG AAA ATC GCG TAC TCG CAA CTG GCT TAT 1841
Ala Ile Asp Tyr Leu Ser Pro Lys Ile Ala Tyr Ser Gln Leu Ala Tyr
330 335 340 345
GAG AAT GAA TTA CAA CAA AAC AAA CCT GTA AAT TCC ATC CCA TGC CTT 1889
Glu Asn Glu Leu Gln Gln Asn Lys Pro Val Asn Ser Ile Pro Cys Leu
350 355 360
25 ATT TTA AAT GGT TAC AAC CCT AGC TGT AAC TAT CGT GAC GTC TTC AAA 1937
Ile Leu Asn Gly Tyr Asn Pro Ser Cys Asn Tyr Arg Asp Val Phe Lys
365 370 375
GAG ATT ACC GAT CTT TTG GTC CCC GCT GAG TTG ACA AGA AGC GAA ACT 1985
30 Glu Ile Thr Asp Leu Leu Val Pro Ala Glu Leu Thr Arg Ser Glu Thr
380 385 390
AAG TAC TGG GGC AAT CAT GTG ATT TTG CAG ATC CAA AAG ATG ATT GAT 2033
Lys Tyr Trp Gly Asn. His Val Ile Leu Gln Ile Gln Lys Met Ile Asp
395 400 405
TTC TAC AAA AAT CAA CCT TTA GAT ATC AAA TTA ATA CTT GTA GTG CAT 2081.
Phe Tyr Lys Asn Gln Pro Leu Asp Ile Lys Leu Ile Leu Val Val His
410 415 420 425
AAT CTG GAT GGT CCT AGC ATA AGG AAA AAC ACT TTT CAG ACG ATG CTA 2129
Asn Leu Asp Gly Pro Ser Ile Arg Lys Asn Thr Phe Gln Thr Met Leu
430 435 440
AGC TTC CTC TCC GTC ATC AGA CAA ATC GCC ATA GTC GCC TCT ACA GAC 2177
Ser Phe Leu Ser Val Ile Arg Gln Ile Ala Ile Val Ala Ser Thr Asp
445 450 455
CAC ATT TAC GCT CCG CTC CTC TGG GAC AAC ATG AAG GCC CAA AAC TAC 2225
S0 His Ile Tyr Ala Pro Leu Leu Trp Asp Asn Met Lys Ala Gln Asn Tyr
460 465 470
AAC TTT GTC TTT CAT GAT ATT TCG AAT TTT GAA CCG TCG ACA GTC GAG 2273
Asn Phe Val Phe His Asp Ile Ser Asn Phe Glu Pro Ser Thr Val Glu
475 480 485
TCT ACG TTC CAA GAT GTG ATG AAG ATG GGT AAA AGC GAT ACC AGC AGT 2321
Ser Thr Phe Gln Asp Val Met Lys Met Gly Lys Ser Asp Thr Ser Ser
490 495 500 505
GGT GCT GAA GGT GCG AAA TAC GTC TTA CAA TCA CTT ACT GTG AAC TCC 2369
Gly Ala Glu Gly Ala Lys Tyr Val Leu Gln Ser Leu Thr Val Asn Ser
510 515 520
65 AAG AAG ATG TAT AAG TTG CTT ATT GAA ACA CAA ATG CAG AAT ATG GGG 2417
Lys Lys Met Tyr Lys Leu Leu Ile Glu Thr Gln Met Gln Asn Met Gly
525 530 535
49
WO 95/16694 2 1 7 8 ~ 6 5 PCT/US94/14563
AAT CTA TCC GCT AAC ACA GGT CCT AAG CGT GGT ACT CAA AGA ACT GGA 246
A~n Leu Ser Ala Asn Thr Gly Pro Ly~ Arg Gly Thr Gln Arg Thr Gly
540 545 550
GTA GAA CTT A~A CTT TTC AAC CAT CTC TGT GCC GCT GAT TTT ATT GCT 2513
Val Glu Leu Lys Leu Phe Asn Hi~ Leu Cys Ala Ala Asp Phe Ile Ala
555 560 565
TCT AAT GAG ATA GCT CTA AGG TCG ATG CTT AGA GAA TTC ATA GAA CAT 2561
Ser Asn Glu Ile Ala Leu Arg Ser Met Leu Arg Glu Phe Ile Glu His
570 575 580 585
AAA ATG GCC AAC ATA ACT AAG AAC AAT TCT GGA ATG GAA ATT ATT TGG 2609
Ly~ Met Ala Asn Ile Thr Lys Asn Asn Ser Gly Met Glu Ile Ile Trp
590 595 600
GTA CCC TAC ACG TAT GCG GAA CTT GAA A~A CTT CTG A~A ACC GTT TTA 2657
Val Pro Tyr Thr Tyr Ala Glu Leu Glu Lys Leu Leu Lys Thr Val Leu
605 610 615
AAT ACT CTA TA~ATGTATA CATATCACGA ACAATTGTAA TAGTACTAGG 2706
Asn Thr Leu
620
25 CTTGCTAGCT TTGCTTTCCC ATAACCAACA ATACTTAGTG ATGTATCTTA AAACGACTAA 2766
AAAACTTCTC ATATAACCCT ACTGA~AAAC GTCTGATGAG CTC 2809
30 ( 2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 620 amino acids
(B) TYPE: amino acid
( D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
; (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Leu Asn Gly Glu Asp Phe Val Glu His Asn Asp Ile Leu Ser Ser
1 5 10 15
Pro Ala Lys Ser Arg Asn Val Thr Pro Lys Arg Val Asp Pro His Gly
20 25 30
Glu Arg Gln Leu Arg Arg Ile His Ser Ser Lys Lys Asn Leu Leu Glu
35~ 40 45
50 Arg Ile Ser Leu Val Gly Asn Glu Arg Lys Asn Thr Ser Pro Asp Pro
50 55 60
Ala Leu Lys Pro Ly~ Thr Pro Ser Lys Ala Pro Arg Lys Arg Gly Arg
65 70 75 80
Pro Arg Lys Ile Gln Glu Glu Leu Thr ARP Arg Ile Lys Lys Asp Glu
85 90 95
Lys A~p Thr Ile Ser Ser Lys Lys Lys Arg Lys Leu Asp Lys Asp Thr
100 105 110
Ser Gly Asn Val Asn Glu Glu Ser Lys Thr Ser Asn Asn Lys Gln Val
115 120 125
65 Met Glu Lys Thr Gly Ile Lys Glu Lys Arg Glu Arg Glu Lys Ile Gln
130 135 140
2 1 78965
WO 95/16694 PCT/US94/14563
Val Ala Thr Thr Thr Tyr Glu Asp Asn Val Thr Pro Gln Thr Asp Asp
145 150 155 160
Asn Phe Val Ser Asn Ser Pro Glu Pro Pro Glu Pro Ala Thr Pro Ser
165 170 175
Lys Lyn Ser Leu Thr Thr Asn His Asp Phe Thr Ser Pro Leu Ly~ Gln
180 185 190
0 Ile Ile Met Asn Asn Leu Lys Glu Tyr Lys Asp Ser Thr Ser Pro Gly
195 200 205
Lys Leu Thr Leu Ser Arg Asn Phe Thr Pro Thr Pro Val Pro LYB Asn
210 215 220
Lys Lys Leu Tyr Gln Thr Ser Glu Thr Lys Ser Ala Ser Ser Phe Leu
225 230 235 240
Asp Thr Phe Glu Gly Tyr Phe Asp Gln Arg Lys Ile Val Arg Thr Asn
245 250 255
Ala Lys Ser Arg His Thr Met Ser Met Ala Pro Asp Val Thr Arg Glu
260 265 270
Glu Phe Ser Leu Val Ser Asn Phe Phe Asn Glu Asn Phe Gln Lys Arg
275 280 285
Pro Arg Gln Lys Leu Phe Glu Ile Gln Lys Lys Met Phe Pro Gln Tyr
290 295 300
Trp Phe Glu Leu Thr Gln Gly Phe Ser Leu Leu Phe Tyr Gly Val Gly
305 310 315 320
Ser Lys Arg Asn Phe Leu Glu Glu Phe Ala Ile Asp Tyr Leu Ser Pro
325 330 335
Lys Ile Ala Tyr Ser Gln Leu Ala Tyr Glu Asn Glu Leu Gln Gln Asn
340 345 350
Lys Pro Val Asn Ser Ile Pro Cys Leu Ile Leu Asn Gly Tyr Asn Pro
355 360 365
Ser Cys Asn Tyr Arg Asp Val Phe Lys Glu Ile Thr Asp Leu Leu Val
370 375 380
Pro Ala Glu Leu Thr Arg Ser Glu Thr Lys Tyr Trp Gly Asn His Val
385 390 395 400
Ile Leu Gln Ile Gln Lys Met Ile Asp Phe Tyr Lys Asn Gln Pro Leu
405 410 415
Asp Ile Lys Leu Ile Leu Val Val His Asn Leu Asp Gly Pro Ser Ile
420 425 430
Arg Lys Asn Thr Phe Gln Thr Met Leu Ser Phe Leu Ser Val Ile Arg
435 440 445
Gln Ile Ala Ile Val Ala Ser Thr Asp His Ile Tyr Ala Pro Leu Leu
450 455 460
Trp Asp Asn Met Lys Ala Gln A~n Tyr Asn Phe Val Phe His Asp Ile
465 470 475 480
Ser Asn Phe Glu Pro Ser Thr Val Glu Ser Thr Phe Gln Asp Val Met
485 490 495
Lys Met Gly Lys Ser Asp Thr Ser Ser Gly Ala Glu Gly Ala Lys Tyr
500 505 510
WO 95/16694 2 1 7 8 9 ~ 5 PCT/US94/14563
Val Leu Gln Ser Leu Thr Val Asn Ser Lys Lys Met Tyr Lys Leu Leu
515 520 525
Ile Glu Thr Gln Met Gln A~n Met Gly Asn Leu Ser Ala Asn Thr Gly
530 535 540
Pro Lys Arg Gly Thr Gln Arg Thr Gly Val Glu Leu Lys Leu Phe Asn
545 550 555 560
0 His Leu Cys Ala Ala ABP Phe Ile Ala Ser Asn Glu Ile Ala Leu Arg
565 570 575
Ser Met Leu Arg Glu Phe Ile Glu His Lys Met Ala Asn Ile Thr Lys
580 585 590
A~n A~n Ser Gly Met Glu Ile Ile Trp Val Pro Tyr Thr Tyr Ala Glu
595 600 605
Leu Glu Lys Leu Leu Lys Thr Val Leu Asn Thr Leu
610 615 620
(2) INFORMATION FOR SEQ ID NO:5:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2759 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
35 TCTGAAATAA AAAGTACAAA AAAGAAAACA ATATACCAGA TATGAACCCT TTTAGTGAGA 60
TTCCAGCATG TCTTTGCGCA GATCCAAATC lll~lll~lC TTGAAATTTA TTCAGTAAAT 120
TAAAAGTCAG l.~ll~AGTA GCATTCATCT TCTTGGTAAG 1~lllll~l L G 1 ~lllGAAA 180
AAGAGTTCCT GAAGlli~lC TACTGTGAAT ATACTTTGCA CAIll~lllA ATTTTTAAAC 240
ACGCTATAAT ll~lCATA AAGAATTTTT TGTAGAATAG ~1.ll L ~1l AATAGGAAAA 300
45 AAAAATAAAA AAAGGTGGAA AAGACAATCT TTTCCAGAAA CTTGAAACTA TACTGGAGAT 360
GAAGGGTTGT CGTTGGTTGC GTTACGAGAC AGGCTTGACA ATTTCACAAG AGTAATGTTT 420
CATTACCTGC l~ll~lATTA TCTTTATATT TAGTAAGACC AG~r-~AACG CTACACGTGA 480
TGATAATGGA ACTAAGCATT CTGTTAGATG GTAAGAATTT TTTTTACCTT CCATTACCAC 540
TAACGCCTTT TTTAGTGTCT TTTTGATATT TACTGACGTA lllllCCGCA CCGTAATTTG 600
AAGAAAAAGA AAAGTGACAA AAGATGGCAT TGTTTACATA CAGAGTCGTA GTATCACAAG 660
AGTAGTCCAA CAGGATGAGC GACCTTAACC AATCr~AAAA GATGAACGTC AGCGAGTTTG 720
CTGACGCCCA AAGGAGCCAC TATACAGTAT ACCCCAGTTT GCCTCAAAGT AACAAAAATG 780
ATAAACACAT lCC~lll~lC AAACTTCTAT CAGGCAAAGA ATCGGAAGTG AACGTGGAAA 840
AAAGATGGGA ATTGTATCAT CAGTTACATT CCCACTTTCA TGATCAAGTA GATCATATTA 900
65 TCGATAATAT TGAAGCAGAC TTGAAAGCAG AGATTTCAGA C~llllATAT AGTGAAACTA 960
CT~A~-~AAAG GCGATGCTTT AACACTATTT TCCTATTAGG TT~-A~-ATAGT ACGAcAAAAA 1020
WO95/16694 : 2 1 78965 PCTJUS94/14563
TTGAACTTAA A~-ACr-~ATCT TCTCGCTACA ACC1l~lGAT TGAATTGACT CC~-AAA~-AAT 1080
CTCCGAATGT AAGAATGATG ~lC6~AGGT CTATGTACAA ACTTTACAGC GCAGCTGATG 1140
CAGAAC-AACA TCCAACTATC AAGTATGAAG ACATTAACGA TGAAGATGGC GATTTTACCG 1200
AGCAAAACAA TGATGTATCA TACGATCTGT CACTTGTGGA AAACTTCAAA AGG~llll~G 1260
~AAAA~-ACTT AGCAATGGTA TTTAATTTTA AAGATGTAGA TTCTATTAAC TTCAACACAT 1320
TGGATAACTT CATAATTCTA TTGAAAAGTG CCTTCAAGTA TGACCATGTT AAAATpA~TT 1380
TAATCTTTAA TATTAATACA AA~l~AA ATATTGAGAA AAATTTGAGA CAATCAACCA 1440
TACGACTTCT GAA~-AGAAAT TAT~ATAAPC TAGACGTGTC GAGTAATAAA GGATTTAAGT 1500
ACGGAAACCA AA~ ~AA AG~ GG ATACGGTTGA TGGCAAACTA AA~ CAG 1560
A,C6,,,,~1 GGAATTCATT CTCAGCAAGA TGGCAAATAA TACTAATCAC AACTTACAAT 1620
TATTGACGAA GATGCTGGAT TA,,C6,1GA l61C6lACTT TTTCCAGAAT GCCl.ll`CAG 1680
TATTCATTGA CC~l6TAAAT GTTGATTTTT TGAACGACGA CTACTTAAAA ATACTGAGCA 1740
25 GAl~,~C~AC ATTCATGTTC l~ CGAAG GTCTTATAAA GCAGCATGCT CCTGCTGACG 1800
AAAl~ .C ATTATTGACA AACAAAAACA GAGGCCTAGA AGAC,~ l GTTGAGTTTT 1860
TGGTAA~-A~-A GAACCCGATT AACGGGCATG CTAAGTTTGT TGCTCGATTC CTCr-AA~-AAG 1920
AATTGAATAT AACCAATTTT AATCTGATAG AATTATATCA TAATTTGCTT ATTGGCAAAC 1980
TAGACTCCTA TCTAGATCGT TGGTCAGCAT GTAAAGAGTA TAAGGATCGG CTTCATTTTG 2040
35 AACCCATTGA TACAATTTTT CAAGAGCTAT TTACTTTGGA ~AACAGAAGT GGATTACTTA 2100
CCCAGTCGAT lllCC~l TACAAGTCAA ATATCGAAGA TAACTTACTA AGTTGGGAGC 2160
AGG~GCTGCC TTCGCTTGAT AAAC-AAAATT ATGATACTCT TTCTGGAGAT TTGGATAAAA 2220
TAATGGCTCC GGTACTGGGT CAGCTATTCA AGCTTTATCG TGAGGCGAAT ATGACTATCA 2280
ACATTTACGA TTTCTACATT GCGTTCAGAG AAACATTACC AAAAGAGGAA ATATTAAATT 2340
45 T~ATAAr.AAA AGAlCC~lCC AACACCAAAC TCTTAGAACT AGCAGAAACA CCGGACGCAT 2400
TTGArAAAGT AGCACTAATT TTATTCATGC AAGCAATCTT CGCCTTTGAA AACATGGGTC 2460
TCATTAAGTT TCAAAGCACC AAGAGTTACG ATCTGGTAGA AAAATGTGTC TGGAGAGGAA 2520
TTTAGATAAA GAATGCACGG ATAAATAAGT AAATAAATAA CCATACATAT ATAGAACCAT 2580
A~A-c~rGT l~ lAATG AACAGTCTAC CTGTATCTCA TCAll~ l GTGTTAACTA 2640
TTATTATTAT TATTATCGAA TGGAGGGTAA TATTATGTAT AGGTAAAATA AATAr.~TAGT 2700
GCCATGATGC GCGAAGATTG GCAATGGGAA ACTCAAGAAG GCAGCAACAA AAAAATAAA 2759
60 ( 2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 615 amino acids
(B TYPE: amino acid
(C STRANDEDNESS: ~ingle
(D TOPOLOGY: 1inear
(ii) MOLECULE TYPE: peptide
WO 95/16694 2 1 7 8 9 6 5 PCI/US94/14563
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ser Asp Leu Asn Gln Ser Lys Lys Met Asn Val Ser Glu Phe Ala
1 5 10 15
Asp Ala Gln Arg Ser His Tyr Thr Val Tyr Pro Ser Leu Pro Gln Ser
20 25 30
Asn Lys Asn Asp Lys His Ile Pro Phe Val Lys Leu Leu Ser Gly Lys
0 35 40 45
Glu Ser Glu Val AEn Val Glu Lys Arg Trp Glu Leu Tyr His Gln Leu
His Ser His Phe His Asp Gln Val Asp His Ile Ile Asp Asn Ile Glu
65 70 75 80
Ala Asp Leu Lys Ala Glu Ile Ser Asp Leu Leu Tyr Ser Glu Thr Thr
85 90 95
Gln Lys Arg Arg Cys Phe Asn Thr Ile Phe Leu Leu Gly Ser Asp Ser
100 105 110
Thr Thr Lys Ile Glu Leu Lys Asp Glu Ser Ser Arg Tyr Asn Val Leu
115 120 125
Ile Glu Leu Thr Pro Lys Glu Ser Pro Asn Val Arg Met Met Leu Arg
130 135 140
Arg Ser Met Tyr Lys Leu Tyr Ser Ala Ala Asp Ala Glu Glu His Pro
145 150 155 160
Thr Ile Lys Tyr Glu Asp Ile Asn Asp Glu Asp Gly Asp Phe Thr Glu
165 170 175
Gln Asn Asn Asp Val Ser Tyr Asp Leu Ser Leu Val Glu Asn Phe Lys
180 185 190
Arg Leu Phe Gly Lys Asp Leu Ala Met Val Phe Asn Phe Lys Asp Val
195 200 205
Asp Ser Ile Asn Phe Asn Thr Leu Asp Asn Phe Ile Ile Leu Leu Lys
210 215 220
Ser Ala Phe Lys Tyr Asp His Val Lys Ile Ser Leu Ile Phe Asn Ile
225 230 235 240
Asn Thr Asn Leu Ser Asn Ile Glu Lys Asn Leu Arg Gln Ser Thr Ile
245 250 255
Arg Leu Leu Lys Arg Asn Tyr His Lys Leu Asp Val Ser Ser Asn Lys
260 265 270
Gly Phe Lys Tyr Gly Asn Gln Ile Phe Gln Ser Phe Leu Asp Thr Val
275 280 285
Asp Gly Lys Leu Asn Leu Ser Asp Arg Phe Val Glu Phe Ile Leu Ser
290 295 300
Lys Met Ala Asn Asn Thr Asn His Asn Leu Gln Leu Leu Thr Lys Met
305 310 315 320
Leu Asp Tyr Ser Leu Met Ser Tyr Phe Phe Gln Asn Ala Phe Ser Val
325 330 335
Phe Ile Asp Pro Val Asn Val Asp Phe Leu Asn Asp Asp Tyr Leu Lys
340 345 350
WO 95/16694 ' 2 1 7 8 9 6 5 PCT/US94/14563
Ile Leu Ser Arg Cys Pro Thr Phe Met Phe Phe Val Glu Gly Leu Ile
355 360 365
Lys Gln His Ala Pro Ala Asp Glu Ile Leu Ser Leu Leu Thr Asn Lys
370 375 380
Asn Arg Gly Leu Glu Glu Phe Phe Val Glu Phe Leu Val Arg Glu Asn
385 390 395 400
Pro Ile Asn Gly His Ala Lys Phe Val Ala Arg Phe Leu Glu Glu Glu
405 410 415
Leu Asn Ile Thr Asn Phe Asn Leu Ile Glu Leu Tyr His Asn Leu Leu
420 425 430
Ile Gly Lys Leu Asp Ser Tyr Leu Asp Arg Trp Ser Ala Cys Lys Glu
435 440 445
Tyr Lys Asp Arg Leu His Phe Glu Pro Ile Asp Thr Ile Phe Gln Glu
450 455 460
Leu Phe Thr Leu Asp Asn Arg Ser Gly Leu Leu Thr Gln Ser Ile Phe
465 470 475 480
Pro Ser Tyr Lys Ser Asn Ile Glu Asp Asn Leu Leu Ser Trp Glu Gln
485 490 49S
Val Leu Pro Ser Leu Asp LYB Glu Asn Tyr Asp Thr Leu Ser Gly Asp
500 505 510
Leu Asp Lys Ile Met Ala Pro Val Leu Gly Gln Leu Phe Lys Leu Tyr
515 520 525
Arg Glu Ala Asn Met Thr Ile Asn Ile Tyr Asp Phe Tyr Ile Ala Phe
Arg Glu Thr Leu Pro Lys Glu Glu Ile Leu Asn Phe Ile Arg Lys Asp
545 550 555 - 560
Pro Ser Asn Thr Lys Leu Leu Glu Leu Ala Glu Thr Pro Asp Ala Phe
565 570 575
Asp Lys Val Ala Leu Ile Leu Phe Met Gln Ala Ile Phe Ala Phe Glu
580 585 590
Asn Met Gly Leu Ile Lys Phe Gln Ser Thr Lys Ser Tyr Asp Leu Val
595 600 605
Glu Lys Cys Val Trp Arg Gly
610 615
~2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2404 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
( ii ) MnT FCUT-~ TYPE: cDNA
(xi) ~Q~N~: DESCRIPTION: SEQ ID NO:7:
65 CTCGAGGCCA C~AA~-AAGAG AAA~-AGA~AGA GC~-ATATT GACTGGAGTG CAGCCAGAGG 60
TTCCAACTTC CAAAGCTCCT CGGAGCCACC AAGAAGAGAA AGAGAAAAGG AAGAACCAGC 120
2 1 7~3 i65
WO 95/16694 PCT/US94/14563
TTTGGATTGG GGTGCTGCCA GAGGTGCTCA GTTTGGTAAG CCTCAACAAA CCAAAAATAC 18
CTACAAGGAT AGG~-~.AA CTAACAAAAA GACTACTGAT GAGCAACCAA AAATCCAGAA 240
5 ~.~..,...AT GA.~,-...AC GTACTGAAGA TGATGATGAA QATGAAGAGG CTGAAAAGCA 300
AAATGGAGAC GCAAAA~-AAA ACAAAGTTGA TGCGGCAGTT GAAAAGCTAC AG~.ATAAAAC 360
TGCTCAATTG ACTGTTGAAG ATGGTGACAA TTGGGAAGTT GTTGGTAAGA AATA~AGTGT 420
TGTATGATGA TAAAATGTAC ATTTGTATTT A~-.-GCT .1....~--- ~..G.l.--C 480
TA~.`.C~.- TCTACCAGGT ATTCTAACTC TATTATATAA TTAAAAAAAA AATAAc~ATA 540
15 TA..--~-AT TAAGTTTCAT ACAl~l~llC AAGTGTATT~ TTGGATTTAT CAll.llClA 600
TGTGAGGTAA Gl..l.GAAT GTCCCATTTT C~l..C6111 TTGGAAAGTT CTAAGAAAAA 660
GCATTAACAA TTAAAAAAAA AAAAAAAATC TAAATAATAC TGATAGAAAT ATCAAATATA 720
AACTACTAAT ATCGGTAATA TTCAAAAGAA GAAGCATGAC TATAAGCGAA G~lCGl~lAT 780
CACCGCAAGT CAA-~l.~-C CCAATAAAGA GGCACTCAAA CGAAGAGGTA GAGGAGACTG 840
25 CAGCGATTCT AAAAAAGCGT ACTATAGATA ATGAAAAGTG TAAAGACAGC GACCCTGGTT 900
TTGGllCC~l TCAAAGAAGG TTACTGCAGC AACTTTATGG CACACTTCCT ACGGACGAAA 960
AGATAATCTT CACATATTTA CAAGATTGTC AACAAGAGAT CGATAGAATC ATTAAACAAT 1020
CCATTATTCA G~A~GAGAGT CATTCAGTAA ll~lCClGGG GCCrA~-A~-AA AGTTACAAAA 1080
CATACTTATT AGACTATGAA ~1~1~11~1 TGCAACAATC TTATAAAGAG CAGTTTATAA 1140
35 CTATCAGGTT GAATGGGTTT ATTCACTCCG AACAAACAGC TATTAACGGT ATAGCAACTC 1200
AATTGGAACA GCAGTTGCAG AAAATTCATG GCAGTGAAGA AAAAATTGAC GATACTTCAT 1260
TAGAGACTAT TAGCAGTGGT TCTTTGACAG AACl-,11.GA GAAAATTCTT TTACTCTTAG 1320
ATTCr-AC~AC GAAGACAAGA AATrAAr-ATA GTGGTGAGGT TGACAGAGAG AGT~TAACAA 1380
Ar-ATAA~A-GT .~...l.ATA TTCGATGAAA TTGATACATT TGCTGGGCCT GTGAGGCAAA 1440
45 CTTTATTATA CAAl~l lll GACATGGTAG AACATTCTCG GGTACCTGTT TGCATTTTTG 1500
GCTGCACAAC GAAATTAAAT ATCTTGGAAT ATTTAGAAAA GAGGGTAAAG AGTAGATTTT 1560
CTCAAAGAGT GATTTATATG CCGCAAATAC AGAATCTAGA CGATATGGTT GACGCCGTCA 1620
GAAATTTACT TACAGTTCGC TCTGAAATCT CCCCCTGGGT TTCACAATGG AATGAAACGT 1680
TGGAAAAAGA ACTATCCGAC CCTCGATCGA ATTTGAATAG ACATATTAGG ATGAATTTCG 1740
55 AAACCTTTAG GTCATTACCT ACATTGAAAA ATAGCATAAT TCCATTAGTA GCGACATCCA 1800
AAAATTTTGG TTCACTCTGC ACTGCCATAA AAlCGl~l.C lll~'.lGAC ATATACAATA 1860
Ar~AA~AAcT ATCTAATAAT TTAACAGGAA GGCTCCAATC TTTATCCGAT TTAGAGTTAG 1920
CCATTTTGAT CTCAGCCGCT AGGGTTGCCT TAAGGGCGAA AGACGGATCT TTTAATTTTA 1980
ATTTAGCTTA TGCAGAGTAT GAAAAGATGA TTAAAGCTAT CAACTCCAGA ATTCCCACCG 2040
65 TGGCTCCTAC TACAAATGTG GGAACAGGTC AAAGTACTTT TTCTATCGAC AATACTATCA 2100
AACTATGGTT GAAAAAGGAC GTCAAGAACG TTTGGGAAAA TTTAGTGCAA CTGGATTTTT 2160
56
~ 217~3965
WO 95/16694 PCT/IJS94/14563
TTA~CGA~-AA ATCAGCCGTT GGTTTGAGAG ATAATGCGAC CGCAGCATTT TACGCTAGCA 2220
ATTATCAATT TCAGGGCACC ATGATCCCGT TTGACTTGAG AAGTTACCAG ATGCAGATCA 2280
~ AGGA ATTAAGAAGA ATTATCCCCA AATCTAATAT GTACTACTCC TGGACACAAC 2340
TGTGAATCTT GG~AA~AATA TAr~ AC~TT TTATTGGCGG TAGCAACTCT GATATTCCAC 2400
TGTT 2404
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A LENGTH: 529 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
Dl TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Thr Ile Ser Glu Ala Arg Leu Ser Pro Gln Val Asn Leu Leu Pro
1 5 10 15
Ile Lys Arg His Ser Asn Glu Glu Val Glu Glu Thr Ala Ala Ile Leu
Lys Lys Arg Thr Ile Asp Asn Glu Lys Cys LYB Asp Ser Asp Pro Gly
Phe Gly Ser Leu Gln Arg Arg Leu Leu Gln Gln Leu Tyr Gly Thr Leu
50 55 60
Pro Thr Asp Glu Lys Ile Ile Phe Thr Tyr Leu Gln Asp Cys Gln Gln
40 Glu Ile Asp Arg Ile Ile Lys Gln Ser Ile Ile Gln Lys Glu Ser His
85 90 95
Ser Val Ile Leu Val Gly Pro Arg Gln Ser Tyr Lys Thr Tyr Leu Leu
100 105 110
Asp Tyr Glu Leu Ser Leu Leu Gln Gln Ser Tyr Lys Glu Gln Phe Ile
115 120 125
Thr Ile Arg Leu Asn Gly Phe Ile His Ser Glu Gln Thr Ala Ile Asn
130 135 140
Gly Ile Ala Thr Gln Leu Glu Gln Gln Leu Gln Lys Ile His Gly Ser
145 150 155 160
55 Glu Glu Lys Ile Asp Asp Thr Ser Leu Glu Thr Ile Ser Ser Gly Ser
165 170 175
Leu Thr Glu Val Phe Glu Lys Ile Leu Leu Leu Leu Asp Ser Thr Thr
180 185 190
Lys Thr Arg Asn Glu Asp Ser Gly Glu Val Asp Arg Glu Ser Ile Thr
195 200 205
Lys Ile Thr Val Val Phe Ile Phe Asp Glu Ile Asp Thr Phe Ala Gly
210 215 220
Pro Val Arg Gln Thr Leu Leu Tyr Asn Leu Phe Asp Met Val Glu His
225 230 235 240
WOg5/16694 ; 2 1 78965 PCT~S94~l4s63
Ser Arg Val Pro Val Cys Ile Phe Gly CYR Thr Thr Lys Leu Asn Ile
245 250 255
Leu Glu Tyr Leu Glu Lys Arg Val Lys Ser Arg Phe Ser Gln Arg Val
260 265 270
Ile Tyr Met Pro Gln Ile Gln Asn Leu Asp Asp Met Val Asp Ala Val
275 280 285
Arg Asn Leu Leu Thr Val Arg Ser Glu Ile Ser Pro Trp Val Ser Gln
290 295 300
Trp Asn Glu Thr Leu Glu Lys Glu Leu Ser Asp Pro Arg Ser Asn Leu
305 310 315 320
Asn Arg His Ile Arg Met Asn Phe Glu Thr Phe Arg Ser Leu Pro Thr
325 330 335
Leu Lys Asn Ser Ile Ile Pro Leu Val Ala Thr Ser Lys Asn Phe Gly
340 345 350
Ser Leu Cys Thr Ala Ile Lys Ser Cys Ser Phe Leu Asp Ile Tyr Asn
355 360 365
Lys Asn Gln Leu Ser Asn Asn Leu Thr Gly Arg Leu Gln Ser Leu Ser
370 375 380
Asp Leu Glu Leu Ala Ile Leu Ile Ser Ala Ala Arg Val Ala Leu Arg
385 390 395 400
Ala Lys Asp Gly Ser Phe Asn Phe Asn Leu Ala Tyr Ala Glu Tyr Glu
405 410 415
Lys Me~t Ile Lys Ala Ile Asn Ser Arg Ile Pro Thr Val Ala Pro Thr
420 425 430
Thr Asn Val Gly Thr Gly Gln Ser Thr Phe Ser Ile Asp Asn Thr Ile
435 440 445
Ly6 Leu Trp Leu Lys Lys Asp Val Lys Asn Val Trp Glu Asn Leu Val
450 455 460
Gln Leu Asp Phe Phe Thr Glu Lys Ser Ala Val Gly Leu Arg Asp Asn
465 470 475 480
Ala Thr Ala Ala Phe Tyr Ala Ser Asn Tyr Gln Phe Gln Gly Thr Met
485 490 495
Ile Pro Phe Asp Leu Arg Ser Tyr Gln Met Gln Ile Ile Leu Gln Glu
500 505 510
Leu Arg Arg Ile Ile Pro Lys Ser Asn Met Tyr Tyr Ser Trp Thr Gln
515 520 525
Leu
(2) INFORMATION FOR SEQ ID NO:9:
~ i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2306 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
WO 9S/16694 2 1 7 8 9 6 5 Pcr/Us94~l4563
(xi) S~QD~ DESCRIPTION: SEQ ID NO:9:
GCTATTTTTT CATGCGTCAG ATGTCACAAA GCCTTTAATC AAGTATTGTT GCAAGAACAC 60
CTGATTCAAA AACTACGTTC TGATATCGAA TCCTATTTAA TTCAAGATTT GAGATGCTCC 120
AGATGTCATA AAGTGAAACG TGACTATATG AGTGCCCACT GTCCATGTGC CGGCGCGTGG 180
GAAGGAACTC TCCCCAGAGA AAGCATTGTT CAAAAGTTAA AlGl~l.lAA GCAAGTAGCC 240
AAGTATTACG GTTTTGATAT ATTATTGAGT TGTATTGCTG ATTTGACCAT ATGAGTAAGC 300
AGTATATAAC GCGAGGTTCA ATGGCCTCTT TACCATGAAA AAJUiUU~AAA AAAAAAAAAA 360
15 AAGGTAAGGA AAAAGAGTAT TTTCAATTCG TTTCTGAACA TATAAATATA AATAACCGAA 420
AAATTAGCCC TT~ArATAA TTAACACTCT TCTTTGATAT TTAAATCACA AGTACTTTTC 480
TTTTATTTTC TTCTTAATAC TTTTGGAAAT AAAATGAATG TGACCACTCC GGAAGTTGCT 540
TTTAGGGAAT ATCAAACCAA ~.G.~.CGCA TCGTATATTT CTGCTGATCC AGACATAACT 600
CCTTCAAATT TAATCTTGCA AGGTTATAGT GGAACAGGAA AAACCTACAC TTTGAAGAAG 660
25 TATTTTAATG CGAATCCAAA TTTGCATGCA GTATGGCTGG AAC~lGl-GA GTTGGTTTCT 720
TGGAAGCCCT TACTGCAGGC GATAGCACGT ACTGTACAAT ATAAATTGAA AACCCTATAT 780
CrPAAr-ATTC CCACCACAGA TTACGATCCT TTACAGGTTG AAGAGCCATT ~ GGTA 840
AAGACGTTGC ACAATATTTT TGTCCAATAT GAATCTTTGC AAGAAAAGAC TTG~ll~l.C 900
TTGATATTGG ATGGTTTCGA TAGTTTACAA GATTTAGACG CCGCACTGTT TAACAAATAT 960
35 ATCAAACTAA ATGAATTACT TCCAAAAGAT TCTAAAATTA ATATAAAATT CATTTACACG 1020
ATGTTAGAGA CATCATTTTT GCAAAGATAT TCTACACATT GCATTCCAAC TGTTATGTTT 1080
CCGAGGTATA ATGTGGACGA A~--l~lACT ATATTAGTGA TGTCTAGATG TGGCGAACTC 1140
ATGGAAGATT ~l.`-.~.ACG TAAGCGTATC ATTGAAGAGC AGATAACGGA CTGTACAGAC 1200
GATCAATTTC AAAATGTAGC TGCGAACTTC ATTCACTTAA TTGTGCAGGC TTTTCATTCT 1260
45 TATACTGGAA ACGACATATT CGCATTGAAT GACTTGATAG ACTTCAAATG GCCCAAGTAT 1320
GTATCTCGCA TTACTAAGGA AAAcATATTT GAACCACTGG ~.~l~lACAA AAGTGCCATC 1380
- AAACTATTTT TAAGCACAGA TGATAATTTA AGTGAAAATG GACAAGGTGA AAGCGCGATA 1440
AC~AAATC GTGATGACCT TGAGAACAGT CAAACTTACG ACTTATCAAT AATTTCGAAG 1500
TATCTGCTCA TAGCCTCATA TAl..Gl.`A TATCTGGAAC CTAGATACGA TGCGAGTATT 1560
55 ..C.~.AGGA AAACACGTAT ~ATA~AGGT AGAGCTGCTT ATGGACGAAG AAA~AAr~AAA 1620
GAAGTTAACC CTAGATATTT ACAGCCTTCT TTATTTGCTA TTGAAAGACT TTTGGCTATT 1680
TTCCAAGCTA TA..CC~lAT TCAAGGTAAG GCGGAGAGTG G..CC~lATC TGCACTTCGT 1740
GAGGAATCCT TAATGAAAGC GAATATCGAG Gl...l~AAA ATTTATCCGA ATTGCATACA 1800
TTGAAATTAA TAGCTACAAC CATGAACAAG AATATCGACT ATTTGAGTCC TAAAGTCAGG 1860
65 TGGAAAGTAA ACGTTCCCTG GGAAATTATT AAAGAAATAT CAGAATCTGT TCATTTCAAT 1920
ATCAGCGATT ACTTCAGCGA TATTCACGAA TGATTATCTC CCTGGAAGGT ATCCAGAGGG 1980
59
W O95/16694 2 i 7 8 9 6 5 pCTrUS94/14S63
CAGGATACGT TCGAAACAAC AACTACGTTA TATAAATATT TATACATAGT GG~-ATAGAAT 204
GAACAATTAT CAAGTAAACC TTGTATTTTT .~CC~ACG CTCTACGCTC iGl-l~llGG 2100
ATATGGTAAT CAAArATTAA TACGTATAAC CGTTATTAAT TCAGTCCACT A~AA~CTATT 2160
AAAAGCGCCC TACTGTATGG AAAAACAATG AATGAGGAGA CTGAACGGCG CAAAATTGTT 2220
AGTTTAGTTG ~C~l~llGG CGGCCGGCGA TAA`~ l CACTTGGTAT TCTTACCAGG 2280
ATTGAGCCTG Al,.. ~..... GTCTTA 2306
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A LENGTH: 479 amino acids
(B TYPE: amino acid
~C STRANDEDNESS: single
(D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asn Val Thr Thr Pro Glu Val Ala Phe Arg Glu Tyr Gln Thr Asn
1 5 10 15
cys Leu Ala Ser Tyr Ile Ser Ala Asp Pro Asp Ile Thr Pro Ser Asn
Leu Ile Leu Gln Gly Tyr Ser Gly Thr Gly Lys Thr Tyr Thr Leu Lys
Lys Tyr Phe Asn Ala Asn Pro Asn Leu His Ala Val Trp Leu Glu Pro
50 55 60
Val Glu Leu Val Ser Trp Lys Pro Leu Leu Gln Ala Ile Ala Arg Thr
65 70 75 8,0
Val Gln Tyr Lys Leu Lys Thr Leu Tyr Pro Asn Ile Pro Thr Thr Asp
85 90 95
Tyr Asp Pro Leu Gln Val Glu Glu Pro Phe Leu Leu Val Lys Thr Leu
100 105 110
His Asn Ile Phe Val Gln Tyr Glu Ser Leu Gln Glu Lys Thr Cys Leu
115 120 125
Phe Leu Ile Leu Asp Gly Phe Asp Ser Leu Gln Asp Leu Asp Ala Ala
130 135 140
Leu Phe Asn Lys Tyr Ile Lys Leu Asn Glu Leu Leu Pro Lys Asp Ser
145 150 155 160
Lys Ile Asn Ile Lys Phe Ile Tyr Thr Met Leu Glu Thr Ser Phe Leu
165 170 175
Gln Arg Tyr Ser Thr His Cys Ile Pro Thr Val Met Phe Pro Arg Tyr
180 185 190
Asn Val Asp Glu Val Ser Thr Ile Leu Val Met Ser Arg Cys Gly Glu
195 200 205
Leu Met Glu Asp Ser Cys Leu Arg Lys Arg Ile Ile Glu Glu Gln Ile
210 215 220
21 7~965
W 095/16694 PCTrUS94/14563
Thr Asp Cy8 Thr Asp Asp Gln Phe Gln Asn Val Ala Ala Asn Phe Ile
225 230 235 240
His Leu Ile Val Gln Ala Phe His Ser Tyr Thr Gly Asn Asp Ile Phe
245 250 255
Ala Leu Asn Asp Leu Ile Acp Phe Lys Trp Pro Lys Tyr Val Ser Arg
260 265 270
Ile Thr Lys Glu Asn Ile Phe Glu Pro Leu Ala Leu Tyr Lys Ser Ala
275 280 285
Ile Lys Leu Phe Leu Ser Thr Asp Asp Asn Leu Ser Glu Asn Gly Gln
290 295 300
Gly Glu Ser Ala Ile Thr Thr Asn Arg Asp Asp Leu Glu Asn Ser Gln
305 310 315 320
Thr Tyr Asp Leu Ser Ile Ile Ser Lys Tyr Leu Leu Ile Ala Ser Tyr
325 330 335
Ile Cys Ser Tyr Leu Glu Pro Arg Tyr Asp Ala Ser Ile Phe Ser Arg
340 345 350
Lys Thr Arg Ile Ile Gln Gly Arg Ala Ala Tyr Gly Arg Arg Lys Lys
355 360 365
Lys Glu Val Asn Pro Arg Tyr Leu Gln Pro Ser Leu Phe Ala Ile Glu
370 375 380
Arg Leu Leu Ala Ile Phe Gln Ala Ile Phe Pro Ile Gln Gly Lys Ala
385 390 395 400
Glu Ser Gly Ser Leu Ser Ala Leu Arg Glu Glu Ser Leu Met Lys Ala
405 410 415
Asn Ile Çlu Val Phe Gln Asn Leu S~r Glu Leu His Thr Leu Lys Leu
420 425 430
0 . Ile Ala Thr Thr Met Asn Lys Asn Ile Asp Tyr Leu Ser Pro Lys Val
435 440 445
Arg Trp Lys Val Asn Val Pro Trp Glu Ile Ile Lys Glu Ile Ser Glu
450 455 460
Ser Val His Phe Asn Ile Ser Asp Tyr Phe Ser Asp Ile His Glu
465 470 475
(2) INFORMATION FOR SEQ ID NO:ll:
( i ) s~:Qu~l._~ CHARACTERISTICS:
A) LENGTH: 1975 base pairs
B) TYPE: nucleic acid
C) STRANDEDNESS: double
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 443..1747
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
CGTGTGCTCT TCTATAGTAA TTTGACATTC TCTAAACGCA GAGACCTCTT ATAAAGATTC 60
AA~AATAAG GAATGTTACC TATGCTAGTC GCAACTCTCT CGTAAGTTGA GGGTTGCTAA 120
61
W O 95/16694 2 1 7 8 9 6 5 PcTrus94ll4563
CAGAAAAACG ATGAGAAGAA ACTTTTGAAA AATATTGTGT GAAAGCAGCA cGA-AA~Ar-AG 18(
TATGAAAAAA GAATGCGGGC ~CCG~AAAG AGCTAGAATC GCAAGTGTCC AGAATATGCA 240
AGGCTTTCGA ATACACTCCT CACGCTTCTC TTCAGCAAAA ATCAACTCTT TGT~-ATAAAA 300
~ ATTT ~ TGCCG--~.. TACGTTAGTA AGAAATCGGC ATTr~AAAAAA- 360
AAAATCTCAC ACTAAAATTG ~AGAAAAAAG TGTACAATAT CAGTAAATAA AATTGGCCAA 420
AArAATAc~A TTAAAACCAG TC ATG TCC ATG CAA CAA GTC CAA CAT TGT GTC 472
Met Ser Met Gln Gln Val Gln His Cys Val
l 5 10
GCA GAA GTA CTT CGA CTA GAT CCA CAA GAA AAA CCG GAC TGG TCG AGC 520
Ala Glu Val Leu Arg Leu Asp Pro Gln Glu Ly~ Pro Asp Trp Ser Ser
GGA TAT TTG AAG AAG TTG ACT AAT GCG ACA TCG ATT TTA TAT AAT ACT 568
20 Gly Tyr Leu Lys Lys Leu Thr Asn Ala Thr Ser Ile Leu Tyr Asn Thr
30 35 40
TCA CTG AAC AAG GTA ATG CTG AAA CAA GAT GAA GAG GTT GCT AGA TGT 616
Ser Leu Asn Lys Val Met Leu Lys Gln Asp Glu Glu Val Ala Arg Cys
45 50 55
CAC ATA TGT GCA TAC ATA GCG TCA CAG AAA ATG AAT GAA AAA CAC ATG 664
His Ile Cys Ala Tyr Ile Ala Ser Gln Lys Met Asn Glu Lys His Met
CCT GAC CTT TGC TAT TAT ATA GAC AGT ATT CCC TTG GAG CCG AAA AAA 712
Pro Asp Leu Cys Tyr Tyr Ile Asp Ser Ile Pro Leu Glu Pro Lys Lys
GCC AAG CAT TTA ATG AAC CTT TTC AGA CAA AGT TTA TCT AAT TCT TCA 760
Ala Lys His Leu Met Asn Leu Phe Arg Gln Ser Leu Ser Asn Ser Ser
lO0 105
CCT ATG AAA CAA TTT GCT TGG ACA CCG AGC CCC AAA AAG AAC AAA CGC 808
Pro Met Lys Gln Phe Ala Trp Thr Pro Ser Pro Lys Lys Asn Lys Arg
110 115 120
AGT CCA GTA AAG AAC GGT GGG AGG TTT ACT TCT TCT GAT CCG AAA GAG 856
Ser Pro Val Lys Asn Gly Gly Arg Phe Thr Ser Ser Asp Pro Lys Glu
125 130 135
TTG AGG AAT CAA CTG TTT GGT ACA CCA ACT AAA GTT AGG AAA AGC CAA 904
Leu Arg Asn Gln Leu Phe Gly Thr Pro Thr Lys Val Arg Lys Ser Gln
140 145 150
AAT AAT GAT TCG TTC GTA ATA CCA GAA CTA CCC CCC ATG CAA ACC AAT 952
Asn Asn Asp Ser Phe Val Ile Pro Glu Leu Pro Pro Met Gln Thr Asn
155 160 165 170
GAA TCG CCG TCT ATT ACT AGG AGA AAG TTA GCA TTT GAA GAG GAT GAG 1000
Glu Ser Pro Ser Ile Thr Arg Arg Lys Leu Ala Phe Glu Glu Asp Glu
175 180 185
60 GAT GAG GAT GAA GAG GAA CCA GGA AAC GAC GGT TTG TCT TTA AAA AGC 1048
Asp Glu Asp Glu Glu Glu Pro Gly Asn Asp Gly Leu Ser Leu Lys Ser
190 195 200
CAT AGT AAT AAG AGC ATT ACT GGA ACC AGA AAT GTA GAT TCT GAT GAG 1096
65 His Ser Asn Lys Ser Ile Thr Gly Thr Arg Asn Val Asp Ser Asp Glu
205 210 215
TAT GAA AAC CAT GAA AGT GAC CCT ACA AGT GAG GAA GAG CCA TTA GGT 1144
62
WO95/16694 ; :. 2 1 78965 PCT/US94/14563
Tyr Glu Asn His Glu Ser Asp Pro Thr Ser Glu Glu Glu Pro Leu Gly
220 225 230
GTG CAA GAA AGC AGA AGC GGG AGA ACG AAA CAA AAT AAG GCA GTT GGA 1192
Val Gln Glu Ser Arg Ser Gly Arg Thr Lys Gln Asn Lys Ala Val Gly
235 240 245 250
AAA CCG CAA TCA GAA TTG AAG ACG GCA AAA GCC CTG AGG AAA AGG GGC 1240
Lys Pro Gln Ser Glu Leu Lys Thr Ala Lys Ala Leu Arg Lys Arg Gly
0 255 260 265
AGA ATA CCA AAT TCT TTG TTA GTA AAG AAG TAT TGC AAA ATG ACT ACT 1288
Arg Ile Pro Asn Ser Leu Leu Val Lys Lys Tyr Cys Lys Met Thr Thr
270 275 280
GAA GAA ATA ATA CGG CTT TGC AAC GAT TTT GAA TTA CCA AGA GAA GTA 1336
Glu Glu Ile Ile Arg Leu Cys Asn Asp Phe Glu Leu Pro Arg Glu Val
285 290 295
20 GCA TAT AAA ATT GTG GAT GAG TAC AAC ATA AAC GCG TCA AGA TTG GTT 1384
Ala Tyr Ly~ Ile Val Asp Glu Tyr Asn Ile Asn Ala Ser Arg Leu Val
300 305 310
TGC CCA TGG CAA TTA GTG TGT GGG TTA GTA TTA AAT TGT ACA TTC ATT 1432
Cy~ Pro Trp Gln Leu Val Cys Gly Leu Val Leu Asn Cys Thr Phe Ile
315 320 325 330
GTA TTT AAT GAA AGA AGA CGC AAG GAT CCA AGA ATT GAC CAT TTT ATA 1480
Val Phe Asn Glu Arg Arg Arg Lys Asp Pro Arg Ile Asp His Phe Ile
335 340 345
GTC AGT AAG ATG TGC AGC TTG ATG TTG ACG TCA AAA GTG GAT GAT GTT 1528
Val Ser Lys Met Cys Ser Leu Met Leu Thr Ser Lys Val Asp Asp Val
~350 355 360
ATT GAA TGT GTA AAA TTA GTG AAG GAA TTA ATT ATC GGT GAA AAA TGG 1576
Ile Glu Cys Val Lys Leu Val Lys Glu Leu Ile Ile Gly Glu Lys Trp
365 370 375
TTC AGA GAT TTG CAA ATT AGG TAT GAT GAT TTT GAT GGC ATC AGA TAC 1624
Phe Arg Asp Leu Gln Ile Arg Tyr Asp Asp Phe Asp Gly Ile Arg Tyr
380 385 390
GAT GAA ATT ATA TTT AGG AAA CTG GGA TCG ATG TTA CAA ACC ACC AAT 1672
Asp Glu Ile Ile Phe Arg Lys Leu Gly Ser Met Leu Gln Thr Thr Asn
395 400 405 410
ATT TTG GTC ACA GAC GAC CAG TAC AAT ATT TGG AAG AAA AGA ATT GAA 1720
Ile Leu Val Thr Asp Asp Gln Tyr Asn Ile Trp Lys Lys Arg Ile Glu
415 420 425
ATG GAT TTG GCA TTA ACA GAA CCT TTA TAACATATCC AGTATTAACT 1767
Met Asp Leu Ala Leu Thr Glu Pro Leu
430 435
AAAAGTATAT ATTTGACCAA TACCTGACAT A~ ~.AAA GCATGCCTTT AGCCCTATAA 1827
CGAGCTAATG TTAGCTCCAT CTTTGCACTT ATGATTGGAT CAGCCCTCAA ACG~L~..~. 1887
60 ATCTTTGCAG CTTCCGCGAA GGTAGTAGCT TGAAGTTTTT CATCCATAGT TCTTGCTAAA 1947
ATTGCAGAAT CTTCAAACAA TTCTATGG 1975
65 ~ 2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 435 amino acids
63
WO 95/16694 ~ 2 ~ 7 8 9 6 5 PCT/US94/]4563
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Ser Met Gln Gln Val Gln His Cys Val Ala Glu Val Leu Arg Leu
1 5 10 15
Asp Pro Gln Glu Lys Pro Asp Trp Ser Ser Gly Tyr Leu Lys Lys Leu
Thr Asn Ala Thr Ser Ile Leu Tyr Asn Thr Ser Leu Asn Lys Val Met
35 40 45
Leu Lys Gln Asp Glu Glu Val Ala Arg Cys His Ile Cys Ala Tyr Ile
20 Ala Ser Gln Lys Met A~n Glu Lys His Met Pro Asp Leu Cys Tyr Tyr
65 70 75 80
Ile A~p Ser Ile Pro Leu Glu Pro Lys Lys Ala Lys His Leu Met Asn
85 90 95
Leu Phe Arg Gln Ser Leu Ser Asn Ser Ser Pro Met Lys Gln Phe Ala
100 105 110
Trp Thr Pro Ser Pro Lys Lys Asn Lys Arg Ser Pro Val Lys Asn Gly
115 120 125
Gly Arg Phe Thr Ser Ser Asp Pro Lys Glu Leu Arg Asn Gln Leu Phe
3 130 135 140
Gly Thr Pro Thr Lys Val Arg Lys Ser Gln Asn Asn Asp Ser Phe Val
145 150 155 160
; Ile Pro Glu Leu Pro Pro Met.Gln Thr Asn Glu Ser Pro Ser Ile Thr
. 165 170 . 175
Arg Arg Lys Leu Ala Phe Glu Glu Asp Glu.Asp Glu Asp Glu Glu Glu
180 185 190
45 Pro Gly Asn Asp Gly Leu Ser Leu Lys Ser His Ser Asn Lys Ser Ile
195 200 205
Thr Gly Thr Arg Asn Val Asp Ser Asp Glu Tyr Glu Asn His Glu Ser
210 215 220
Asp Pro Thr Ser Glu Glu Glu Pro Leu Gly Val Gln Glu Ser Arg Ser
225 230 235 240
Gly Arg Thr Lys Gln Asn Lys Ala Val Gly Lys Pro Gln Ser Glu Leu
245 250 255
Lys Thr Ala Lys Ala Leu Arg Lys Arg Gly Arg Ile Pro Asn Ser Leu
260 265 270
60 Leu Val Lys Lys Tyr Cys Lys Met Thr Thr Glu Glu Ile Ile Arg Leu
275 280 285
Cys Asn Asp Phe Glu Leu Pro Arg Glu Val Ala Tyr Lys Ile Val Asp
290 295 300
Glu Tyr Asn Ile Asn Ala Ser Arg Leu Val Cys Pro Trp Gln Leu Val
305 310 315 320
2 t 78965
WO 95/16694 ~ . . PCTtUS94tl4563
Cys Gly Leu Val Leu Asn Cys Thr Phe Ile Val Phe Asn Glu Arg Arg
325 330 335
Arg Lys Asp Pro Arg Ile Asp His Phe Ile Val Ser Lys Met Cys Ser
340 345 350
Leu Met Leu Thr Ser Lys Val Asp Asp Val I le Glu Cys Val Lys Leu
355 360 365
0 Val LYB Glu Leu Ile Ile Gly Glu Lys Trp Phe Arg Asp Leu Gln Ile
370 375 380
Arg Tyr ABP Asp Phe ABP Gly Ile Arg Tyr ABP Glu Ile Ile Phe Arg
385 390 395 400
LYB Leu Gly Ser Met Leu Gln Thr Thr Asn Ile Leu Val Thr Asp Asp
405 410 415
Gln Tyr Asn I le Trp LYB LYB Arg I le Glu Met Asp Leu Ala Leu Thr
420 425 430
Glu Pro Leu
435
