Ubiquitin conjugating enzymes

The present invention relates to drug screening assays which provide a systematic and practical approach for the identification of candidate agents able to inhibit ubiquitin-mediated degradation of a cell-cycle regulatory protein, such as p53, p27, myc, fos, MAT.alpha.2, or cyclins. The invention further relates to novel ubiquitin-conjugating enzymes, and uses related thereto.

BACKGROUND OF THE INVENTION 
The ubiquitin-mediated proteolysis system is the major pathway for the 
selective, controlled degradation of intracellular proteins in eukaryotic 
cells. Ubiquitin modification of a variety of protein targets within the 
cell appears to be important in a number of basic cellular functions such 
as regulation of gene expression, regulation of the cell-cycle, 
modification of cell surface receptors, biogenesis of ribosomes, and DNA 
repair. One major function of the ubiquitin-mediated system is to control 
the half-lives of cellular proteins. The half-life of different proteins 
can range from a few minutes to several days, and can vary considerably 
depending on the cell-type, nutritional and environmental conditions, as 
well as the stage of the cell-cycle. 
Targeted proteins undergoing selective degradation, presumably through the 
actions of a ubiquitin-dependent proteosome, are covalently tagged with 
ubiquitin through the formation of an isopeptide bond between the 
C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the 
substrate protein. This process is catalyzed by a ubiquitin-activating 
enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances 
may also require auxiliary substrate recognition proteins (E3s). Following 
the linkage of the first ubiquitin chain, additional molecules of 
ubiquitin may be attached to lysine side chains of the previously 
conjugated moiety to form branched multi-ubiquitin chains. 
The conjugation of ubiquitin to protein substrates is a multi-step process. 
In an initial ATP requiring step, a thioester is formed between the 
C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. 
Activated ubiquitin is then transferred to a specific cysteine on one of 
several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein 
substrates. Substrates are recognized either directly by 
ubiquitin-conjugated enzymes or by associated substrate recognition 
proteins, the E3 proteins, also known as ubiquitin ligases. 
Ubiquitin is itself a substrate for ubiquitination. Depending on the 
ubiquitin-conjugating enzyme and the nature of the substrate, specific 
lysine residues of ubiquitin are used as acceptor sites for further 
ubiquitinations. This can lead to either a linear multi-ubiquitin chain 
(when a single lysine residue of ubiquitin is used) or multi-ubiquitin 
"trees" (when more than one lysine reside of ubiquitin is used). Although 
the attachment of a single ubiquitin moiety to a substrate can be 
sufficient for degradation, multi-ubiquitination appears to be required in 
most cases. 
Many proteins that control cell-cycle progression are short-lived. For 
example, regulation of oncoproteins and anti-oncoproteins clearly plays an 
important role in determining steady-state levels of protein expression, 
and alterations in protein degradation are as likely as changes in 
transcription and/or translation to cause either the proliferative arrest 
of cells, or alternatively, the transformation of cells. 
For instance, the p53 protein is a key regulator of mammalian cell growth 
and its gene is frequently mutated in a wide range of human tumors 
(Hollstein et al. (1991) Science 253:49-53). Furthermore, many DNA tumor 
viruses encode viral antigens that inactivate p53 (e.g., see Vogelstein et 
al. (1992) Cell 70:523-526). The high risk human papillomaviruses, such as 
HPV-16 and -18, are strongly implicated in the pathogenesis of cervical 
carcinoma (zur Hansen et al. (1991) Science 254:1167-1173). These viruses 
encode two transforming proteins, E6 and E7, that target the cellular 
growth regulators p53 and pRb respectively. The mode of inactivation of 
p53 by E6 is apparently mediated by a ubiquitin-dependent pathway. Viral 
E6 and a cellular E6 -associated protein (E6AP) combine to stimulate the 
ubiquitination of p53, thus targeting p53 for degradation (Scheffner et 
al. (1990) Cell 63:1129-1136. In this reaction, E6 and E6 AP are thought 
to be providing a ubiquitin ligase, or E3-like activity (Scheffner et al. 
(1993) Cell 75:495-505). However, the ubiquitin-conjugating enzyme (E2) 
involved in p53 ubiquitination has not previously been characterized. 
SUMMARY OF THE INVENTION 
The present invention relates to the discovery of novel ubiquitin 
conjugating enzymes (hereinafter UBC's, e.g., UbCE's or rapUBC). 
One aspect of the present invention relates to the discovery in eukaryotic 
cells, particularly human cells and certain yeast cells, of a novel 
ubiquitin conjugating enzyme (hereinafter "UbCE"). In human cells, the 
enzyme can function to mediate ubiquitination of cell check regulatory 
proteins, e.g. p53, and is therefore involved in regulating cell cycle 
progression, e.g. cell growth. 
Another aspect of the present invention relates to the discovery in human 
cells of novel ubiquitin conjugating enzyme (rapUBC), which was discovered 
based on its ability to bind FKBP/rapamycin complexes. This enzyme can 
function to mediate ubiquitination of cell check regulatory proteins, e.g. 
p53, and is therefore involved in regulating eucaryotic cell cycle 
progression, e.g. cell growth. 
Another aspect of the invention features a substantially pure preparation 
of a human UbCE polypeptide ("hUbCE"), or a fragment thereof, which can 
function as a ubiquitin conjugating enzyme. In a preferred embodiment: the 
polypeptide has an amino acid sequence at least 90% homologous to the 
amino acid sequence of SEQ ID No. 2; the polypeptide has an amino acid 
sequence at least 95% homologous to the amino acid sequence of SEQ ID No. 
2; the polypeptide has an amino acid sequence at least 97% homologous to 
the amino acid sequence of SEQ ID No. 2; the polypeptide has an amino acid 
sequence identical to the amino acid sequence of SEQ ID No. 2. In a 
preferred embodiment: the fragment comprises at least 5 contiguous amino 
acid residues of SEQ ID No. 2; the fragment comprises at least 20 
contiguous amino acid residues of SEQ ID No. 2; the fragment comprises at 
least 50 contiguous amino acid residues of SEQ ID No. 2. In a preferred 
embodiment, the fragment comprises at least a portion of amino acid 
residues Cys-107 through Met-147, e.g. 5 amino acid residues, e.g. 15 
amino acid residues, e.g. 25 amino acid residues. 
Another aspect of the invention features a substantially pure preparation 
of a Candida UbCE polypeptide ("caUbCE"), or a fragment thereof, which can 
function as a ubiquitin conjugating enzyme. In a preferred embodiment: the 
polypeptide has an amino acid sequence at least 90% homologous to the 
amino acid sequence of SEQ ID No. 4; the polypeptide has an amino acid 
sequence at least 95% homologous to the amino acid sequence of SEQ ID No. 
4; the polypeptide has an amino acid sequence at least 97% homologous to 
the amino acid sequence of SEQ ID No. 4; the polypeptide has an amino acid 
sequence identical to the amino acid sequence of SEQ ID No. 4. In a 
preferred embodiment: the fragment comprises at least 5 contiguous amino 
acid residues of SEQ ID No. 4; the fragment comprises at least 20 
contiguous amino acid residues of SEQ ID No. 4; the fragment comprises at 
least 50 contiguous amino acid residues of SEQ ID No. 4. In a preferred 
embodiment, the fragment comprises at least a portion of amino acid 
residues Cys-107 through Val-147, e.g. 5 amino acid residues, e.g. 15 
amino acid residues, e.g. 25 amino acid residues. 
Another aspect of the invention features a substantially pure preparation 
of a Schizosaccharomyces UbCE polypeptide ("spUbCE"), or a fragment 
thereof, which can function as a ubiquitin conjugating enzyme. In a 
preferred embodiment: the polypeptide has an amino acid sequence at least 
90% homologous to the amino acid sequence of SEQ ID No. 6; the polypeptide 
has an amino acid sequence at least 95% homologous to the amino acid 
sequence of SEQ ID No. 6; the polypeptide has an amino acid sequence at 
least 97% homologous to the amino acid sequence of SEQ ID No. 6; the 
polypeptide has an amino acid sequence identical to the amino acid 
sequence of SEQ ID No. 6. In a preferred embodiment: the fragment 
comprises at least 5 contiguous amino acid residues of SEQ ID No. 6; the 
fragment comprises at least 20 contiguous amino acid residues of SEQ ID 
No. 6; the fragment comprises at least 50 contiguous amino acid residues 
of SEQ ID No. 6. In a preferred embodiment, the fragment comprises at 
least a portion of amino acid residues Cys-107 through Ile-147, e.g. 5 
amino acid residues, e.g. 15 amino acid residues, e.g. 25 amino acid 
residues. 
Another aspect of the invention features a substantially pure preparation 
of a human UBC polypeptide ("rapUBC"), or a fragment thereof, which can 
function as a ubiquitin conjugating enzyme. In a preferred embodiment: the 
polypeptide has an amino acid sequence at least 90% homologous to the 
amino acid sequence of SEQ ID No. 13; the polypeptide has an amino acid 
sequence at least 95% homologous to the amino acid sequence of SEQ ID No. 
13; the polypeptide has an amino acid sequence at least 97% homologous to 
the amino acid sequence of SEQ ID No. 13; the polypeptide has an amino 
acid sequence identical to the amino acid sequence of SEQ ID No. 13. In a 
preferred embodiment: the fragment comprises at least 5 contiguous amino 
acid residues of SEQ ID No. 13; the fragment comprises at least 20 
contiguous amino acid residues of SEQ ID No. 13; the fragment comprises at 
least 50 contiguous amino acid residues of SEQ ID No. 13. 
Another aspect of the present invention features an hUbCE polypeptide which 
functions in one of either role of an agonist of cell-cycle regulation or 
an antagonist of cell-cycle regulation. In a preferred embodiment the 
hUbCE polypeptide has: an ability to mediate ubiquitination of cellular 
proteins, e.g. cell-cycle regulatory proteins, e.g. p53; an ability to 
mediate ubiquitin-dependent degradation of cellular proteins, e.g. 
cell-cycle regulatory proteins, e.g. p53; an ability to affect the 
cellular half-life of a cell-cycle regulatory protein, e.g. a cell-cycle 
checkpoint protein, e.g. p53, e.g. in normal cells, e.g. in normal 
proliferating cells, e.g. in virally-infected cells, e.g. in 
papillomavirus infected cells, e.g. in HPV-infected cells, e.g. in HPV-16, 
HPV-18, HPV-31, or HPV-33 infected cells, e.g. in cells expressing a 
papillomavirus E6 protein, e.g. in transformed cells, e.g. in cancerous 
cells. The biological activity can further include the ability to bind and 
conjugate ubiquitin, as well as bind and transfer ubiquitin to E6AP. 
Another aspect of the present invention features a rapUBC polypeptide which 
functions in one of either role of an agonist of cell-cycle regulation or 
an antagonist of cell-cycle regulation. In a preferred embodiment the 
rapUBC polypeptide has: an ability to bind a FKBP/rapamycin complex, an 
ability to mediate ubiquitination of cellular proteins, e.g. cell-cycle 
regulatory proteins, e.g. p53 or p27; an ability to mediate 
ubiquitin-dependent degradation of cellular proteins, e.g. cell-cycle 
regulatory proteins, e.g. p53; an ability to affect the cellular half-life 
of a cell-cycle regulatory protein, e.g. a cell-cycle checkpoint protein, 
e.g. p53, e.g. in normal cells, e.g. in cancerous cells. Given that 
rapamycin causes a block in the cell-cycle during G1 phase, it is probable 
that the spectrum of biological activity of the subject rapUBC enzyme 
includes control of half-lives of certain cell cycle regulatory proteins, 
particularly relatively short lived proteins (e.g. proteins which have 
half-lives on the order of 30 minutes to 2 hours). For example, the 
subject rapUBC may have the ability to mediate ubiquitination of, for 
example, p53, p27, myc and/or cyclins, and therefore affects the cellular 
half-life of a cell-cycle regulatory protein in proliferating cells. The 
binding of the rapUBC to the FKBP/rapamycin complex may result in 
sequestering of the enzyme away from its substrate proteins. Thus, 
rapamycin may interfere with the ubiquitin-mediated degradation of p53 in 
a manner which causes cellular p53 levels to rise which in turn inhibits 
progression of the G1 phase. 
Yet another aspect of the present invention concerns an immunogen 
comprising an UBC polypeptide, or a fragment thereof, in an immunogenic 
preparation, the immunogen being capable of eliciting an immune response 
specific for the UBC polypeptide; e.g. a humoral response, eg. an antibody 
response; e.g. a cellular response. 
A still further aspect of the present invention features an antibody 
preparation specifically reactive with an epitope of the UBC immunogen, 
e.g., reactive with rapUBC, e.g. reactive with hUbCE, e.g. reactive with 
caUbC, e.g. reactive with spUbCE. 
Another aspect of the present invention features recombinant hUbCE 
polypeptide, or a fragment thereof, having an amino acid sequence 
preferably: at least 90% homologous to SEQ ID No. 2; at least 95% 
homologous to SEQ ID No: 2; at least 97% homologous to SEQ ID No. 2. In a 
preferred embodiment, the recombinant hUbCE protein functions in one of 
either role of an agonist of cell cycle regulation or an antagonist of 
cell cycle regulation. In a more preferred embodiment: the hUbCE 
polypeptide mediates ubiquitination of cellular proteins, e.g. cell-cycle 
regulatory proteins, e.g. p53; the hUbCE polypeptide mediates 
ubiquitin-dependent degradation of cellular proteins, e.g. cell-cycle 
regulatory proteins, e.g. p53; the hUbCE polypeptide affects the cellular 
half-life of a cell-cycle regulatory protein, e.g. a cell-cycle checkpoint 
protein, e.g. p53, e.g. in normal cells, e.g. in normal proliferating 
cells, e.g. in virally-infected cells, e.g. in papillomavirus infected 
cells, e.g. in HPV-infected cells, e.g. in HPV-16, HPV-18, HPV-31, or 
HPV-33 infected cells, e.g. in cells expressing a papillomavirus E6 
protein, e.g. in transformed cells, e.g. in cancerous cells. 
Another aspect of the present invention features recombinant caUbCE 
polypeptide, or a fragment thereof, having an amino acid sequence 
preferably: at least 90% homologous to SEQ ID No. 4; at least 95% 
homologous to SEQ ID No. 4; at least 97% homologous to SEQ ID No. 4. In a 
preferred embodiment, the recombinant caUbCE protein functions in one of 
either role of an agonist of cell cycle regulation or an antagonist of 
cell cycle regulation. In a more preferred embodiment the caUbCE 
polypeptide mediates ubiquitination of cellular proteins of candida cells. 
Another aspect of the present invention features recombinant spUbCE 
polypeptide, or a fragment thereof, having an amino acid sequence 
preferably: at least 90% homologous to SEQ ID No. 6; at least 95% 
homologous to SEQ ID No. 6; at least 97% homologous to SEQ ID No. 6. In a 
preferred embodiment, the recombinant spUbCE protein functions in one of 
either role of an agonist of cell cycle regulation or an antagonist of 
cell cycle regulation. In a more preferred embodiment the spUbCE 
polypeptide mediates ubiquitination of cellular proteins of 
Schizosaccharomyces cells. 
Another a frag of the present invention features recombinant rapUBC 
polypeptide, or a fragment thereof, having an amino acid sequence 
preferably: at least 90% homologous to SEQ ID No. 13; at least 95% 
homologous to SEQ ID No: 13; at least 97% homologous to SEQ ID No. 13. In 
a preferred embodiment, the recombinant rapUBC protein functions in one of 
either role of an agonist of cell cycle regulation or an antagonist of 
cell cycle regulation. In a more preferred embodiment: the rapUBC 
polypeptide mediates ubiquitination of cellular proteins, e.g. cell-cycle 
regulatory proteins, e.g. p53; the rapUBC polypeptide mediates 
ubiquitin-dependent degradation of cellular proteins, e.g. cell-cycle 
regulatory proteins, e.g. p53; the rapUBC polypeptide affects the cellular 
half-life of a cell-cycle regulatory protein, e.g. a cell-cycle checkpoint 
protein, e.g. p53, e.g. in normal cells, e.g. in cancerous cells. 
In yet other preferred embodiments, the recombinant UBC protein is a fusion 
protein further comprising a second polypeptide portion having an amino 
acid sequence from a protein unrelated the protein of SEQ ID No. 2, 4, 6 
or 13. Such fusion proteins can be functional in a two-hybrid assay. 
Another aspect of the present invention provides a substantially pure 
nucleic acid having a nucleotide sequence which encodes an hUbCE 
polypeptide, or a fragment thereof, having an amino acid sequence at least 
90% homologous to SEQ ID NO. 2. In a more preferred embodiment, the 
nucleic acid encodes a protein having an amino acid sequence at least 95% 
homologous to SEQ ID No. 2; and more preferably at least 97% homologous to 
SEQ ID No. 2. The nucleic preferably encodes: a hUbCE polypeptide which 
mediates ubiquitination of cellular proteins, e.g. cell-cycle regulatory 
proteins, e.g. p53; a hUbCE polypeptide which mediates ubiquitin-dependent 
degradation of cellular proteins, e.g. cell-cycle regulatory proteins, 
e.g. p53; a hUbCE polypeptide which affects the cellular half-life of a 
cell-cycle regulatory protein, e.g. a cell-cycle checkpoint protein, e.g. 
p53, e.g. in normal cells, e.g. in normal proliferating cells, e.g. in 
virally-infected cells, e.g. in papillomavirus infected cells, e.g. in 
HPV-infected cells, e.g. in HPV-16, HPV-18, HPV-31, or HPV-33 infected 
cells, e.g. in cells expressing a papillomavirus E6 protein, e.g. in 
transformed cells, e.g. in cancerous cells. 
Another aspect of the present invention provides a substantially pure 
nucleic acid having a nucleotide sequence which encodes a caUbCE 
polypeptide, or a fragment thereof, having an amino acid sequence at least 
90% homologous to SEQ ID NO. 4. In a more preferred embodiment, the 
nucleic acid encodes a protein having an amino acid sequence at least 95% 
homologous to SEQ ID No. 4; and more preferably at least 97% homologous to 
SEQ ID No. 4. 
Another aspect of the present invention provides a substantially pure 
nucleic acid having a nucleotide sequence which encodes an spUbCE 
polypeptide, or a fragment thereof, having an amino acid sequence at least 
90% homologous to SEQ ID NO. 4. In a more preferred embodiment, the 
nucleic acid encodes a protein having an amino acid sequence at least 95% 
homologous to SEQ ID No. 4; and more preferably at least 97% homologous to 
SEQ ID No. 4. 
Another aspect of the present invention provides a substantially pure 
nucleic acid having a nucleotide sequence which encodes a rapUBC 
polypeptide, or a fragment thereof, having an amino acid sequence at least 
90% homologous to SEQ ID NO. 13. In a more preferred embodiment, the 
nucleic acid encodes a protein having an amino acid sequence at least 95% 
homologous to SEQ ID No. 13; and more preferably at least 97% homologous 
to SEQ ID No. 13. The nucleic acid preferably encodes: a rapUBC 
polypeptide which mediates ubiquitination of cellular proteins, e.g. 
cell-cycle regulatory proteins, e.g. p53; a rapUBC polypeptide which 
mediates ubiquitin-dependent degradation of cellular proteins, e.g. 
cell-cycle regulatory proteins, e.g. p53; a rapUBC polypeptide which 
affects the cellular half-life of a cell-cycle regulatory protein, e.g. a 
cell-cycle checkpoint protein, e.g. p53, e.g. in normal cells, e.g. in 
cancerous cells. 
In yet a further preferred embodiment, the nucleic acid which encodes a UBC 
polypeptide of the present invention, or a fragment thereof, hybridizes 
under stringent conditions to a nucleic acid probe corresponding to at 
least 12 consecutive nucleotides of one of SEQ ID Nos. 1, 3, 5 or 12; more 
preferably to at least 20 consecutive nucleotides of said sequences; more 
preferably to at least 40 consecutive nucleotides. In yet a further 
preferred embodiment, the UbCE encoding nucleic acid hybridizes to a 
nucleic acid probe corresponding to a subsequence encoding at least 4 
consecutive amino acids between residues 107 and 147 of SEQ ID No. 2, 4 or 
6, more preferably at least 10 consecutive amino acid residues, and even 
more preferably at least 20 amino acid residues. In yet a preferred 
embodiment the nucleic acid encodes an hUbCE polypeptide which includes 
Cys-107 through Cys-111. 
Furthermore, in certain preferred embodiments, UBC encoding nucleic acid 
will comprise a transcriptional regulatory sequence, e.g. at least one of 
a transcriptional promoter or transcriptional enhancer sequence, operably 
linked to the UBC gene sequence so as to render the UBC gene sequence 
suitable for use as an expression vector. In one embodiment, the UBC gene 
is provided as a sense construct. In another embodiment, the UBC gene is 
provided as an anti-sense construct. 
The present invention also features transgenic non-human animals, e.g. 
mice, which either express a heterologous hUbCE or rapUBC gene, e.g. 
derived from humans, or which mis-express their own homolog of the subject 
human gene, e.g. expression of the mouse hUbCE or rapUBC homolog is 
disrupted. Such a transgenic animal can serve as an animal model for 
studying cellular disorders comprising mutated or mis-expressed hUbCE opr 
rapUBC alleles. 
The present invention also provides a probe/primer comprising a 
substantially purified oligonucleotide, wherein the oligonucleotide 
comprises a region of nucleotide sequence which hybridizes under stringent 
conditions to at least 10 consecutive nucleotides of sense or antisense 
sequence of SEQ ID No. 1 or SEQ ID No:12, or naturally occurring mutants 
thereof. In preferred embodiments, the probe/primer further comprises a 
label group attached thereto and able to be detected, e.g. the label group 
is selected from a group consisting of radioisotopes, fluorescent 
compounds, enzymes, and enzyme co-factors. Such probes can be used as a 
part of a diagnostic test kit for identifying transformed cells, such as 
for measuring a level of a hUbCE or a rapUBC nucleic acid in a sample of 
cells isolated from a patient; e.g. measuring the hUbCE or rapUBC mRNA 
level in a cell; e.g. determining whether the genomic hUbCE or rapUBC gene 
has been mutated or deleted. 
The present invention also provides a method for treating an animal having 
unwanted cell growth characterized by a loss of wild-type p53 function, 
comprising administering a therapeutically effective amount of an agent 
able to inhibit a ubiquitin conjugating activity of the subject hUbCE or 
rapUBC protein. 
The present invention also provides a method for treating an animal having 
an unwanted mycotic infection, comprising administering a therapeutically 
effective amount of an agent able to inhibit a ubiquitin conjugating 
activity of a fungal ubiquitin-conjugating enzyme, such as the subject 
caUbCE protein or spUBC protein, without substantially inhibiting the 
hUbCE protein. 
Another aspect of the present invention provides a method of determining if 
a subject, e.g. a human patient, is at risk for a disorder characterized 
by unwanted cell proliferation, comprising detecting, in a tissue of the 
subject, the presence or absence of a genetic lesion characterized by at 
least one of (i) a mutation of a gene encoding a protein represented by 
SEQ ID No. 2 or SEQ ID No:13, or a homolog thereof; or (ii) the 
mis-expression of the hUbCE or rapUBC gene. In preferred embodiments: 
detecting the genetic lesion comprises ascertaining the existence of at 
least one of a deletion of one or more nucleotides from the gene, an 
addition of one or more nucleotides to the gene, an substitution of one or 
more nucleotides of the gene, a gross chromosomal rearrangement of the 
gene, a gross alteration in the level of a messenger RNA transcript of the 
gene, the presence of a non-wild type splicing pattern of a messenger RNA 
transcript of the gene, or a non-wild type level of the protein. For 
example, detecting the genetic lesion can comprise (i) providing a 
probe/primer comprising an oligonucleotide containing a region of 
nucleotide sequence which hybridizes to a sense or antisense sequence of 
SEQ ID No. 1 or SEQ ID No:12, or naturally occurring mutants thereof or 5' 
or 3' flanking sequences naturally associated with the gene; (ii) exposing 
the probe/primer to nucleic acid of the tissue; and (iii) detecting, by 
hybridization of the probe/primer to the nucleic acid, the presence or 
absence of the genetic lesion; e.g. wherein detecting the lesion comprises 
utilizing the probe/primer to determine the nucleotide sequence of the 
hUbCE or rapUBC gene and, optionally, of the flanking nucleic acid 
sequences; e.g. wherein detecting the lesion comprises utilizing the 
probe/primer in a polymerase chain reaction (PCR); e.g. wherein detecting 
the lesion comprises utilizing the probe/primer in a ligation chain 
reaction (LCR). In alternate embodiments, the level of the protein is 
detected in an immunoassay. 
The present invention also provides a systematic and practical approach for 
the identification of candidate agents able to inhibit ubiquitin-mediated 
degradation of a cell-cycle regulatory protein, such as p53, p27, myc, 
fos, MAT.alpha.2, or cyclins. One aspect of the present invention relates 
to a method for identifying an inhibitor of ubiquitin-mediated proteolysis 
of a cell-cycle regulatory protein by (i) providing a 
ubiquitin-conjugating system that includes the regulatory protein and 
ubiquitin under conditions which promote the ubiquitination of the target 
protein, and (ii) measuring the level of ubiquitination of the subject 
protein brought about by the system in the presence and absence of a 
candidate agent. A decrease in the level of ubiquitin conjugation is 
indicative of an inhibitory activity for the candidate agent. The level of 
ubiquitination of the regulatory protein can be measured by determining 
the actual concentration of protein:ubiquitin conjugates formed; or 
inferred by detecting some other quality of the subject protein affected 
by ubiquitination, including the proteolytic degradation of the protein. 
In certain embodiments, the present assay comprises an in vivo 
ubiquitin-conjugating system, such as a cell able to conduct the 
regulatory protein through at least a portion of a ubiquitin-mediated 
proteolytic pathway. In other embodiments, the present assay comprises an 
in vitro ubiquitin-conjugating system comprising a reconstituted protein 
mixture in which at least the ability to transfer ubiquitin to the 
regulatory protein is constituted. Moreover, the present assay may further 
comprise auxiliary proteins which influence the level of 
ubiquitin-mediated degradation, including viral oncogenic proteins, such 
as the E6 protein of high-risk HPVs, which influence the level of the 
regulatory protein in an infected cell by enhancing or otherwise altering 
the proteolysis of the protein. 
Other features and advantages of the invention will be apparent from the 
following detailed description, and from the claims. 
The practice of the present invention will employ, unless otherwise 
indicated, conventional techniques of cell biology, cell culture, 
molecular biology, transgenic biology, microbiology, recombinant DNA, and 
immunology, which are within the skill of the art. Such techniques are 
explained fully in the literature. See, for example, Molecular Cloning A 
Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold 
Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. 
N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); 
Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. 
Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. 
Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, 
Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 
1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the 
treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene 
Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 
155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology 
(Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of 
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, 
eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory 
Press, Cold Spring Harbor, N.Y., 1986).

DETAILED DESCRIPTION OF THE INVENTION 
The ubiquitin system is essential for a wide spectrum of cellular 
phenomena, and is a component of many biological regulatory mechanisms, 
including aspects of growth control, metabolic regulation, tissue 
differentiation and development, and cell-cycle progression. 
The present invention relates to the discovery of ubiquitin-conjugating 
enzymes (UBC's) involved in regulating cell cycle progression. 
One aspect of the present invention relates to the discovery of a family of 
related ubiquitin-conjugating enzymes ("UbCE"?). In particular, members of 
this family have been cloned from various eukaryotic sources, and include, 
for example, a human ubiquitin-conjugating enzyme ("hUbCE"), a C. albican 
ubiquitin-conjugating enzyme ("caUbCE"), and an S. pombe 
ubiquitin-conjugating enzyme ("spUbCE"). The nucleotide sequences for the 
human UbCE, the C. albican UbCE, and the S. pombe UbCE coding sequences 
are provided in SEQ ID Nos. 1, 3 and 5, respectively. The corresponding 
amino acid sequences are represented in SEQ ID Nos. 2, 4 and 6. 
Another aspect of the invention relates to the discovery of a novel human 
ubiquitin-conjugating enzyme ("rapUBC"). rapUBC has been cloned based on 
its ability to bind FKBP/rapamycin complexes. The human rapUBC coding 
sequence is provided in SEQ ID No:12. The corresponding amino acid 
sequence is represented in SEQ ID No:13. 
The biological activity of the UBCE (e.g., UbCE and rapUBC) proteins of the 
present invention is evidently to be important in a number of basic 
cellular functions, such as regulation of gene expression, regulation of 
the cell-cycle, modification of cell surface receptors, biogenesis of 
ribosomes, and DNA repair. An apparent function of these enzymes in 
ubiquitin-mediated systems is to control the cellular half-lives of 
vasrious proteins. For instance, as demonstrated in the Examples, hUbCE is 
implicated in the ubiquitin-mediated inactivation of cell-cycle regulatory 
proteins, particularly p53. As is generally known, p53 is a checkpoint 
protein that plays an important role in sensing DNA damage or regulating 
cellular response to stress. Moreover, lesions in the p53 gene have been 
shown to be associated with a wide variety of proliferative diseases. 
Consequently, the present invention identifies a potential molecular 
target, e.g., hUbCE, for regulating the cellular half-life of p53 and 
thereby modulating, for instance, cell proliferation, apoptosis and 
cellular sensitivity to chemotherapeutics and DNA damaging agents. 
Accordingly, the present invention makes available diagnostic and 
therapeutic assays, reagents and kits for detecting and treating 
proliferative disorders arising from, for example, tumorogenic 
transformation of cells, or other hyperplastic or neoplastic 
transformation processes. For example, the present invention makes 
available reagents, such as antibodies and nucleic acid probes, for 
detecting altered complex formation, and/or altered levels of hUbCE or 
rapUBC expression, and/or hUbCE or rapUBC-gene deletion or mutation, in 
order to identify transformed cells. Moreover, the present invention 
provides a method of treating wide variety of pathological cell 
proliferative conditions, such as by gene therapy utilizing recombinant 
gene constructs encoding the subject UBC proteins, by providing 
peptidomimetics which either inhibit or potentiate the interaction between 
the UBC and other cellular proteins, or by providing inhibitors of the 
catalytic activity of the enzyme. Such methods can also be used in cell 
and tissue culture, such as to regulate the transformation of cells in 
vitro. 
In similar fashion, the present invention also makes available diagnostic 
and therapeutic assays for detecting and treating yeast/fungal infections, 
where such infections occur in an animal, e.g. humans, or on a non-living 
object, such as food or medical instruments. For example, given the 
apparent role of the subject UbCEs, namely caUbCE and spUbCE, in 
regulation of proteins involved in growth, mating and proliferation of 
yeast, inhibitors of the subject ubiquitin conjugating enzyme can be used 
to treat mycotic infections, as disinfectants, or as food preservatives. 
For convenience, certain terms employed in the specification, examples, and 
appended claims are collected here. 
As used herein, the term "nucleic acid" refers to polynucleotides such as 
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid 
(RNA). The term should also be understood to include, as equivalents, 
analogs of either RNA or DNA made from nucleotide analogs, and, as 
applicable to the embodiment being described, single (sense or antisense) 
and double-stranded polynucleotides. 
As used herein, the terms "gene", "recombinant gene" and "gene construct" 
refer to a nucleic acid comprising an open reading frame encoding a UBC 
polypeptide of the present invention, including both exon and (optionally) 
intron sequences. In preferred embodiments, the nucleic acid is DNA or 
RNA. Exemplary recombinant genes include nucleic acids which encode all or 
a catalytically active portion of the hUbCE protein represented in SEQ ID 
No. 2, the caUbCE protein represented in SEQ ID No. 4, the spUbCE protein 
represented in SEQ ID No. 6, or the rapUBC protein represented in SEQ ID 
No: 13. The term "intron" refers to a DNA sequence present in a given 
UBC-gene which is not translated into protein and is generally found 
between exons. 
The term "transfection" refers to the introduction of a nucleic acid, e.g., 
an expression vector, into a recipient cell by nucleic acid-mediated gene 
transfer. "Transformation", as used herein, refers to a process in which a 
cell's genotype is changed as a result of the cellular uptake of exogenous 
nucleic acid, and, for example, the transformed cell expresses a 
recombinant form of one of the subject UBC proteins. 
"Cells" or "cell cultures" or "recombinant host cells" or "host cells" are 
often used interchangeably as will be clear from the context. These terms 
include the immediate subject cell which expresses a ubiquitin-conjugating 
enzyme of the present invention, and, of course, the progeny thereof. It 
is understood that not all progeny are exactly identical to the parental 
cell, due to chance mutations or difference in environment. However, such 
altered progeny are included in these terms, so long as the progeny retain 
the characteristics relevant to those conferred on the originally 
transformed cell. In the present case, such a characteristic might be the 
ability to produce a recombinant UBC-protein. 
As used herein, the term "vector" refers to a nucleic acid molecule capable 
of transporting another nucleic acid to which it has been linked. The term 
"expression vector" includes plasmids, cosmids or phages capable of 
synthesizing the subject proteins encoded by their respective recombinant 
genes carried by the vector. Preferred vectors are those capable of 
autonomous replication and/expression of nucleic acids to which they are 
linked. In the present specification, "plasmid" and "vector" are used 
interchangeably as the plasmid is the most commonly used form of vector. 
Moreover, the invention is intended to include such other forms of 
expression vectors which serve equivalent functions and which become known 
in the art subsequently hereto. 
"Transcriptional regulatory sequence" is a generic term used throughout the 
specification to refer to DNA sequences, such as initiation signals, 
enhancers, and promoters, as well as polyadenylation sites, which induce 
or control transcription of protein coding sequences with which they are 
operably linked. In preferred embodiments, transcription of a recombinant 
UBC-gene is under the control of a promoter sequence (or other 
transcriptional regulatory sequence) which controls the expression of the 
recombinant gene in a cell-type in which expression is intended. It will 
also be understood that the recombinant gene can be under the control of 
transcriptional regulatory sequences which are the same or which are 
different from those sequences which control transcription of the 
naturally-occurring form of the regulatory protein. 
The term "tissue-specific promoter" means a DNA sequence that serves as a 
promoter, i.e., regulates expression of a selected DNA sequence operably 
linked to the promoter, and which effects expression of the selected DNA 
sequence in specific cells of a tissue, such as cells of an epithelial 
lineage, e.g. cervical squamous cells. In an illustrative embodiment of 
epithelial-specific promoters, gene constructs can be used as a part of 
gene therapy to deliver, for example, genes encoding a domaint negative 
hUbCE or rapUBC mutant, in order to inhibit degradation of p53 required 
for the pathogenesis of certain papillomavirus-mediated disorders, e.g. 
papillomas, or to direct expression of an antisense construct of the 
subject ubiquitin-conjugating enzyme in only epithelial tissue. The term 
also covers so-called "leaky" promoters, which regulate expression of a 
selected DNA primarily in one tissue, but cause expression in other 
tissues as well. 
As used herein, a "transgenic animal" is any animal, preferably a non-human 
mammal in which one or more of the cells of the animal contain 
heterologous nucleic acid introduced by way of human intervention, such as 
by trangenic techniques well known in the art. The nucleic acid is 
introduced into the cell, directly or indirectly by introduction into a 
precursor of the cell, by way of deliberate genetic manipulation, such as 
by microinjection or by infection with a recombinant virus. The term 
genetic manipulation does not include classical cross-breeding, or in 
vitro fertilization, but rather is directed to the introduction of a 
recombinant DNA molecule. This molecule may be integrated within a 
chromosome, or it may be extrachromosomally replicating DNA. In the 
typical transgenic animals described herein, the transgene causes cells to 
express a recombinant form of the subject UBC protein, e.g. either 
agonistic or antagonistic forms, or in which the endogenous UBC gene has 
been disrupted. However, transgenic animals in which the recombinant UBC 
gene is silent are also contemplated, as for example, the FLP or CRE 
recombinase dependent constructs described below. The "non-human animals" 
of the invention include vertebrates such as rodents, non-human primates, 
sheep, dog, cow, amphibians, reptiles, etc. Preferred non-human animals 
are selected from the rodent family including rat and mouse, most 
preferably mouse. The term "chimeric animal" is used herein to refer to 
animals in which the recombinant gene is found, or in which the 
recombinant is expressed in some but not all cells of the animal. The term 
"tissue-specific chimeric animal" indicates that the recombinant UBC gene 
is present and/or expressed in some tissues but not others. 
As used herein, the term "transgene" means a nucleic acid sequence 
(encoding, e.g., a UBC polypeptide), which is partly or entirely 
heterologous, i.e., foreign, to the transgenic animal or cell into which 
it is introduced, or, is homologous to an endogenous gene of the 
transgenic animal or cell into which it is introduced, but which is 
designed to be inserted, or is inserted, into the animal's genome in such 
a way as to alter the genome of the cell into which it is inserted (e.g., 
it is inserted at a location which differs from that of the natural gene 
or its insertion results in a knockout). A transgene can include one or 
more transcriptional regulatory sequences and any other nucleic acid, such 
as introns, that may be necessary for optimal expression of a selected 
nucleic acid. 
"Homology" refers to sequence similarity between two peptides or between 
two nucleic acid molecules. Homology can be determined by comparing a 
position in each sequence which may be aligned for purposes of comparison. 
When a position in the compared sequence is occupied by the same base or 
amino acid, then the molecules are homologous at that position. A degree 
of homology between sequences is a function of the number of matching or 
homologous positions shared by the sequences. 
The term "evolutionarily related to", with respect to nucleic acid 
sequences encoding the subject ubiquitin-conjugating enzymes, refers to 
nucleic acid sequences which have arisen naturally in an organism, 
including naturally occurring mutants. The term also refers to nucleic 
acid sequences which, while derived from a naturally occurring enzymes, 
have been altered by mutagenesis, as for example, combinatorial 
mutagenesis described below, yet still encode polypeptides which have at 
least one activity of a UBC protein. 
As described below, one aspect of this invention pertains to an isolated 
nucleic acid comprising a nucleotide sequence encoding one of the subject 
UBC proteins, fragments thereof encoding polypeptides having at least one 
biological activity of the UBC protein, and/or equivalents of such nucleic 
acids. The term "nucleic acid" as used herein is intended to include such 
fragments and equivalents. The term "equivalent" is understood to include 
nucleotide sequences encoding functionally equivalent UBC proteins or 
functionally equivalent peptides having an activity of a 
ubiquitin-conjugating enzyme such as described herein. Equivalent 
nucleotide sequences will include sequences that differ by one or more 
nucleotide substitutions, additions or deletions, such as allelic 
variants; and will also include sequences that differ from the nucleotide 
sequence encoding the hUbCE gene shown in SEQ ID No: 1, the caUbCE gene 
shown in SEQ ID No: 3, the spUbCE gene shown in SEQ ID No: 5, or the 
rapUBC gene shown in SEQ ID No: 12, due to the degeneracy of the genetic 
code. Equivalents will also include nucleotide sequences which hybridize 
under stringent conditions (i.e., equivalent to about 20-27.degree. C. 
below the melting temperature (T.sub.m) of the DNA duplex formed in about 
1M salt) to the nucleotide sequence represented in at least one of SEQ ID 
Nos: 1, 3, 5 or 12. In one embodiment, equivalents will further include 
nucleic acid sequences derived from and evolutionarily related to the 
nucleotide sequences shown in any of SEQ ID Nos: 1, 3, 5 or 12. 
The term "isolated" as also used herein with respect to nucleic acids, such 
as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, 
respectively, that are present in the natural source of the macromolecule. 
For example, an isolated nucleic acid encoding on of the subject 
UBC-proteins preferably includes no more than 10 kilobases (kb) of nucleic 
acid sequence which naturally immediately flanks the UBC gene in genomic 
DNA, more preferably no more than 5 kb of such naturally occurring 
flanking sequences, and most preferably less than 1.5 kb of such naturally 
occurring flanking sequence. The term isolated as used herein also refers 
to a nucleic acid or peptide that is substantially free of cellular 
material or culture medium when produced by recombinant DNA techniques, or 
chemical precursors or other chemicals when chemically synthesized. 
Moreover, an "isolated nucleic acid" is meant to include nucleic acid 
fragments which are not naturally occurring as fragments and would not be 
found in the natural state. 
Polypeptides referred to herein as possessing the activity of a 
ubiquitin-conjugating enzyme (UBC), e.g. are UBC agonists, are understood 
to have an amino acid sequence identical to or homologous with the amino 
acid sequences shown in any on of SEQ ID Nos: 2, 4, 6 or 13, and which are 
capable of forming a thiol ester adduct with the C-terminal carboxyl group 
of ubiquitin and transferring the ubiquitin to an i-amino group in an 
acceptor protein by formation of an isopeptide bond. The biological 
activity of the subject UBC proteins can include participation in 
degradative pathways for selective proteolysis of constitutively or 
conditionally short-lived proteins as well as abnormal proteins. 
Antagonistic forms of the subject UBC proteins are defined as proteins 
that are homologous, but not identical, to the UBC proteins represented in 
SEQ ID Nos: 2, 4, 6 or 13, or that are fragments of the wild-type 
proteins, which inhibit the transfer of ubiquitin by the naturally 
occurring form of the ubiquitin-conjugating enzyme. For instance, as 
described below, mutations in the active site of the enzyme, e.g. Cys-85, 
can produce dominant negative forms of the subject UbCEs which antagonize 
the action of the wild-type form of the protein. 
Polypeptides referred to in particular as having an activity of an hUbCE 
protein are defined as peptides that have an amino acid sequence 
corresponding to all or a portion of the amino acid sequence of the human 
ubiquitin conjugating enzyme shown in SEQ ID No: 2 and which have at least 
one biological activity of an hUbCE protein: such as an ability to mediate 
ubiquitination of cellular proteins, e.g. cell-cycle regulatory proteins, 
e.g. p53; an ability to mediate ubiquitin-dependent degradation of 
cellular proteins, e.g. cell-cycle regulatory proteins, e.g. p53; an 
ability to affect the cellular half-life of a cell-cycle regulatory 
protein, e.g. a cell-cycle checkpoint protein, e.g. p53, e.g. in normal 
cells, e.g. in normal proliferating cells, e.g. in virally-infected cells, 
e.g. in papillomavirus infected cells, e.g. in HPV-infected cells, e.g. in 
HPV-16, HPV-18, HPV-31, or HPV-33 infected cells, e.g. in cells expressing 
a papillomavirus E6 protein, e.g. in transformed cells, e.g. in cancerous 
cells. Other biological activities of the subject hUbCE proteins are 
described herein or will be reasonably apparent to those skilled in the 
art. 
Polypeptides referred to in particular as having an activity of a rapUBC 
protein are defined as peptides that have an amino acid sequence 
corresponding to all or a portion of the amino acid sequence of the human 
ubiquitin conjugating enzyme shown in SEQ ID No:13 and which have at least 
one biological activity of a rapUBC protein: such as an ability to bind a 
FKBP/rapamycin complex, an ability to mediate ubiquitination of cellular 
proteins, e.g. cell-cycle regulatory proteins, e.g. p53; an ability to 
mediate ubiquitin-dependent degradation of cellular proteins, e.g. 
cell-cycle regulatory proteins, e.g. p53; an ability to affect the 
cellular half-life of a cell-cycle regulatory protein, e.g. a cell-cycle 
checkpoint protein, e.g. p53, e.g. in normal cells, e.g. in cancerous 
cells. Given that rapamycin causes a block in the cell-cycle during G1 
phase, the spectrum of biological activity of the subject rapUBC enzyme is 
believed to include control of half-lives of certain cell cycle regulatory 
proteins, particularly relatively short lived proteins (e.g. proteins 
which have half-lives on the order of 30 minutes to 2 hours). For example, 
the subject rapUBC may mediate ubiquitination of, for example, p53, myc, 
p27 and/or cyclins, and therefore affects the cellular half-life of a 
cell-cycle regulatory protein in proliferating cells. The binding of the 
rapUBC to the FKBP/rapamycin complex may result in sequestering of the 
enzyme away from its substrate proteins. Thus, rapamycin may interfere 
with the ubiquitin-mediated degradation of p53 in a manner which causes 
cellular p53 levels to rise which in turn inhibits progression of the G1 
phase. 
Moreover, it will be generally appreciated that, under certain 
circumstances, it will be advantageous to provide homologs of 
naturally-occurring forms of the subject UBC proteins which are either 
agonists or antagonists of only a subset of that protein's biological 
activities. Thus, specific biological effects can be elicited by treatment 
with a homolog of limited function, and with fewer side effects relative 
to treatment with agonists or antagonists which are directed to all of the 
biological activities of that protein. For example, hUbCE and rapUBC 
homologs can be generated which bind to and inhibit activation of other 
proteins in the ubiquitin pathway of p53 without substantially interfering 
with the ubiquitination of other cellular proteins. 
In one embodiment, the nucleic acid of the invention encodes a polypeptide 
which is either an agonist or antagonist the human UBC protein and 
comprises an amino acid sequence represented by SEQ ID No: 2. Preferred 
nucleic acids encode a peptide having an hUbCE protein activity, or which 
is an antagonist thereof, and being at least 90% homologous, more 
preferably 95% homologous and most preferably 97% homologous with an amino 
acid sequence shown in SEQ ID No: 2. Nucleic acids which encode agonist or 
antagonist forms of an hUbCE protein and having at least about 98-99% 
homology with a sequence shown in SEQ ID No: 2 are also within the scope 
of the invention. Preferably, the nucleic acid is a cDNA molecule 
comprising at least a portion of the nucleotide sequence encoding an hUbCE 
protein shown in SEQ ID No. 1. A preferred portion of the cDNA molecule 
shown in SEQ ID No. 1 includes the coding region of the molecule. 
In another embodiment, the nucleic acid of the invention encodes a 
polypeptide which is either an agonist or antagonist a Candida UbCE 
protein, e.g. a C. albican UbCE, and comprises an amino acid sequence 
represented by SEQ ID No: 4. Preferred nucleic acids encode a peptide 
having an caUbCE protein activity, or which is an antagonist thereof, and 
being at least 90% homologous, more preferably 95% homologous and most 
preferably 97% homologous with an amino acid sequence shown in SEQ ID No: 
4. Nucleic acids which encode agonist or antagonist forms of an caUbCE 
protein and having at least about 98-99% homology with a sequence shown in 
SEQ ID No: 4 are also within the scope of the invention. Preferably, the 
nucleic acid is a cDNA molecule comprising at least a portion of the 
nucleotide sequence encoding an caUbCE protein shown in SEQ ID No. 3. A 
preferred portion of the cDNA molecule shown in SEQ ID No. 3 includes the 
coding region of the molecule. The present invention contemplates closely 
related homologs (orthologs) from other species of Candida, e.g. Candida 
stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, 
Candida pseudotropicalis, Candida quillermondii, or Candida rugosa. 
In yet another embodiment, the nucleic acid of the invention encodes a 
polypeptide which is either an agonist or antagonist of a 
Schizosaccharomyces UbCE protein, e.g. an S. pombe UbCE, and comprises an 
amino acid sequence represented by SEQ ID No: 6. Preferred nucleic acids 
encode a peptide having an spUbCE protein activity, or which is an 
antagonist thereof, and being at least 90% homologous, more preferably 95% 
homologous and most preferably 97% homologous with an amino acid sequence 
shown in SEQ ID No: 6. Nucleic acids which encode agonist or antagonist 
forms of an spUbCE protein and having at least about 98-99% homology with 
a sequence shown in SEQ ID No: 6 are also within the scope of the 
invention. Preferably, the nucleic acid is a cDNA molecule comprising at 
least a portion of the nucleotide sequence encoding an spUbCE protein 
shown in SEQ ID No. 5. A preferred portion of the cDNA molecule shown in 
SEQ ID No. 5 includes the coding region of the molecule. 
In yet another embodiment, the nucleic acid of the invention encodes a 
polypeptide which is either an agonist or antagonist of the human UBC 
protein and comprises an amino acid sequence represented by SEQ ID No:13. 
Preferred nucleic acids encode a peptide having a rapUBC protein activity, 
or which is an antagonist thereof, and being at least 90% homologous, more 
preferably 95% homologous and most preferably 97% homologous with an amino 
acid sequence shown in SEQ ID No:13. Nucleic acids which encode agonist or 
antagonist forms of a rapUBC protein and having at least about 98-99% 
homology with a sequence shown in SEQ ID No: 13 are also within the scope 
of the invention. Preferably, the nucleic acid is a cDNA molecule 
comprising at least a portion of the nucleotide sequence encoding a rapUBC 
protein shown in SEQ ID No:12. A preferred portion of the cDNA molecule 
shown in SEQ ID No: 12 includes the coding region of the molecule. 
Another aspect of the invention provides a nucleic acid which hybridizes 
under high or low stringency conditions to a nucleic acid which encodes a 
peptide having all or a portion of an amino acid sequence shown in one of 
SEQ ID Nos: 2, 4, 6 or 13. Appropriate stringency conditions which promote 
DNA hybridization, for example, 6.0.times.sodium chloride/sodium citrate 
(SSC) at about 45.degree. C., followed by a wash of 2.0.times.SSC at 
50.degree. C., are known to those skilled in the art or can be found in 
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 
6.3.1-6.3.6. For example, the salt concentration in the wash step can be 
selected from a low stringency of about 2.0.times.SSC at 50.degree. C. to 
a high stringency of about 0.2.times.SSC at 50.degree. C. In addition, the 
temperature in the wash step can be increased from low stringency 
conditions at room temperature, about 22.degree. C., to high stringency 
conditions at about 65.degree. C. 
Isolated nucleic acids which differ in sequence from the nucleotide 
sequences represented in SEQ ID Nos: 1, 3, 5 or 12 due to degeneracy in 
the genetic code are also within the scope of the invention. Such nucleic 
acids can encode functionally equivalent peptides (i.e., a peptide having 
a biological activity of a UBC protein) but differ in sequence from the 
sequence shown in SEQ ID No: 1, 3, 5 or 12 due to degeneracy in the 
genetic code. For example, a number of amino acids are designated by more 
than one triplet. Codons that specify the same amino acid, or synonyms 
(for example, CAU and CAC are synonyms for histidine) may result in 
"silent" mutations which do not affect the amino acid sequence of the 
subject UBC protein. However, it is expected that DNA sequence 
polymorphisms that do lead to changes in the amino acid sequences of the 
present hUbCE or rapUBC proteins will exist from one human subject to the 
next. One skilled in the art will appreciate that these variations in one 
or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic 
acids encoding peptides having an activity of, for example, an hUbCE or a 
rapUBC protein may exist among individuals due to natural allelic 
variation. Any and all such nucleotide variations and resulting amino acid 
polymorphisms are within the scope of this invention. 
Fragments of the nucleic acid encoding an active portion of one of the 
subject ubiquitin-conjugating enzymes are also within the scope of the 
invention. As used herein, a fragment of the nucleic acid encoding an 
active portion of a UBC protein refers to a nucleotide sequence having 
fewer nucleotides than the nucleotide sequence encoding the entire amino 
acid sequence of the protein but which encodes a peptide which possess 
agonistic or antagonistic activity relative to a naturally occurring form 
of the enzyme. 
Nucleic acid fragments within the scope of the invention also include those 
capable of hybridizing under high or low stringency conditions with 
nucleic acids from other species for use in screening protocols to detect 
UBC homologs. 
Nucleic acids within the scope of the invention may also contain linker 
sequences, modified restriction endonuclease sites and other sequences 
useful for molecular cloning, expression or purification of recombinant 
peptides having at least one biological activity of the subject UbCE 
ubiquitin-conjugating enzymes. In a preferred embodiment, the nucleic acid 
fragment comprises at least a portion of the nucleic acid sequence 
represented by nucleotide residues 319 through 441 of SEQ ID No. 1, 
corresponding to amino acid residues Cys-107 through Met-147. In preferred 
embodiments, the nucleic acid encodes an hUbCE polypeptide which includes 
Cys-107 through Cys-111, and more preferably includes Cys-107 through 
Asp-117. As apparent from our computer modeling, certain of the residues 
from Cys-107 to Asp-111 are important members of the ubiquitin-binding 
site of hUbCE. Correspondingly, nucleic acid encoding caUbCE or spUbCE 
preferably include Cys-107 through Val-147 and Cys-107 through Ile-107, 
respectively. 
As indicated by the examples set out below, a nucleic acid encoding a 
peptide having an activity of the subject ubiquitin-conjugating enzymes 
may be obtained from mRNA or genomic DNA present in any of a number of 
eukaryotic cells in accordance with protocols described herein, as well as 
those generally known in the art. A cDNA encoding a homolog of one of the 
human UBC proteins, for example, can be obtained by isolating total mRNA 
from a cell, e.g. a mammalian cell. Double stranded cDNAs can then be 
prepared from the total mRNA, and subsequently inserted into a suitable 
plasmid or bacteriophage vector using any one of a number of known 
techniques. A gene encoding a UBC protein can also be cloned using 
established polymerase chain reaction techniques in accordance with the 
nucleotide sequence information provided herein. 
Another aspect of the invention relates to the use of the isolated nucleic 
acid in "antisense" therapy. As used herein, "antisense" therapy refers to 
administration or in situ generation of oligonucleotide probes or their 
derivatives which specifically hybridizes (e.g. binds) under cellular 
conditions, with the cellular mRNA and/or genomic DNA encoding a UBC 
protein so as to inhibit expression of that protein, e.g. by inhibiting 
transcription and/or translation. The binding may be by conventional base 
pair complementarity, or, for example, in the case of binding to DNA 
duplexes, through specific interactions in the major groove of the double 
helix. In general, "antisense" therapy refers to the range of techniques 
generally employed in the art, and includes any therapy which relies on 
specific binding to oligonucleotide sequences. 
An antisense construct of the present invention can be delivered, for 
example, as an expression plasmid which, when transcribed in the cell, 
produces RNA which is complementary to at least a unique portion of the 
cellular mRNA which encodes one of the subject UBC-proteins, e.g. the 
human hUbCE gene represented in SEQ ID No. 1 or the rapUBC gene 
represented in SEQ ID No:12. Alternatively, the antisense construct can be 
an oligonucleotide probe which is generated ex vivo and which, when 
introduced into the cell causes inhibition of expression by hybridizing 
with the mRNA and/or genomic sequences encoding one of the UBC proteins. 
Such oligonucleotide probes are preferably modified oligonucleotide which 
are resistant to endogenous nucleases, e.g. exonucleases and/or 
endonucleases, and are therefore stable in vivo. Exemplary nucleic acid 
molecules for use as antisense oligonucleotides are phosphoramidate, 
phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. 
Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general 
approaches to constructing oligomers useful in antisense therapy have been 
reviewed, for example, by van der Krol et al. (1988) Biotechniques 
6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. 
Accordingly, the modified oligomers of the invention are useful in 
therapeutic, diagnostic, and research contexts. In therapeutic 
applications, the oligomers are utilized in a manner appropriate for 
antisense therapy in general. For such therapy, the oligomers of the 
invention can be formulated for a variety of loads of administration, 
including systemic and topical or localized administration. Techniques and 
formulations generally may be found in Remmington's Pharmaceutical 
Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, 
injection is preferred, including intramuscular, intravenous, 
intraperitoneal, and subcutaneuos for injection, the oligomers of the 
invention can be formulated in liquid solutions, preferably in 
physiologically compatible buffers such as Hank's solution or Ringer's 
solution. In addition, the oligomers may be formulated in solid form and 
redissolved or suspended immediately prior to use. Lyophilized forms are 
also included. 
Systemic administration can also be by transmucosal or transdermal means, 
or the compounds can be administered orally. For transmucosal or 
transdermal administration, penetrants appropriate to the barrier to be 
permeated are used in the formulation. Such penetrants are generally known 
in the art, and include, for example, for transmucosal administration bile 
salts and fusidic acid derivatives. In addition, detergents may be used to 
facilitate permeation. Transmucosal administration may be through nasal 
sprays or using suppositories. For oral administration, the oligomers are 
formulated into conventional oral administration forms such as capsules, 
tablets, and tonics. For topical administration, the oligomers of the 
invention are formulated into ointments, salves, gels, or creams as 
generally known in the art. 
In addition to use in therapy, the oligomers of the invention may be used 
as diagnostic reagents to detect the presence or absence of the target DNA 
or RNA sequences to which they specifically bind. Such diagnostic tests 
are described in further detail below. 
This invention also provides expression vectors containing a nucleic acid 
encoding the subject UBC proteins, operably linked to at least one 
transcriptional regulatory sequence. Operably linked is intended to mean 
that the nucleic acid is linked to a transcriptional regulatory sequence 
in a manner which allows expression of the enzyme encoded by the nucleic 
acid, and that expression is, for example, either constitutively or 
inducibly controlled by the transcriptional regulatory sequence. 
Regulatory sequences are art-recognized. Accordingly, the term regulatory 
sequence includes promoters, enhancers and other expression control 
elements. Such regulatory sequences are described in Goeddel; Gene 
Expression Technology: Methods in Enzymology 185, Academic Press, San 
Diego, Calif. (1990). 
For instance, any of a wide variety of expression control 
sequences-sequences that control the expression of a DNA sequence when 
operatively linked to it may be used in these vectors to express DNA 
sequences encoding the UBC proteins of this invention. Such useful 
expression control sequences, include, for example, the early and late 
promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, 
the lac system, the trp system, the TAC or TRC system, T7 promoter whose 
expression is directed by T7 RNA polymerase, the major operator and 
promoter regions of phage lambda, the control regions for fd coat protein, 
the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, 
the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast 
alpha-mating factors, the polyhedron promoter of the baculovirus system 
and other sequences known to control the expression of genes of 
prokaryotic or eukaryotic cells or their viruses, and various combinations 
thereof. It should be understood that the design of the expression vector 
may depend on such factors as the choice of the host cell to be 
transformed and/or the type of protein desired to be expressed. Moreover, 
the vector's copy number, the ability to control that copy number and the 
expression of any other proteins encoded by the vector, such as antibiotic 
markers, should also be considered. 
In one embodiment, the expression vector includes DNA encoding one of the 
subject hUbCE proteins, e.g. a recombinant hUbCE protein. Similar 
expression vectors for producing recombinant forms of the rapUBC protein 
are also contemplated. Such expression vectors can be used to transfect 
cells to thereby produce proteins or peptides, including fusion proteins 
or peptides, encoded by nucleic acids as described herein. 
Moreover, hUbCE or rapUBC-expression vectors can be used as a part of a 
gene therapy protocol to reconstitute hUbCE or rapUBC function in a 
mammalian cell in which hUbCE or rapUBC is misexpressed, or alternatively, 
to provide an antagonist of the naturally-occurring hUbCE or rapUBC, or an 
antisense construct--such as to inhibit hUbCE or rapUBC-mediated 
degradation of a cell-cycle regulatory protein. For instance, expression 
constructs of the subject hUbCE or rapUBC-proteins may be administered in 
any biologically effective carrier, e.g. any formulation or composition 
capable of effectively transfecting cells in vivo with a recombinant hUbCE 
or rapUBC-gene. Approaches include insertion of the subject gene in viral 
vectors including recombinant retroviruses, adenovirus, adeno-associated 
virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic 
plasmids. Viral vectors can be used to transfect cells directly; plasmid 
DNA can be delivered with the help of, for example, cationic liposomes 
(lipofectin) or derivatized (e.g. antibody conjugated), polylysine 
conjugates, gramacidin S, artificial viral envelopes or other such 
intracellular carriers, as well as direct injection of the gene construct 
or CaPO.sub.4 precipitation carried out in vivo. It will be appreciated 
that because transduction of appropriate target cells represents the 
critical first step in gene therapy, choice of the particular gene 
delivery system will depend on such factors as the phenotype of the 
intended target and the route of administration, e.g. locally or 
systemically. 
A preferred approach for in vivo introduction of nucleic acid encoding one 
of the subject proteins into a cell is by use of a viral vector containing 
nucleic acid, e.g. a cDNA, encoding the gene product. Infection of cells 
with a viral vector has the advantage that a large proportion of the 
targeted cells can receive the nucleic acid. Additionally, molecules 
encoded within the viral vector, e.g., by a cDNA contained in the viral 
vector, are expressed efficiently in cells which have taken up viral 
vector nucleic acid. 
Retrovirus vectors and adeno-associated virus vectors are generally 
understood to be the recombinant gene delivery system of choice for the 
transfer of exogenous genes in vivo, particularly into humans. These 
vectors provide efficient delivery of genes into cells, and the 
transferred nucleic acids are stably integrated into the chromosomal DNA 
of the host. A major prerequisite for the use of retroviruses is to ensure 
the safety of their use, particularly with regard to the possibility of 
the spread of wild-type virus in the cell population. The development of 
specialized cell lines (termed "packaging cells") which produce only 
replication-defective retroviruses has increased the utility of 
retroviruses for gene therapy, and defective retroviruses are well 
characterized for use in gene transfer for gene therapy purposes (for a 
review see Miller, A. D. (1990) Blood 76:271). Thus, recombinant 
retrovirus can be constructed in which part of the retroviral coding 
sequence (gag, pol, env) has been replaced by nucleic acid encoding one of 
the subject hUbCE or rapUBC-proteins rendering the retrovirus replication 
defective. The replication defective retrovirus is then packaged into 
virions which can be used to infect a target cell through the use of a 
helper virus by standard techniques. Protocols for producing recombinant 
retroviruses and for infecting cells in vitro or in vivo with such viruses 
can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et 
al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and 
other standard laboratory manuals. Examples of suitable retroviruses 
include pLJ, pZIP, pWE and pEM which are well known to those skilled in 
the art. Examples of suitable packaging virus lines for preparing both 
ecotropic and amphotropic retroviral systems include .psi.Crip, .psi.Cre, 
.psi.2 and .psi.Am. Retroviruses have been used to introduce a variety of 
genes into many different cell types, including neural cells, epithelial 
cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow 
cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) 
Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. 
USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 
85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 
87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; 
Ferry et al. (1991) Proc. Natl. Acad Sci. USA 88:8377-8381; Chowdhury et 
al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. 
Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 
3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; 
Hwu et al. (1993) J Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. 
Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 
89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). 
Furthermore, it has also been shown that it is possible to limit the 
infection spectrum of retroviruses and consequently of retroviral-based 
vectors, by modifying the viral packaging proteins on the surface of the 
viral particle (see, for example PCT publications WO93/25234, WO94/06920, 
and WO94/11524). For instance, strategies for the modification of the 
infection spectrum of retroviral vectors include: coupling antibodies 
specific for cell surface antigens to the viral env protein (Roux et al. 
(1989) PNAS 86:9079-9083; Julan et al. (1992) J Gen Virol 73:3251-3255; 
and Goud et al. (1983) Virology 163:251-254); or coupling cell surface 
ligands to the viral env proteins (Neda et al. (1991) J Biol Chem 
266:14143-14146). Coupling can be in the form of the chemical 
cross-linking with a protein or other variety (e.g. lactose to convert the 
env protein to an asialoglycoprotein), as well as by generating fusion 
proteins (e.g. single-chain antibody/env fusion proteins). This technique, 
while useful to limit or otherwise direct the infection to certain tissue 
types, and can also be used to convert an ecotropic vector in to an 
amphotropic vector. 
Moreover, use of retroviral gene delivery can be further enhanced by the 
use of tissue or cell-specific transcriptional regulatory sequences which 
control expression of the hUbCE or rapUBC-gene of the retroviral vector. 
Another viral gene delivery system useful in the present invention 
utilitizes adenovirus-derived vectors. The genome of an adenovirus can be 
manipulated such that it encodes a gene product of interest, but is 
inactivate in terms of its ability to replicate in a normal lytic viral 
life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; 
Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) 
Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus 
strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 
etc.) are well known to those skilled in the art. Recombinant adenoviruses 
can be advantageous in certain circumstances in that they are not capable 
of infecting nondividing cells and can be used to infect a wide variety of 
cell types, including airway epithelium (Rosenfeld et al. (1992) cited 
supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. 
USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. 
Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. 
Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is 
relatively stable and amenable to purification and concentration, and as 
above, can be modified so as to affect the spectrum of infectivity. 
Additionally, introduced adenoviral DNA (and foreign DNA contained 
therein) is not integrated into the genome of a host cell but remains 
episomal, thereby avoiding potential problems that can occur as a result 
of insertional mutagenesis in situations where introduced DNA becomes 
integrated into the host genome (e.g., retroviral DNA). Moreover, the 
carrying capacity of the adenoviral genome for foreign DNA is large (up to 
8 kilobases) relative to other gene delivery vectors (Berkner et al., 
supra; Haj-Ahmand and Graham (1986) J Virol. 57:267). Most 
replication-defective adenoviral vectors currently in use and therefore 
favored by the present invention are deleted for all or parts of the viral 
E1 and E3 genes but retain as much as 80% of the adenoviral genetic 
material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et al., 
supra; and Graham et al. in Methods in Molecular Biolo, E. J. Murray, Ed. 
(Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127). Expression of the 
inserted hUbCE or rapUBC-gene can be under control of, for example, the 
E1A promoter, the major late promoter (MLP) and associated leader 
sequences, the E3 promoter, or exogenously added promoter sequences. 
Yet another viral vector system useful for delivery of the subject hUbCE or 
rapUBC-genes is the adeno-associated virus (AAV). Adeno-associated virus 
is a naturally occurring defective virus that requires another virus, such 
as an adenovirus or a herpes virus, as a helper virus for efficient 
replication and a productive life cycle. (For a review see Muzyczka et al. 
Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of 
the few viruses that may integrate its DNA into non-dividing cells, and 
exhibits a high frequency of stable integration (see for example Flotte et 
al. (1992) Am. J Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. 
(1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 
62:1963-1973). Vectors containing as little as 300 base pairs of AAV can 
be packaged and can integrate. Space for exogenous DNA is limited to about 
4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) 
Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A 
variety of nucleic acids have been introduced into different cell types 
using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. 
Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 
4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin 
et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. 
Chem. 268:3781-3790). 
Other viral vector systems that may have application in gene therapy have 
been derived from herpes virus, vaccinia virus, and several RNA viruses. 
In particular, herpes virus vectors may provide a unique strategy for 
persistence of the recombinant hUbCE or rapUBC-genes in cells of the 
central nervous system and occular tissue (Pepose et al. (1994) Invest 
Ophthalmol Vis Sci 35:2662-2666). 
In addition to viral transfer methods, such as those illustrated above, 
non-viral methods can also be employed to cause expression of an hUbCE or 
rapUBC-protein, or an hUbCE or a rapUBC antisense molecule, in the tissue 
of an animal. Most nonviral methods of gene transfer rely on normal 
mechanisms used by mammalian cells for the uptake and intracellular 
transport of macromolecules. In preferred embodiments, non-viral gene 
delivery systems of the present invention rely on endocytic pathways for 
the uptake of the subject hUbCE or rapUBC-gene by the targeted cell. 
Exemplary gene delivery systems of this type include liposomal derived 
systems, poly-lysine conjugates, and artificial viral envelopes. 
In a representative embodiment, a gene encoding one of the subject 
ubiquitin-conjugating enzymes can be entrapped in liposomes bearing 
positive charges on their surface (e.g., lipofectins) and (optionally) 
which are tagged with antibodies against cell surface antigens of the 
target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT 
publication WO91/06309; Japanese patent application 1047381; and European 
patent publication EP-A-43075). For example, lipofection of 
papilloma-virus infected epithelial cells can be carried out using 
liposomes tagged with monoclonal antibodies against, for example, squamous 
cells. 
In similar fashion, the gene delivery system comprises an antibody or cell 
surface ligand which is cross-linked with a gene binding agent such as 
poly-lysine (see, for example, PCT publications WO93/04701, WO92/22635, 
WO92/20316, WO92/19749, and WO92/06180). For example, the subject UBC-gene 
construct can be used to transfect HPV-infected squamous cells in vivo 
using a soluble polynucleotide carrier comprising an HPV viral caot 
protein conjugated to a polycation, e.g. poly-lysine (see U.S. Pat. No. 
5,166,320). It will also be appreciated that effective delivery of the 
subject nucleic acid constructs via--mediated endocytosis can be improved 
using agents which enhance escape of the gene from the endosomal 
structures. For instance, whole adenovirus or fusogenic peptides of the 
influenza HA gene product can be used as part of the delivery system to 
induce efficient disruption of DNA-containing endosomes (Mulligan et al. 
(1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano 
et al. (1993) PNAS 90:2122). 
In clinical settings, the gene delivery systems can be introduced into a 
patient by any of a number of methods, each of which is familiar in the 
art. For instance, a pharmaceutical preparation of the gene delivery 
system can be introduced systemically, e.g. by intravenous injection, and 
specific transduction of the in the target cells occurs predominantly from 
specificity of transfection provided by the gene delivery vehicle, 
cell-type or tissue-type expression due to the transcriptional regulatory 
sequences controlling expression of the gene, or a combination thereof. In 
other embodiments, initial delivery of the recombinant gene is more 
limited with introduction into the animal being quite localized. For 
example, the gene delivery vehicle can be introduced by catheter (see U.S. 
Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) 
PNAS 91:3054-3057). 
Moreover, the pharmaceutical preparation can consist essentially of the 
gene delivery system in an acceptable diluent, or can comprise a slow 
release matrix in which the gene delivery vehicle is imbedded. 
Alternatively, where the complete gene delivery system can be produced 
intact from recombinant cells, e.g. retroviral packages, the 
pharmaceutical preparation can comprise one or more cells which produce 
the gene delivery system. In the case of the latter, methods of 
introducing the viral packaging cells may be provided by, for example, 
rechargeable or biodegradable devices. The generation of such implants is 
generally known in the art. See, for example, Concise Encyclopedia of 
Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, 
Mass., 1990); Sabel et al. U.S. Pat. No. 4,883,666; Aebischer et al. U.S. 
Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Lim U.S. 
Pat. No. 4,391,909; Sefton U.S. Pat. No. 4,353,888; and Aebischer et al. 
(1991) Biomaterials 12:50-55). 
This invention also pertains to a host cell transfected or transformed to 
express a recombinant forms of the subject UBC proteins. The host cell may 
be any prokaryotic or eukaryotic cell. For example, an hUbCE or rapUBC 
polypeptide of the present invention may be expressed in bacterial cells 
such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. 
Other suitable host cells are known to those skilled in the art. 
The term "recombinant protein" refers to a protein of the present invention 
which is produced by recombinant DNA techniques, wherein generally DNA 
encoding the UBC protein is inserted into a suitable expression vector 
which is in turn used to transform a host cell to produce the heterologous 
protein. Moreover, the phrase "derived from", with respect to a 
recombinant gene encoding the recombinant UBC, is meant to include within 
the meaning of "recombinant protein" those proteins having an amino acid 
sequence of a native UBC, e.g. hUbCE, caUbCE, spUbCE, or rapUBC, or an 
amino acid sequence similar thereto which is generated by mutations 
including substitutions and deletions of a naturally occurring form of the 
protein. Recombinant proteins preferred by the present invention, in 
addition to native proteins, are at least 90% homologous, more preferably 
95% homologous and most preferably 97% homologous with an amino acid 
sequence shown in one of SEQ ID Nos: 2, 4, 6 or 13. Polypeptides having an 
activity of a UBC protein, or which are antagonistic thereto, and which 
are at least about 90%, more preferably at least about 95%, and most 
preferably at least about 98-99% homologous with a sequence shown in SEQ 
ID No: 2, 4, 6 or 13 are also within the scope of the invention. 
The present invention further pertains to recombinant UBC homologs which 
are encoded by genes derived from other non-human mammals, e.g. mouse, 
rat, rabbit, or pig, and which have amino acid sequences evolutionarily 
related to an hUbCE or rapUBC protein. As described above, such 
recombinant UBC or rapUBC proteins preferably are capable of functioning 
in one of either role of an agonist or antagonist of at least one 
biological activity of an hUbCE or rapUBC. The term "evolutionarily 
related to", as set out above, refers to ubiquitin-conjugating enzymes 
having amino acid sequences which have arisen naturally, or which are 
mutationally derived, for example, by combinatorial mutagenesis or 
scanning mutagenesis, but which proteins are homologous to either the 
hUbCE protein represented in SEQ ID No: 2 or rapUBC protein represented in 
SEQ ID No:13. 
The present invention further pertains to methods of producing the subject 
proteins. For example, a host cell transfected with an expression vector 
encoding one of the subject UBC proteins can be cultured under appropriate 
conditions to allow expression of the polypeptide to occur. The peptide 
may be secreted (e.g. through use of recombinantly added signal sequence) 
and isolated from a mixture of cells and medium containing the secreted 
protein. Alternatively, the peptide may be retained cytoplasmically, as it 
presumably is its naturally occurring form, and the cells harvested, lysed 
and the protein isolated. A cell culture includes host cells, media and 
other byproducts. Suitable media for cell culture are well known in the 
art. The subject UBC polypeptides can be isolated from cell culture 
medium, host cells, or both using techniques known in the art for 
purifying proteins including ion-exchange chromatography, gel filtration 
chromatography, ultrafiltration, electrophoresis, and immunoaffinity 
purification with antibodies raised against the protein. In a preferred 
embodiment, the UBC protein is a fusion protein containing a domain which 
facilitates its purification, such as the hUbCE-GST fusion protein 
described below. 
Thus, a nucleotide sequence derived from the cloning of a UBC protein of 
the present invention, encoding all or a selected portion of the protein, 
can be used to produce a recombinant form of the enzyme via microbial or 
eukaryotic cellular processes. Ligating the polynucleotide sequence into a 
gene construct, such as an expression vector, and transforming or 
transfecting into hosts, either eukaryotic (yeast, avian, insect or 
mammalian) or prokaryotic (bacterial cells), are standard procedures used 
in producing other well-known proteins, e.g. p53, C-myc, cyclins, cdks and 
the like. Similar procedures, or modifications thereof, can be employed to 
prepare recombinant proteins, or portions thereof, by microbial means or 
tissue-culture technology in accord with the subject invention. 
The recombinant protein can be produced by ligating the cloned gene, or a 
portion thereof, into a vector suitable for expression in either 
prokaryotic cells, eukaryotic cells, or both. Expression vehicles for 
production of recombinant UBCs include plasmids and other vectors. For 
instance, suitable vectors for the expression of the subject proteins 
include plasmids of the types: pBR322-derived plasmids, pEMBL-derived 
plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived 
plasmids for expression in prokaryotic cells, such as E. coli. 
A number of vectors exist for the expression of recombinant proteins in 
yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are 
cloning and expression vehicles useful in the introduction of genetic 
constructs into S. cerevisiae (see, for example, Broach et al. (1983) in 
Experimental Manipulation of Gene Expression, ed. M. Inouye Academic 
Press, p. 83, incorporated by reference herein). These vectors can 
replicate in E. coli due the presence of the pBR322 ori, and in S. 
cerevisiae due to the replication determinant of the yeast 2 micron 
plasmid. In addition, drug resistance markers such as ampicillin can be 
used. 
The preferred mammalian expression vectors contain both prokaryotic 
sequences to facilitate the propagation of the vector in bacteria, and one 
or more eukaryotic transcription units that are expressed in eukaryotic 
cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, 
pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples 
of mammalian expression vectors suitable for transfection of eukaryotic 
cells. Some of these vectors are modified with sequences from bacterial 
plasmids, such as pBR322, to facilitate replication and drug resistance 
selection in both prokaryotic and eukaryotic cells. Alternatively, 
derivatives of viruses such as the bovine papilloma virus (BPV-1), or 
Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for 
transient expression of proteins in eukaryotic cells. The various methods 
employed in the preparation of the plasmids and transformation of host 
organisms are well known in the art. For other suitable expression systems 
for both prokaryotic and eukaryotic cells, as well as general recombinant 
procedures, see Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by 
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) 
Chapters 16 and 17. 
In some instances, it may be desirable to express the recombinant UBc by 
the use of a baculovirus expression system. Examples of such baculovirus 
expression systems include pVL-derived vectors (such as pVL1392, pVL1393 
and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived 
vectors (such as the 13-gal containing pBlueBac III). 
When expression of a portion of the ubiquitin-conjugating enzyme is 
desired, i.e. a truncation mutant, it may be necessary to add a start 
codon (ATG) to the oligonucleotide fragment containing the desired 
sequence to be expressed. It is well known in the art that a methionine at 
the N-terminal position can be enzymatically cleaved by the use of the 
enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli 
(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonella 
typhimurium and its in vitro activity has been demonstrated on recombinant 
proteins (Miller et al. (1987) PNAS 84:2718-1722). Therefore, removal of 
an N-terminal methionine, if desired, can be achieved either in vivo by 
expressing UBC-derived polypeptides in a host which produces MAP (e.g., E. 
coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., 
procedure of Miller et al.). 
Alternatively, the coding sequences for the polypeptide can be incorporated 
as a part of a fusion gene including a nucleotide sequence encoding a 
different polypeptide. This type of expression system can be useful under 
conditions where it is desirable to produce an immunogenic fragment of a 
UBC protein. In an exemplary embodiment, the VP6 capsid protein of 
rotavirus can be used as an immunologic carrier protein for portions of 
the hUbCE or rapUBC polypeptide, either in the monomeric form or in the 
form of a viral particle. The nucleic acid sequences corresponding to the 
portion of the hUbCE or rapUBC protein to which antibodies are to be 
raised can be incorporated into a fusion gene construct which includes 
coding sequences for a late vaccinia virus structural protein to produce a 
set of recombinant viruses expressing fusion proteins comprising a portion 
of the protein hUbCE or rapUBC as part of the virion. It has been 
demonstrated with the use of immunogenic fusion proteins utilizing the 
Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B 
virions can be utilized in this role as well. Similarly, chimeric 
constructs coding for fusion proteins containing a portion of an UBC 
protein and the poliovirus capsid protein can be created to enhance 
immunogenicity of the set of polypeptide antigens (see, for example, EP 
Publication No. 0259149; and Evans et al. (1989) Nature 339:385; Huang et 
al. (1988) J. Virol. 62:3855; and Schlienger et al. (1992) J. Virol. 
66:2). 
The Multiple Antigen Peptide system for peptide-based immunization can also 
be utilized, wherein a desired portion of a UBC protein is obtained 
directly from organochemical synthesis of the peptide onto an oligomeric 
branching lysine core (see, for example, Posnett et al. (1988) J Biol Chem 
263:1719 and Nardelli et al. (1992) J. Immunol. 148:914). Antigenic 
determinants of the UBC proteins can also be expressed and presented by 
bacterial cells. 
In addition to utilizing fusion proteins to enhance immunogenicity, it is 
widely appreciated that fusion proteins can also facilitate the expression 
of proteins, such as the UBC proteins of the present invention. For 
example, as described below, the hUbCE protein can be generated as a 
glutathione-S-transferase (GST) fusion protein. Such GST fusion proteins 
can enable purification of the hUbCE protein, such as by the use of 
glutathione-derivatized matrices (see, for example, Current Protocols in 
Molecular Biology, eds. Ausubel et al. (NY: John Wiley & Sons, 1991); 
Smith et al. (1988) Gene 67:31; and Kaelin et al. (1992) Cell 70:351). In 
another embodiment, a fusion gene coding for a purification leader 
sequence, such as a poly-(His)/enterokinase cleavage site sequence at the 
N-terminus of the desired portion of the hUbCE protein, can allow 
purification of the expressed hUbCE -fusion protein by affinity 
chromatography using a Ni.sup.2+ metal resin. The purification leader 
sequence can then be subsequently removed by treatment with enterokinase 
(e.g., see Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht 
et al. PNAS 88:8972). Similar constructs can be generated for expression 
of rapUBC, caUbCE, or spUbCE. 
Techniques for making fusion genes are well known. Essentially, the joining 
of various DNA fragments coding for different polypeptide sequences is 
performed in accordance with conventional techniques, employing 
blunt-ended or stagger-ended termini for ligation, restriction enzyme 
digestion to provide for appropriate termini, filling-in of cohesive ends 
as appropriate, alkaline phosphatase treatment to avoid undesirable 
joining, and enzymatic ligation. In another embodiment, the fusion gene 
can be synthesized by conventional techniques including automated DNA 
synthesizers. Alternatively, PCR amplification of gene fragments can be 
carried out using anchor primers which give rise to complementary 
overhangs between two consecutive gene fragments which can subsequently be 
annealed to generate a chimeric gene sequence (see, for example, Current 
Protocols in Molecular Biology, eds. Ausabel et al. John Wiley & Sons: 
1992). 
Various modifications of the hUbCE protein to produce these and other 
functionally equivalent peptides are described in detail herein. In 
similar fashion, homologs of the subject rapUBC, caUBC and spUBC 
polypeptides are contemplated, including both agonistic and antagonistic 
forms. The term peptide, as used herein, refers to peptides, proteins, and 
polypeptides. 
The present invention also makes available isolated UBC proteins, which 
proteins are isolated from or otherwise substantially free of other 
extracellular proteins, especially other proteins of the ubiquitin 
conjugating system (i.e. other E1 or E2 enzymes, as well as E3 proteins or 
ubiquitin) normally associated with the ubiquitin-conjugating enzyme in 
the cellular milleau. The term "substantially free of other extracellular 
proteins" (also referred to herein as "contaminating proteins") is defined 
as encompassing preparations of the subject UBC protein comprising less 
than 20% (by dry weight) contaminating protein, and preferably comprising 
less than 5% contaminating protein. Functional forms of the subject UBC 
proteins can be prepared, for the first time, as purified preparations by 
using a cloned gene as described herein. By "purified", it is meant, when 
referring to a peptide or DNA or RNA sequence, that the indicated molecule 
is present in the substantial absence of other biological macromolecules, 
such as other proteins (particularly other enzymes of the ubiquitin system 
such as other E1 or E2 proteins, as well as other contaminating proteins). 
The term "purified" as used herein preferably means at least 80% by dry 
weight, more preferably in the range of 95-99% by weight, and most 
preferably at least 99.8% by weight, of biological macromolecules of the 
same type present (but water, buffers, and other small molecules, 
especially molecules having a molecular weight of less than 5000, can be 
present). The term "pure" as used herein preferably has the same numerical 
limits as "purified" immediately above. "Isolated" and "purified" do not 
encompass either natural materials in their native state or natural 
materials that have been separated into components (e.g., in an acrylamide 
gel) but not obtained either as pure (e.g. lacking contaminating proteins 
or chromatography reagents such as denaturing agents and polymers, e.g. 
acrylamide or agarose) substances or solutions. 
Isolated peptides having an activity of an UBC protein, or which can 
function as antagonists of a naturally occurring form of the UBC protein 
described herein can also be obtained by screening peptides recombinantly 
produced from the corresponding fragment of the nucleic acids encoding 
such peptides. In addition, fragments can be chemically synthesized using 
techniques known in the art such as conventional Merrifield solid phase 
f-Moc or t-Boc chemistry. For example, the hUbCE protein may be 
arbitrarily divided into fragments of desired length with no overlap of 
the fragments, or preferably divided into overlapping fragments of a 
desired length. The fragments can be produced (recombinantly or by 
chemical synthesis) and tested to identify those peptides having an hUbCE 
protein activity or alternatively to identify antagonists. Similar 
manipulation of the rapUBC, caUbCE and soUbCE proteins can be carried out. 
Furthermore, it is also possible to modify the structure of a UBC 
polypeptide for such purposes as enhancing therapeutic or prophylactic 
efficacy, or stability (e.g., shelf life ex vivo and resistance to 
proteolytic degradation in vivo). Such modified peptides are considered 
functional equivalents of peptides having an activity of, or which 
antagonize, a UBC protein as defined herein. A modified polypeptide can be 
produced in which the amino acid sequence has been altered, such as by 
amino acid substitution, deletion, or addition. 
For example, it is reasonable to expect that an isolated replacement of a 
leucine with an isoleucine or valine, an aspartate with a glutamate, a 
threonine with a serine, or a similar replacement of an amino acid with a 
structurally related amino acid (i.e. conservative mutations) will not 
have a major effect on the biological activity of the resulting molecule. 
Conservative replacements are those that take place within a family of 
amino acids that are related in their side chains. Genetically encoded 
amino acids are can be divided into four families: (1) acidic=aspartate, 
glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, 
valine, leucine, isoleucine, proline, phenylalanine, methionine, 
tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, 
cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and 
tyrosine are sometimes classified jointly as aromatic amino acids. In 
similar fashion, the amino acid repertoire can be grouped as (1) 
acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) 
aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, 
threonine, with serine and threonine optionally be grouped separately as 
aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) 
amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and 
methionine. (see, for example, Biochemistry, 2nd ed, Ed. by L. Stryer, WH 
Freeman and Co.: 1981). Whether a change in the amino acid sequence of a 
peptide results in a functional UBC homolog can be readily determined by 
assessing the ability of the variant peptide to, for instance, mediate 
ubiquitination in a fashion similar to the wild-type UBC. Peptides in 
which more than one replacement has taken place can readily be tested in 
the same manner. 
The invention also includes a method of generating sets of combinatorial 
mutants of the subject UBC proteins, as well as truncation and 
fragmentation mutants, and is especially useful for identifying potential 
variant sequences which are functional in ubiquitinating cellular 
proteins. One purpose for screening such combinatorial libraries is, for 
example, to isolate novel UBC homologs which act as antagonist of the 
wild-type ("authentic") UBC activity, e.g. an hUbCE homolog which inhibits 
p53 ubiquitination, or alternatively, possess novel activities all 
together. Such proteins, when expressed from recombinant DNA constructs, 
can be used in gene therapy protocols. 
Likewise, mutagenesis can give rise to UBC homologs which have 
intracellular half-lives dramatically different than the corresponding 
wild-type protein. For example, the altered protein can be rendered either 
more stable or less stable to proteolytic degradation or other cellular 
process which result in destruction of, or otherwise inactivation of, a 
naturally occurring form of the subject hUbCE or rapUBC proteins. Such 
hUbCE or rapUBC homologs (either agonist or antagonist homologs), and the 
genes which encode them, can be utilized to alter the envelope of 
recombinant hUbCE or rapUBC expression by modulating the half-life of the 
protein. For instance, a short half-life for the recombinant hUbCE or 
rapUBC can give rise to more transient biological effects associated with 
that homolog and, when part of an inducible expression system, can allow 
tighter control of recombinant hUbCE or rapUBC levels within the cell. As 
above, such proteins, and particularly their recombinant nucleic acid 
constructs, can be used in gene therapy protocols. 
In one aspect of this method, the amino acid sequences for a population of 
UBC homologs or other related proteins are aligned, preferably to promote 
the highest homology possible. Such a population of variants can include, 
for example, hUbCE or rapUBC homologs from one or more species, or UBC 
homologs from the same species but which differ due to mutation. Amino 
acids which appear at each position of the aligned sequences are selected 
to create a degenerate set of combinatorial sequences. For instance, 
alignment of the hUbCE, caUbCE and spUbCE sequences provided in the 
appended sequence listing (see also FIG. 1) can be used to generate a 
degenerate library of UbCE proteins represented by the general formula: 
EQU Met Xaa(1) Leu Lys Arg Ile Xaa(2) Xaa(3) Glu Leu Xaa(4) Asp Leu Xaa(5) 
EQU Xaa(6) Asp Pro Pro Xaa(7) Xaa(8) Cys Ser Ala Gly Pro Val Gly Asp Asp Xaa(9) 
EQU Xaa(10) His Trp Gln Ala Xaa(11) Ile Met Gly Pro Asn Asp Ser Pro Tyr Xaa(12) 
Gly Gly Val Phe Phe Leu Xaa(13) Ile His Phe Pro Thr Asp Tyr Pro Xaa(14) 
EQU Lys Pro Pro Lys Xaa(15) Xaa(16) Xaa(17) Thr Thr Xaa(18) Ile Tyr His Pro Asn 
Ile Asn Ser Asn Gly Xaa(19) 
EQU Ile Cys Leu Asp Ile Leu Xaa(20) Xaa(21) Gln Trp Ser Pro Ala Leu Thr Ile Ser 
Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Xaa(22) 
EQU Asp Xaa(23) Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala Xaa(24) 
EQU Xaa(25) Tyr Xaa(26) Xaa(27) Asp Arg Xaa(28) Xaa(29) Tyr Xaa(30) Xaa(31) 
Xaa(32) Ala Xaa(33) Glu Trp Thr Xaa(34) Lys Tyr Ala Xaa(35)(SEQ ID No. 7) 
wherein Xaa(1) represents Ala or Ser; Xaa(2) represents His or Asn; Xaa(3) 
represents Lys or Arg; Xaa(4) represents Ala, Ser or Asn; Xaa(5) 
represents Gly or Ala; Xaa(6) represents Arg or Lys; Xaa(7) represents Ala 
or Ser; Xaa(8) represents Gln or Ser; Xaa(9) represents Leu or Met; 
Xaa(10) represents Phe or Tyr; Xaa(11) represents Ser or Thr; Xaa(12) 
represents Gln or Ala; Xaa(13) represents Ser or Thr; Xaa(14) represents 
Leu or Phe; Xaa(15) represents Val or Ile; Xaa(16) represents Ala or Asn; 
Xaa(17) represents Leu or Phe; Xaa(18) represents Arg or Lys; Xaa(19) 
represents Ser or Asn; Xaa(20) represents Arg or Lys; Xaa(21) represents 
Ser or Asp; Xaa(22) represents Thr or Cys; Xaa(23) represents Ala or Pro; 
Xaa(24) represents Arg or His; Xaa(25) represents Val or Ile; Xaa(26) 
represents Lys or Gln; Xaa(27) represents Thr or Gln; Xaa(28) represents 
Ser, Lys or Glu; Xaa(29) represents Arg or Lys; Xaa(30) represents Asn or 
Gln; Xaa(31) represents Ala, Leu or Arg; Xaa(32) represents Ile, Ser or 
Thr; Xaa(33) represents Arg or Lys; Xaa(34) represents Arg, Lys or Gln; 
Xaa(35) represents Val, Ile or Met. 
To further expand the library, each of the degenerate positions (Xaa) can 
be rendered even more degenerate by including other amino acid residues 
which are of the same "family" as the residues which appear in each of the 
UbCEs, e.g. Xaa(1) can be Gly, Ala, Val, Leu, Ile Ser or Thr (e.g. 
aliphatic), Xaa(22) can be Ser, Thr, Cys or Met (aliphatic-hydroxyl and 
sulfur-containing), etc. Alternatively, isosteric substitutions can be 
made without regard to, for example, charge or polarity of the amino acid 
sidechain. For instance, Xaa(17) can be Leu, Ile, Asn, Met, Phe or Tyr, as 
the sidechains of Ile, Asn and Met each occupy approximately the same 
steric space as Leu, and Tyr is isosteric for Phe. Likewise, where the 
degeneracy is conserved from the human and yeast homologs, the degenerate 
library can, at that position, only include, for example, the amino acid 
residue which occurs in the human UbCE. To illustrate, Xaa(3) is a Lysine 
in hUbCE and caUbCE, and Arginine in spUbCE. In a library which rejects 
conservative mutations of the human UbCE as equivalent, Xaa(3) would be 
Lys. 
In a preferred embodiment, the combinatorial UBC library is produced by way 
of a degenerate library of genes encoding a library of polypeptides which 
each include at least a portion of potential UBC sequences. A mixture of 
synthetic oligonucleotides can be enzymatically ligated into gene 
sequences such that the degenerate set of potential UBC sequences are 
expressible as individual polypeptides, or alternatively, as a set of 
larger fusion proteins (e.g. for phage display) containing the set of UBC 
sequences therein. 
There are many ways by which the library of potential UBC homologs can be 
generated from a degenerate oligonucleotide sequence. Chemical synthesis 
of a degenerate gene sequence can be carried out in an automatic DNA 
synthesizer, and the synthetic genes then be ligated into an appropriate 
gene for expression. The purpose of a degenerate set of genes is to 
provide, in one mixture, all of the sequences encoding the desired set of 
potential UBC sequences. The synthesis of degenerate oligonucleotides is 
well known in the art (see, for example, Narang, SA (1983) Tetrahedron 
39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. 
Macromolecules, ed. A. G. Walton, Amsterdam: E1sevier pp273-289; Itakura 
et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 
198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have 
been employed in the directed evolution of other proteins (see, for 
example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) 
PNAS 89:2429-2433; Devlin et al. (1990) Science 249:404-406; Cwirla et al. 
(1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, 
and 5,096,815). 
A wide range of techniques are known in the art for screening gene products 
of combinatorial libraries made by point mutations, and for screening cDNA 
libraries for gene products having a certain property. Such techniques 
will be generally adaptable for rapid screening of the gene libraries 
generated by the combinatorial mutagenesis of UBC homologs. The most 
widely used techniques for screening large gene libraries typically 
comprises cloning the gene library into replicable expression vectors, 
transforming appropriate cells with the resulting library of vectors, and 
expressing the combinatorial genes under conditions in which detection of 
a desired activity facilitates relatively easy isolation of the vector 
encoding the gene whose product was detected. Each of the illustrative 
assays described below are amenable to high through-put analysis as 
necessary to screen large numbers of, for example, degenerate UBC 
sequences created by combinatorial mutagenesis techniques. 
In one illustrative screening assay, the candidate hUbCE or rapUBC gene 
products are displayed on the surface of a cell or viral particle, and the 
ability of particular cells or viral particles to bind other components of 
the ubiquitin pathway, such as E1 or E3 proteins (e.g. E6AP or E6AP 
complexes), ubiquitin, or a cell-cycle regulatory protein, via this gene 
product is detected in a "panning assay". For instance, the gene library 
can be cloned into the gene for a surface membrane protein of a bacterial 
cell, and the resulting fusion protein detected by panning (Ladner et al., 
WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et 
al. (1992) TIBS 18:136-140). In a similar fashion, fluorescently labeled 
molecules which bind hUbCE or rapUBC can be used to score for potentially 
functional hUbCE or rapUBC homologs. Cells can be visually inspected and 
separated under a fluorescence microscope, or, where the morphology of the 
cell permits, separated by a fluorescence-activated cell sorter. 
In an alternate embodiment, the gene library is expressed as a fusion 
protein on the surface of a viral particle. For instance, in the 
filamentous phage system, foreign peptide sequences can be expressed on 
the surface of infectious phage, thereby conferring two significant 
benefits. First, since these phage can be applied to affinity matrices at 
very high concentrations, a large number of phage can be screened at one 
time. Second, since each infectious phage displays the combinatorial gene 
product on its surface, if a particular phage is recovered from an 
affinity matrix in low yield, the phage can be amplified by another round 
of infection. The group of almost identical E. coli filamentous phages 
M13, fd, and f1 are most often used in phage display libraries, as either 
of the phage gIII or gVIII coat proteins can be used to generate fusion 
proteins without disrupting the ultimate packaging of the viral particle 
(Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT 
publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 
267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et 
al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 
89:4457-4461). 
In an illustrative embodiment, the recombinant phage antibody system (RPAS, 
Pharmacia Catalog number 27-9400-01) can be easily modified for use in 
expressing and screening hUbCE or rapUBC combinatorial libraries. For 
instance, the pCANTAB 5 phagemid of the RPAS kit contains the gene which 
encodes the phage gIII coat protein. The hUbCE or rapUBC combinatorial 
gene library can be cloned into the phagemid adjacent to the gIII signal 
sequence such that it will be expressed as a gIII fusion protein. After 
ligation, the phagemid is used to transform competent E. coli TG1 cells. 
Transformed cells are subsequently infected with M13KO7 helper phage to 
rescue the phagemid and its candidate hUbCE or rapUBC gene insert. The 
resulting recombinant phage contain phagemid DNA encoding a specific 
candidate hUbCE or rapUBC, and display one or more copies of the 
corresponding fusion coat protein. The phage-displayed candidate hUbCE or 
rapUBC which are capable of binding a particular target protein, such as 
an E1 enzyme, an E3 protein (i.e. E6 or E6-AP), or a particular regulatory 
protein (such as p53 or p27), are selected or enriched by panning. For 
instance, the phage library can be panned on glutathione immobilized 
p53-GST fusion proteins or E6-GST or E6-AP-GST fusion proteins and unbound 
phage washed away from the cells. The bound phage is then isolated, and if 
the recombinant phage express at least one copy of the wild type gIII coat 
protein, they will retain the ability to infect E. coli. Thus, successive 
rounds of reinfection and panning can be employed to greatly enrich for 
UBC homologs that retain some ability to interact with normal targets of 
the wild-type enzyme and which can then be screened for further biological 
activities in order to differentiate agonists and antagonists. In an 
exemplary embodiment, by use of two or more target proteins in sequential 
panning steps, the phage display library can be used to isolate hUbCE or 
rapUBC homologs which are candidate antagonists of the normal cellular 
function of the naturally occurring UBC. For instance, isolating from the 
library those variants which retain the ability to bind, for example, 
either the papillomavirus E6 protein or the cellular E6-AP protein, but 
which are unable to bind p53, provides a set of hUbCE or rapUBC homologs 
some of which may be capable of antagonizing the ability of the 
corresponding wild-type enzyme to mediate ubiquitination of p53. 
In yet another illustrative embodiment, the p53-dependent reporter 
construct described in the 08/176,937 application can be used to identify 
antagonists through their ability to enhance expression of the reporter 
gene by inhibiting the degradation of p53 wild-type hUbCE or rapUBC. Thus, 
a combinatorial library can screened by a detecting expression of the 
reporter gene, and appropriate clones isolated for further manipulation. 
Other forms of mutagenesis can also be utilized to generate a combinatorial 
library from the subject UBC proteins. For example, hUbCE or rapUBC 
homologs (both agonist and antagonist forms) can be generated and isolated 
from a library by screening using, for example, alanine scanning 
mutagenesis and the like (Ruf et al. (1994) Biochemistry 33:1565-1572; 
Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 
137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; 
Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) 
Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 
244:1081-1085), by linker scanning mutagenesis (Gustin et al. (1993) 
Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; 
McKnight et al. (1982) Science 232:316); by saturation mutagenesis (Meyers 
et al. (1986) Science 232:613); by PCR mutagenesis (Leung et al. (1989) 
Method Cell Mol Biol 1:11-19); or by random mutagenesis (Miller et al. 
(1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring 
Harbor, N.Y.; and Greener et al. (1994) Strategies in Mol Biol 7:32-34). 
An important goal of the present invention is to provide reduction of the 
UBC proteins to small functional units that can be ultimately used to 
generate UBC mimetics, e.g. peptide or non-peptide agents, which are able 
to disrupt binding of UBC with other cellular and/or viral proteins. Thus, 
such mutagenic techniques as described herein are particularly useful to 
map the determinants of the hUbCE or rapUBC protein which participate in 
protein-protein interactions involved in, for example, binding of the 
subject hUbCE or rapUBC to other proteins of the ubiquitin-conjugating 
system (both cellular and viral), as well as the target protein itself 
(e.g. a cell-cycle regulatory protein). To illustrate, the critical 
residues of hUbCE involved in molecular recognition of E6 and/or E6-AP can 
be determined and used to generate hUbCE-derived peptidomimetics which 
competitively inhibit hUbCE binding. By employing, for example, scanning 
mutagenesis to map the amino acid residues of hUbCE involved in binding 
E6AP, peptidomimetic compounds can be generated which mimic those residues 
in binding to E6AP, and which therefore can inhibit binding of the hUbCE 
to E6AP and interfere with the function of E6AP in regulating the cellular 
half-life of p53. For instance, non-hydrolyzable peptide analogs of such 
residues can be generated using benzodiazepine (e.g., see Freidinger et 
al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM 
Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffinan et al. 
in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: 
Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. 
in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: 
Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. 
(1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and 
Function (Proceedings of the 9th American Peptide Symposium) Pierce 
Chemical Co. Rockland, Ill., 1985), .beta.-turn dipeptide cores (Nagai et 
al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc 
Perkin Trans 1:1231), and .beta.-aminoalcohols (Gordon et al. (1985) 
Biochem Biophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys 
Res Commun 134:71). Such peptidomimetics can serve as drugs which prevent 
the action of hUbCE in the destruction of, for example, p53. In like 
manner, peptidomimetics of caUbCE and spUbCE can be derived which may be 
useful in, for example, the generation of anti-mycotic agents. 
Another aspect of the invention pertains to an antibody specifically 
reactive with the subject UBC proteins. For example, by using immunogens 
derived from the hUbCE or rapUBC protein of the present invention, 
anti-protein/anti-peptide antisera or monoclonal antibodies can be made by 
standard protocols (See, for example, Antibodies: A Laboratory Manual ed. 
by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a 
mouse, a hamster or rabbit can be immunized with an immunogenic form of 
the peptide (e.g., the whole UBC protein or an antigenic fragment which is 
capable of eliciting an antibody response). Techniques for conferring 
immunogenicity on a protein or peptide include conjugation to carriers or 
other techniques well known in the art. An immunogenic portion of the 
subject UBC protein can be administered in the presence of adjuvant. The 
progress of immunization can be monitored by detection of antibody titers 
in plasma or serum. Standard ELISA or other immunoassays can be used with 
the immunogen as an antigen to assess the levels of antibodies. In a 
preferred embodiment, the subject antibodies are immunospecific for hUbCE 
antigenic determinants, e.g. antigenic determinants of a protein 
represented by SEQ ID No. 2 or a closely related human or non-human 
mammalian homolog (e.g. 90 percent homologous to SEQ ID No. 2, preferably 
at least 95 percent homologous and more preferably at least 97 percent 
homologous to SEQ ID No.2). In yet a further preferred embodiment of the 
present invention, the anti-hUbCE antibodies does not substantially cross 
react with a protein which is: e.g. less than 90 percent homologous with 
SEQ ID No. 2; e.g. less than 95 percent homologous with SEQ ID No. 2; e.g. 
less than 98-99 percent homologous with SEQ ID No.2. By "does not 
substantially cross-react", it is meant that: the antibody has a binding 
affinity for a non-homologous E2 enzyme which is less than 10 percent, 
more preferably less than 5 percent, and most preferably less than about 
1-2 percent of the binding affinity of that antibody for the protein of 
SEQ ID No. 2; the antibody does not specifically bind a protein which is 
non-homologous to SEQ ID No. 2. Preferred antibodies against the subject 
caUbCE, spUbCE and rapUBC proteins have similar criteria, e.g., antibodies 
specific for caUbCE, spUbCE or rapUBC do not specifically bind proteins 
which do not share high sequence homology with SEQ ID No. 4, 6, or 13 
respectively. 
Following immunization, antisera selectively reactive with one or more of 
the subject UBCs can be obtained and, if desired, polyclonal anti-UBC 
antibodies isolated from the serum. To produce monoclonal antibodies, 
antibody producing cells (lymphocytes) can be harvested from an immunized 
animal and fused by standard somatic cell fusion procedures with 
immortalizing cells such as myeloma cells to yield hybridoma cells. Such 
techniques are well known in the art, an include, for example, the 
hybridoma technique (originally developed by Kohler and Milstein, (1975) 
Nature, 256:495-497), the human B cell hybridoma technique (Kozbar et al., 
(1983) Immunology Today, 4:72), and the EBV-hybridoma technique to produce 
human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be 
screened immunochemically for production of antibodies specifically 
reactive with the subject proteins and monoclonal antibodies isolated from 
a culture comprising such hybridoma cells. 
The term antibody as used herein is intended to include fragments thereof 
which are also specifically reactive with the UBC proteins of the present 
invention. Antibodies can be fragmented using conventional techniques and 
the fragments screened for utility in the same manner as described above 
for whole antibodies. For example, F(ab').sub.2 fragments can be generated 
by treating antibody with pepsin. The resulting F(ab').sub.2 fragment can 
be treated to reduce disulfide bridges to produce Fab' fragments. The 
antibody of the present invention is further intended to include 
bispecific and chimeric molecules having an anti-UBC portion. 
Both monoclonal and polyclonal antibodies (Ab) directed against the subject 
ubiquitin conjugating enzymes, and antibody fragments such as Fab' and 
F(ab').sub.2, can be used as specialty chemicals to block the action of 
the enzyme and allow the study of, for example, the cell cycle or cell 
proliferation when the subject UBC is inhibited, e.g. by microinjection of 
anti-UBC antibodies. 
Antibodies which specifically bind hUbCE or rapUBC epitopes can also be 
used in immunohistochemical staining of tissue samples in order to 
evaluate the abundance and pattern of expression of hUbCE or rapUBC. 
Anti-hUbCE or anti-rapUBC antibodies can be used diagnostically in 
immuno-precipitation and immuno-blotting to detect and evaluate hUbCE or 
rapUBC levels in tissue or bodily fluid as part of a clinical testing 
procedure. For instance, such measurements can be useful in predictive 
valuations of the onset or progression of tumors. Likewise, the ability to 
monitor hUbCE or rapUBC levels in an individual can allow determination of 
the efficacy of a given treatment regimen for an individual afflicted with 
such a disorder. The level of each of the subject UBCs can be measured in 
cells isolated from bodily fluid, such as in samples of cerebral spinal 
fluid or blood, or can be measured in tissue, such as produced by biopsy. 
Diagnostic assays using anti-hUbCE or anti-rapUBC antibodies can include, 
for example, immunoassays designed to aid in early diagnosis of a 
neoplastic or hyperplastic disorder, e.g. the presence of cancerous cells 
in the sample, e.g. to detect cells in which a lesion of the hUbCE or 
rapUBC gene has occurred. 
Another application of anti-UBC antibodies is in the immunological 
screening of cDNA libraries constructed in expression vectors, such as 
.lambda.gt11, .lambda.gt18-23, .lambda.ZAP, and .lambda.ORF8. Messenger 
libraries of this type, having coding sequences inserted in the correct 
reading frame and orientation, can produce fusion proteins. For instance, 
.lambda.gt11 will produce fusion proteins whose amino termini consist of 
.beta.-galactosidase amino acid sequences and whose carboxy termini 
consist of a foreign polypeptide. Antigenic epitopes of UBc can then be 
detected with antibodies, as, for example, reacting nitrocellulose filters 
lifted from infected plates with anti-UBC antibodies. Phage, scored by 
this assay, can then be isolated from the infected plate. Thus, the 
presence of hUbCE or rapUBC homologs can be detected and cloned from other 
human sources, i.e. to identify other closely homologous human isoforms, 
as well as to identify hUbCE or rapUBC homologs in other mammals. 
Moreover, the nucleotide sequence determined from the cloning of the 
subject hUbCE or rapUBC from a human cell line will further allow for the 
generation of probes designed for use in identifying hUbCE or rapUBC 
homologs in other human cell-types, particularly cancer or other 
transformed or immortalized cells, as well as hUbCE or rapUBC homologs 
from other non-human mammals. Probes based on the yeast UbCE sequences, 
caUbCE and spUbCE, can be generated and used to identify and phenotype 
mycotic infections. 
In addition, nucleotide probes can be generated from the cloned sequence of 
the hUbCE or rapUBC protein, which allow for histological screening of 
intact tissue and tissue samples for the presence of hUbCE or rapUBC mRNA. 
Similar to the diagnostic uses of anti-hUbCE or anti-rapUBC antibodies, 
the use of probes directed to hUbCE or rapUBC mRNA, or to genomic hUbCE or 
rapUBC sequences, can be used for both predictive and therapeutic 
evaluation of allelic mutations which might be manifest in, for example, 
neoplastic or hyperplastic disorders (e.g. unwanted cell growth). Used in 
conjunction with anti-hUbCE or anti-rapUBC antibody immunoassays, the 
nucleotide probes can help facilitate the determination of the molecular 
basis for a developmental disorder which may involve some abnormality 
associated with expression (or lack thereof) of an hUbCE or a rapUBC 
protein. For instance, variation in hUbCE or rapUBC synthesis can be 
differentiated from a mutation in the hUbCE or rapUBC coding sequence. 
For example, the present method provides a method for determining if a 
subject is at risk for a disorder characterized by unwanted cell 
proliferation. In preferred embodiments, the subject method can be 
generally characterized as comprising detecting, in a tissue of a subject 
(e.g. a human patient), the presence or absence of a genetic lesion 
characterized by at least one of (i) a mutation of a gene encoding hUbCE 
or rapUBC, or (ii) the mis-expression of the UBC gene. To illustrate, such 
genetic lesions can be detected by ascertaining the existence of at least 
one of (i) a deletion of one or more nucleotides from the UBC gene, (ii) 
an addition of one or more nucleotides to the UBC gene, (iii) a 
substitution of one or more nucleotides of the UBC gene, (iv) a gross 
chromosomal rearrangement of the hUbCE or rapUBC gene, (v) a gross 
alteration in the level of a messenger RNA transcript of the hUbCE or 
rapUBC gene, (vi) the presence of a non-wild type splicing pattern of a 
messenger RNA transcript of the hUbCE or rapUBC gene, and (vii) a non-wild 
type level of the hUbCE or rapUBC protein. In one aspect of the invention 
there is provided a probe/primer comprising an oligonucleotide containing 
a region of nucleotide sequence which is capable of hybridizing to a sense 
or antisense sequence of SEQ ID No: 1 or SEQ ID No:12, or naturally 
occurring mutants thereof, or 5' or 3' flanking sequences, or intronic 
sequences naturally associated with the hUbCE or rapUBC gene. The probe is 
exposed to nucleic acid of a tissue sample; and the hybridization of the 
probe to the sample nucleic acid is detected. In certain embodiments, 
detection of the lesion comprises utilizing the probe/primer in, for 
example, a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 
4,683,195 and 4,683,202), or, alternatively, in a ligation chain reaction 
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and 
Nakazawa et al. (1994) PNAS 91:360-364), the later of which can be 
particularly useful for detecting even point mutations in the hUbCE or 
rapUBC gene. Alternatively, or additionally, the level of hUbCE or rapUBC 
protein can be detected in an immunoassay. 
Also, the use of anti-sense techniques (e.g. microinjection of antisense 
molecules, or transfection with plasmids whose transcripts are anti-sense 
with regard to, e.g. UBC mRNA) can be used to investigate the role of each 
of the subject UBC proteins in the cell cycle and cell proliferation, by 
inhibiting endogenous production of that protein. Such techniques can be 
utilized in cell culture, but can also be used in the creation of 
transgenic animals. 
Another aspect of the present invention concerns transgenic animals, e.g. 
as animal models for developmental and proliferative diseases, which are 
comprised of cells (of that animal) which contain a transgene of the 
present invention and which preferably (though optionally) express a 
recombinant form (agonist or antagonist) of one or more of the subject UBC 
enzymes in one or more cells in the animal. In preferred embodiments, the 
expression of the transgene is restricted to specific subsets of cells, 
tissues or developmental stages utilizing, for example, cis-acting 
sequences that control expression in the desired pattern. In the present 
invention, such mosiac expression of the subject UBC proteins can be 
essential for many forms of lineage analysis and can additionally provide 
a means to assess the effects of UBC mutations or overexpression that 
might grossly alter development in small patches of tissue within an 
otherwise normal embryo. Toward this and, tissue-specific regulatory 
sequences and conditional regulatory sequences can be used to control 
expression of the transgene in certain spatial patterns. Moreover, 
temporal patterns of expression can be provided by, for example, 
conditional recombination systems or prokaryotic transcriptional 
regulatory sequences. 
Genetic techniques which allow for the expression of transgenes can be 
regulated via site-specific genetic manipulation in vivo are known to 
those skilled in the art. For instance, genetic systems are available 
which allow for the regulated expression of a recombinase that catalyzes 
the genetic recombination a target sequence. As used herein, the phrase 
"target sequence" refers to a nucleotide sequence that is genetically 
recombined by a recombinase. The target sequence is flanked by recombinase 
recognition sequences and is generally either excised or inverted in cells 
expressing recombinase activity. Recombinase catalyzed recombination 
events can be designed such that recombination of the target sequence 
results in either the activation or repression of expression of the 
subject receptor. For example, excision of a target sequence which 
interferes with the expression of the receptor can be designed to activate 
expression of that protein. This interference with expression of the 
subject protein can result from a variety of mechanisms, such as spatial 
separation of the UBC gene from the promoter element or an internal stop 
codon. Moreover, the transgene can be made wherein the coding sequence of 
the UBC gene is flanked by recombinase recognition sequences and is 
initially transfected into cells in a 3' to 5' orientation with respect to 
the promoter element. In such an instance, inversion of the target 
sequence will reorient the subject UBC gene by placing the 5' end of the 
coding sequence in an orientation with respect to the promoter element 
which allow for promoter driven transcriptional activation. 
In an illustrative embodiment, either the cre/loxP recombinase system of 
bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. 
(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces 
cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCT publication 
WO 92/15694) can be used to generate in vivo site-specific genetic 
recombination systems. Cre recombinase catalyzes the site-specific 
recombination of an intervening target sequence located between loxP 
sequences. loxP sequences are 34 base pair nucleotide repeat sequences to 
which the Cre recombinase binds and are required for Cre recombinase 
mediated genetic recombination. The orientation of loxP sequences 
determines whether the intervening target sequence is excised or inverted 
when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 
259:1509-1514); catalyzing the excision of the target sequence when the 
loxP sequences are oriented as direct repeats and catalyzes inversion of 
the target sequence when loxP sequences are oriented as inverted repeats. 
Accordingly, genetic recombination of the target sequence is dependent on 
expression of the Cre recombinase. Expression of the recombinase can be 
regulated by promoter elements which are subject to regulatory control, 
e.g., tissue-specific, developmental stage-specific, inducible or 
repressible by externally added agents. This regulated control will result 
in genetic recombination of the target sequence only in cells where 
recombinase expression is mediated by the promoter element. Thus, the 
activation of expression of the recombinant UBC gene can be regulated via 
regulation of recombinase expression. 
Use of the these recombinase system to regulate expression of, for example, 
a dominant negative UBC gene, such as the Cys85Ser mutant or an antisense 
gene, requires the construction of a transgenic animal containing 
transgenes encoding both the Cre recombinase and the subject gene. Animals 
containing both the Cre recombinase and the UBC genes can be provided 
through the construction of "double" transgenic animals. A convenient 
method for providing such animals is to mate two transgenic animals each 
containing a transgene, e.g., the UBC gene and recombinase gene. 
One advantage derived from initially constructing transgenic animals 
containing a UBC transgene in a recombinase-mediated expressible format 
derives from the likelihood that the subject UBC protein, whether 
antagonistic or agonistic, will be deleterious upon expression in the 
transgenic animal. In such an instance, a founder population, in which the 
subject transgene is silent in all tissues, can be propagated and 
maintained. Individuals of this founder population can be crossed with 
animals expressing the recombinase in, for example, one or more tissues. 
Thus, the creation of a founder population in which the UBC transgene is 
silent will allow the study of, for example, the role of the p53 
checkpoint in tissue or at developmental stages which can confer, for 
example, a lethal phenotype. 
Similar conditional transgenes can be provided using prokaryotic promoter 
sequences which require prokaryotic proteins to be simultaneous expressed 
in order to facilitate expression of the transgene. Exemplary promoters 
and the corresponding trans-activating prokaryotic proteins are given in 
U.S. Pat. No. 4,833,080. Moreover, expression of the conditional 
transgenes can be induced by gene therapy-like methods wherein a gene 
encoding the trans-activating protein, e.g. a recombinase or a prokaryotic 
protein, is delivered to the tissue and caused to be expressed, such as in 
a cell-type specific manner. By this method, the transgene could remain 
silent into adulthood until "turned on" by the introduction of the 
trans-activator. 
Methods of making knock-out or disruption transgenic animals are also 
generally known. See, for example, Manipulating the Mouse Embryo, (Cold 
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). 
Furthermore, the present invention, by making available purified and 
recombinant forms of the subject UBC proteins, facilitates the development 
of assays that can be used to screen for drugs which inhibit the 
conjugating activity. For instance, in addition to agents which disrupt 
binding of a UBC protein to other cellular (or viral) proteins, inhibitors 
of the enzymatic activity of the subject E2 enzymes may prevent transfer 
of ubiquitin to the enzyme (by an E1 enzyme) or inhibit any downstream 
transfer of ubiquitin from the E2 enzyme to a cellular substrate or an 
intermediary E3 complex, e.g., an E6/E6-AP. In a preferred embodiment, the 
UBC inhibitor is a mechanism based inhibitor which chemically alters the 
enzyme, e.g. covalently binds Cys-85 of hUbCE or Cys-93 of rapUBC, and 
which is a specific inhibitor of that enzyme, e.g. has an inhibition 
constant 10-fold, 100-fold, or more preferably, 1000-fold different for 
human E2 enzymes other than the subject UBC enzyme. Inhibitor specificity 
can be improved, for example, by utilizing specificity subsites of the 
hUbCE enzyme involved in interactions between hUbCE and an E6 /E6AP 
complex, or hUbCE and an E1 enzyme, which are unique to one of those 
complexes relative to other human E2 enzymes. Similar approaches can also 
be used to screen for drugs agonistic or antagonistic to rapUBC 
activities. 
Assays for the measurement of ubiquitination can be generated in many 
different forms, and include assays based on cell-free systems, e.g. 
purified proteins or cell lysates, as well as cell-based assays which 
utilize intact cells. Assays as described herein can be used in 
conjunction with the subject E2 enzymes to generate a 
ubiquitin-conjugating system for detecting agents able to inhibit 
particular E2-mediated ubiquitination of a cellular or viral regulatory 
proteins. Such inhibitors can be used, for example, in the treatment of 
proliferative and/or differentiative disorders, to modulate apoptosis, and 
in the treatment of viral infections, such by adenoviruses or 
papillomaviruses. Similar assay systems can be constructed for the fungal 
homologs in order to detect inhibitors which may serve as anti-fungal 
agents. In preferred embodiments, the assay system employed for 
identifying anti-fungal agents are run side-by-side with the analogous 
assay system derived with the mammalian homolog of the UBC, e.g. hUbCE or 
rapUBC. Differential screening assays can be used to exploit any 
difference in mechanism or specificity between mammalian UBCs and yeast 
UBCs (including other yeast E2 enzymes) in order to identify agents which 
display a statistically significant increase in specificity for inhibiting 
the yeast enzymes relative to the mammalian enzymes. Thus, lead compounds 
which act specifically on pathogens, such as fungus involved in mycotic 
infections, can be developed. 
In many drug screening programs which test libraries of compounds and 
natural extracts, high throughput assays are desirable in order to 
maximize the number of compounds surveyed in a given period of time. 
Assays which are performed in cell-free systems, such as may be derived 
with purified or semi-purified proteins or with lysates, are often 
preferred as "primary" screens in that they can be generated to permit 
rapid development and relatively easy detection of an alteration in a 
molecular target which is mediated by a test compound. Moreover, the 
effects of cellular toxicity and/or bioavailability of the test compound 
can be generally ignored in the in vitro system, the assay instead being 
focused primarily on the effect of the drug on the molecular target as may 
be manifest in an alteration of binding affinity with other proteins or 
change in enzymatic properties of the molecular target. Accordingly, 
potential E2 inhibitors can be detected in a cell-free assay generated by 
constitution of a functional ubiquitin-protein ligase system in a cell 
lysate, such as generated by charging a ubiquitin-depleted reticulocyte 
lysate (Hersko et al. (1983) J Biol Chem 258:8206-6214) with one of the 
subject UBC enzymes and, as needed, an E1 enzyme, an E3 enzyme (cellular 
or viral in origin), ubiquitin, and a substrate for UBC-dependent 
ubiquitination. In an alternative format, the assay can be derived as a 
reconstituted protein mixture which, as described below, offers a number 
of benefits over lysate-based assays. 
In yet other embodiments, the present assay comprises an in vivo 
ubiquitin-conjugating system, such as a cell able to conduct the 
regulatory protein through at least a portion of a ubiquitin-mediated 
proteolytic pathway. 
The level of ubiquitination of the substrate protein brought about by the 
system is measured in the presence and absence of a candidate agent, and a 
decrease in the level ubiquitin conjugation is indicative of an inhibitory 
activity for the candidate agent. As described below, the level of 
ubiquitination of the regulatory protein can be measured by determining 
the actual concentration of protein:ubiquitin conjugates formed; or 
inferred by detecting some other quality of the subject protein affected 
by ubiquitination, including the proteolytic degradation of the protein. A 
statistically significant decrease in ubiquitination of the target protein 
in the presence of the test compound is indicative of the test compound 
being an inhibitor of E2-dependent ubiquitin conjugation. 
In preferred in vitro embodiments of the present assay, the 
ubiquitin-conjugating system comprises a reconstituted protein mixture of 
at least semi-purified proteins. By semi-purified, it is meant that the 
proteins utilized in the reconstituted mixture have been previously 
separated from other cellular or viral proteins. For instance, in contrast 
to cell lysates, the proteins involved in conjugation of ubiquitin to a 
target protein, together with the target protein, are present in the 
mixture to at least 50% purity relative to all other proteins in the 
mixture, and more preferably are present at 90-95% purity. In certain 
embodiments of the subject method, the reconstituted protein mixture is 
derived by mixing highly purified proteins such that the reconstituted 
mixture substantially lacks other proteins (such as of cellular or viral 
origin) which might interfere with or otherwise alter the ability to 
measure specific ubiquitination or ubiquitin-mediated degradation of the 
target regulatory protein. 
Each of the protein components utilized to generate the reconstituted 
ubiquitin-conjugating system are preferably isolated from, or otherwise 
substantially free of, other proteins normally associated with the 
proteins in a cell or cell lysate. The term "substantially free of other 
cellular proteins" (also referred to herein as "contaminating proteins") 
is defined as encompassing individual preparations of each of the 
component proteins comprising less than 20% (by dry weight) contaminating 
protein, and preferably comprises less than 5% contaminating protein. 
Functional forms of each of the component proteins can be prepared as 
purified preparations by using a cloned gene as described in the attached 
examples. By "purified", it is meant, when referring to the component 
proteins preparations used to generate the reconstituted protein mixture, 
that the indicated molecule is present in the substantial absence of other 
biological macromolecules, such as other proteins (particularly other 
proteins which may substantially mask, diminish, confuse or alter the 
characteristics of the component proteins either as purified preparations 
or in their function in the subject reconstituted mixture). The term 
"purified" as used herein preferably means at least 80% by dry weight, 
more preferably in the range of 95-99% by weight, and most preferably at 
least 99.8% by weight, of biological macromolecules of the same type 
present (but water, buffers, and other small molecules, especially 
molecules having a molecular weight of less than 5000, can be present). 
The term "pure" as used herein preferably has the same numerical limits as 
"purified" immediately above. "Isolated" and "purified" do not encompass 
either protein in its native state (e.g. as a part of a cell), or as part 
of a cell lysate, or that have been separated into components (e.g., in an 
acrylamide gel) but not obtained either as pure (e.g. lacking 
contaminating proteins) substances or solutions. The term isolated as used 
herein also refers to a component protein that is substantially free of 
cellular material or culture medium when produced by recombinant DNA 
techniques, or chemical precursors or other chemicals when chemically 
synthesized. 
With respect to measuring ubiquitination, the purified protein mixture can 
substantially lack any proteolytic activity which would degrade the target 
protein and/or components of the ubiquitin conjugating system. For 
instance, the reconstituted system can be generated to have less than 10% 
of the proteolytic activity associated with a typical reticulocyte lysate, 
and preferably no more than 5%, and most preferably less than 2%. 
Alternatively, the mixture can be generated to include, either from the 
onset of ubiquitination or from some point after ubiquitin conjugation of 
the regulatory protein, a ubiquitin-dependent proteolytic activity, such 
as a purified proteosome complex, that is present in the mixture at 
measured amounts. 
In the subject method, ubiquitin conjugating systems derived from purified 
proteins hold a number of significant advantages over cell lysate or wheat 
germ extract based assays (collectively referred to hereinafter as 
"lysates"). Unlike the reconstituted protein system, the synthesis and 
destruction of the target protein cannot be readily controlled for in 
lysate-based assays. Without knowledge of particular kinetic parameters 
for Ub-independent and Ub-dependent degradation of the target protein in 
the lysate, discerning between the two pathways can be extremely 
difficult. Measuring these parameters, if at all possible, is further made 
tedious by the fact that cell lysates tend to be inconsistent from batch 
to batch, with potentially significant variation between preparations. 
Evaluation of a potential inhibitor using a lysate system is also 
complicated in those circumstances where the lysate is charged with mRNA 
encoding the target protein, as such lysates may continue to synthesize 
the protein during the assay, and will do so at unpredictable rates. 
Using similar considerations, knowledge of the concentration of each 
component of the ubiquitin conjugation pathway can be required for each 
lysate batch, along with the degradative kinetic data, in order to 
determine the necessary time course and calculate the sensitivity of 
experiments performed from one lysate preparation to the next. 
Furthermore, the lysate system can be unsatisfactory where the target 
protein itself has a relatively short half-life, especially if due to 
degradative processes other than the ubiquitin-mediated pathway to which 
an inhibitor is sought. For example, in assays for an inhibitor of 
HPV-induced ubiquitination of p53, lysate based systems can be difficult 
to use, in addition to the reasons set forth above, due to the short 
half-life of p53 even in extracts which lack HPV proteins. In such 
systems, the ability to measure HPV-mediated ubiquitination of p53 is made 
difficult by the already rapid, ongoing degradation of p53 presumably 
occurring by proteolytic processes which are not mediated by any HPV 
proteins. 
The use of reconstituted protein mixtures allows more careful control of 
the reaction conditions in the ubiquitin-conjugating system. Moreover, the 
system can be derived to favor discovery of inhibitors of particular steps 
of the ubiquitination process. For instance, a reconstituted protein assay 
can be generated which does not facilitate degradation of the 
ubiquitinated protein. The level of ubiquitin conjugated protein can 
easily be measured directly in such as system, both in the presence and 
absence of a candidate agent, thereby enhancing the ability to detect a 
ubiquitination inhibitor. Alternatively, the Ub-conjugating system can be 
allowed to develop a steady state level of regulatory protein:Ub 
conjugates in the absence of a proteolytic activity, but then shifted to a 
degradative system by addition of purified Ub-dependent proteases. Such 
degradative systems would be amenable to identifying proteosome 
inhibitors. 
The purified protein mixture includes a purified preparation of the 
regulatory protein and ubiquitin under conditions which drive the 
conjugation of the two molecules. For instance, the mixture can include a 
ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and 
a nucleotide triphosphate (e.g. ATP). Alternatively, the E1 enzyme, the 
ubiquitin, and the nucleotide triphosphate can be substituted in the 
system with a pre-activated ubiquitin in the form of an E1::Ub conjugate. 
Likewise, a pre-activated ubiquitin can instead comprise an E2::Ub 
conjugate which can directly transfer the pre-activated ubiquitin to the 
target protein substrate. 
Furthermore, the reconstituted mixture can also be generated to include at 
least one auxiliary substrate recognition protein (E3) which may be, for 
example, of cellular or viral origin. In illustrative embodiments 
described below, in order to generate an assay which approximates the 
ubiquitination of p53 in HPV-16 or HPV-18 infected cells, the 
reconstituted ubiquitin conjugating system may further include an E6 
protein of HPV origin, as well as an E6-associated protein (E6-AP) of 
cellular origin. 
Ubiquitination of the target regulatory protein via an in vitro 
ubiquitin-conjugating system, in the presence and absence of a candidate 
inhibitor, can be accomplished in any vessel suitable for containing the 
reactants. Examples include microtitre plates, test tubes, and 
micro-centrifuge tubes. In certain embodiments of the present assay, the 
in vitro assay system is generated to lack the ability to degrade the 
ubiquitinated target protein. In such an embodiments, a wide range of 
detection means can be practiced to score for the presence of the 
ubiquitinated protein. 
In one embodiment of the present assay, the products of a non-degradative 
ubiquitin-conjugating system are separated by gel electrophoresis, and the 
level of ubiquitinated target protein assessed, using standard 
electrophoresis protocols, by measuring an increase in molecular weight of 
the target protein that corresponds to the addition of one or more 
ubiquitin chains. For example, one or both of the target protein and 
ubiquitin can be labeled with a radioisotope such as .sup.35 S, .sup.14 C, 
or .sup.3 H, and the isotopically labeled protein bands quantified by 
autoradiographic techniques. Standardization of the assay samples can be 
accomplished, for instance, by adding known quantities of labeled proteins 
which are not themselves subject to ubiquitination or degradation under 
the conditions which the assay is performed. Similarly, other means of 
detecting electrophoretically separated proteins can be employed to 
quantify the level of ubiquitination of the regulatory protein, including 
immunoblot analysis using antibodies specific for either the regulatory 
protein or ubiquitin, or derivatives thereof. As described below, the 
antibody can be replaced with another molecule able to bind one of either 
the regulatory protein or ubiquitin. By way of illustration, one 
embodiment of the present assay comprises the use of biotinylated 
ubiquitin in the conjugating system. The biotin label is detected in a gel 
during a subsequent detection step by contacting the electrophoretic 
products (or a blot thereof) with a streptavidin-conjugated label, such as 
a streptavidin linked fluorochrome or enzyme, which can be readily 
detected by conventional techniques. Moreover, where a reconstituted 
protein mixture is used (rather than a lysate) as the conjugating system, 
it may be possible to simply detect the regulatory protein and ubiquitin 
conjugates in the gel by standard staining protocols, including coomassie 
blue and silver staining. 
In another embodiment, an immunoassay or similar binding assay, is used to 
detect and quantify the level of ubiquitinated regulatory protein produced 
in the ubiquitin-conjugating system. Many different immunoassay techniques 
are amenable for such use and can be employed to detect and quantitate the 
regulatory protein:Ub conjugates. For example, the wells of a microtitre 
plate (or other suitable solid phase) can be coated with an antibody which 
specifically binds one of either the regulatory protein or ubiquitin. 
After incubation of the ubiquitin-conjugated system with and without the 
candidate agent, the products are contacted with the matrix bound 
antibody, unbound material removed by washing, and ubiquitin conjugates of 
the regulatory protein specifically detected. To illustrate, if an 
antibody which binds the regulatory protein is used to sequester the 
protein on the matrix, then a detectable anti-ubiquitin antibody can be 
used to score for the presence of ubiquitinated regulatory protein on the 
matrix. 
However, it will be clear to those skilled in the art that the use of 
antibodies in these binding assays is merely illustrative of binding 
molecules in general, and that the antibodies are readily substituted in 
the assay with any suitable molecule that can specifically detect one of 
either the substrate protein or the ubiquitin. As described below, a 
biotin-derivative of ubiquitin can be used, and streptavidin (or avidin) 
employed to bind the biotinylated ubiquitin. In an illustrative 
embodiment, wells of a microtitre plate are coated with streptavidin and 
contacted with the developed ubiquitin-conjugating system under conditions 
wherein the biotinylated ubiquitin binds to and is sequestered in the 
wells. Unbound material is washed from the wells, and the level of 
regulatory protein (bound to the matrix via a conjugated ubiquitin moiety) 
is detected in each well. Alternatively, the microtitre plate wells can be 
coated with an antibody (or other binding molecule) which binds and 
sequesters the regulatory protein on the solid support, and detection of 
ubiquitinated conjugates of the matrix-bound regulatory protein are 
derivatively carried out using a detectable streptavidin derivative, such 
as an alkaline phosphatase/streptavidin complex. 
In similar fashion, epitope-tagged ubiquitin, such as myc-ub (see Ellison 
et al. (1991) J. Biol. Chem. 266:21150-21157; ubiquitin which includes a 
10-residue sequence encoding a protein of c-myc) can be used in 
conjunction with antibodies to the epitope tag. A major advantage of using 
such an epitope-tagged ubiquitin approach for detecting Ub:protein 
conjugates is the ability of an N-terminal tag sequences to inhibit 
ubiquitin-mediated proteolysis of the conjugated regulatory protein. 
Other ubiquitin derivatives include detectable labels which do not 
interfere greatly with the conjugation of ubiquitin to the regulatory 
protein. Such detectable lables can include fluorescently-labeled (e.g. 
FITC) or enzymatically-labeled ubiquitin fusion proteins. These 
derivatives can be produced by chemical cross-linking, or, where the label 
is a protein, by generation of a fusion protein. Several labeled ubiquitin 
derivatives are commercially available. 
Likewise, other binding molecules can be employed in place of the 
antibodies that bind the regulatory protein. For example, the regulatory 
protein can be generated as a glutathione-S-transferase (GST) fusion 
protein. As a practical matter, such GST fusion protein can enable easy 
purification of the regulatory protein in the preparation of components of 
the ubiquitin-conjugating system (see, for example, Current Protocols in 
Molecular Biology, eds. Ausubel et al. (NY: John Wiley & Sons, 1991); 
Smith et al. (1988) Gene 67:31; and Kaelin et al. (1992) Cell 70:351) 
Moreover, glutathione derivatized matrices (e.g. glutathione-sepharose or 
glutathione-coated microtitre plates) can be used to sequester free and 
ubiquitinated forms of the regulatory protein from the 
ubiguitin-conjugating system, and the level of ubiquitin immobilized can 
be measured as described. Likewise, where the matrix is generated to bind 
ubiquitin, the level of sequestered GST-regulatory protein can be detected 
using agents which bind to the GST moiety (such as anti-GST antibodies), 
or, alternatively, using agents which are enzymatically acted upon by GST 
to produce detectable products (e.g. 1-chloro-2,4-dinitrobenzene; Habig et 
al. (1974) J Biol Chem 249:7130). Similarly, other fusion proteins 
involving the regulatory protein and an enzymatic activity are 
contemplated by the present method. For example, fusion proteins 
containing .beta.-galactosidase or luciferase, to name but a few, can be 
employed as labels to determine the amount of regulatory protein 
sequestered on a matrix by virtue of a conjugated ubiquitin chain. 
Moreover, such enzymatic fusion proteins can be used to detect and 
quantitate ubiquitinated regulatory protein in a heterogeneous assay, that 
is one which does not require separation of the components of the 
conjugating system. For example, ubiquitin conjugating systems can be 
generated to have a ubiquitin-dependent protease which degrades the 
regulatory protein. The enzymatic activity of the fusion protein provides 
a detectable signal, in the presence of substrate, for measuring the level 
of the regulatory protein ubiquitination. Similarly, in a non-degradative 
conjugating system, ubiquitination of the regulatory protein portion of 
the fusion protein can allosterically influence the enzymatic activity 
associated with the fusion the protein and thereby provides a means for 
monitoring the level of ubiquitin conjugation. 
In binding assay-type detection steps set out above, the choice of which of 
either the regulatory protein or ubiquitin should be specifically 
sequestered on the matrix will depend on a number of factors, including 
the relative abundance of both components in the conjugating system. For 
instance, where the reaction conditions of the ubiquitin conjugating 
system provide ubiquitin at a concentration far in excess of the level of 
the regulatory protein, (e.g., one order of magnitude or greater) 
sequestering the ubiquitin and detecting the amount of regulatory protein 
bound with the ubiquitin can provide less dynamic range to the detection 
step of the present method than the converse embodiment of sequestering 
the regulatory protein and detecting ubiquitin conjugates from the total 
regulatory protein bound to the matrix. That is, where ubiquitin is 
provided in great excess relative to the regulatory protein, the 
percentage of ubiquitin conjugated regulatory protein in the total 
ubiquitin bound to the matrix can be small enough that any diminishment in 
ubiquitination caused by an inhibitor can be made difficult to detect by 
the fact that, for example, the statistical error of the system (e.g. the 
noise) can be a significant portion of the measured change in 
concentration of bound regulatory protein. Furthermore, it is clear that 
manipulating the reaction conditions and reactant concentrations in the 
ubiquitin-conjugating system can be carried out to provide, at the 
detection step, greater sensitivity by ensuring that a strong 
ubiquitinated protein signal exists in the absence of any inhibitor. 
Furthermore, drug screening assays can be generated which do not measure 
ubiquitination per se, but rather detect inhibitory agents on the basis of 
their ability to interfere with binding of one of the subject UBC proteins 
with any other immediate upstream or downstream component of the ubiquitin 
conjugation pathway. In an exemplary binding assay, the compound of 
interest is contacted with a mixture generated from an isolated and 
purified E2 protein, such as hUbCE or rapUBC, and another component of the 
ubiquitin conjugation pathway which binds to one of the UBC proteins (e.g. 
a "UBC-associated protein"), such as an E1 or E3 protein, or other 
cellular substrates of the subject UBC. Detection and quantification of E2 
complexes provides a means for determining the compound's efficacy at 
inhibiting (or potentiating) complex formation between the UBC-associated 
protein and the UBC protein. The efficacy of the compound can be assessed 
by generating dose response curves from data obtained using various 
concentrations of the test compound. Moreover, a control assay can also be 
performed to provide a baseline for comparison. In the control assay, 
isolated and purified UBC is added to a composition containing the 
UBC-associated protein, and the formation of UBC-containing complexes is 
quantitated in the absence of the test compound. 
Complex formation between the UBC protein and UBC-associated protein may be 
detected by a variety of techniques, many of which are effectively 
described above. For instance, modulation in the formation of complexes 
can be quantitated using, for example, detectably labelled proteins (e.g. 
radiolabelled, fluorescently labelled, or enzymatically labelled), by 
immunoassay, or by chromatographic detection. 
Typically, it will be desirable to immobilize either UBC or the 
UBC-associated protein to facilitate separation of complexes from 
uncomplexed forms of one of the proteins, as well as to accommodate 
automation of the assay. In an illustrative embodiment, a fusion protein 
can be provided which adds a domain that permits the protein to be bound 
to an insoluble matrix. For example, GST/UBC fusion proteins can be 
adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) 
or glutathione derivatized microtitre plates, which are then combined with 
the UBC-associated protein, e.g. an .sup.35 S-labeled UBC-associated 
protein, and the test compound and incubated under conditions conducive to 
complex formation. Following incubation, the beads are washed to remove 
any unbound UBC-associated protein, and the matrix bead-bound radiolabel 
determined directly (e.g. beads placed in scintilant), or in the 
supernatant after the UBC complexes are dissociated, e.g. when microtitre 
plaste is used. Alternatively, after washing away unbound protein, the 
complexes can be dissociated from the matrix, separated by SDS-PAGE gel, 
and the level of UBC-associated protein found in the matrix-bound fraction 
quantitated from the gel using standard electrophoretic techniques. 
In still further embodiments of the present assay, the 
ubiquitin-conjugating system is generated in whole cells, taking advantage 
of cell culture techniques to support the subject assay. For example, as 
described below, the ubiquitin-conjugating system (including the target 
protein and detection means) can be constituted in a eukaryotic cell 
culture system, including mammalian and yeast cells. Advantages to 
generating the subject assay in an intact cell include the ability to 
detect inhibitors which are functional in an environment more closely 
approximating that which therapeutic use of the inhibitor would require, 
including the ability of the agent to gain entry into the cell. 
Furthermore, certain of the in vivo embodiments of the assay, such as 
examples given below, are amenable to high through-put analysis of 
candidate agents. 
The components of the ubiquitin-conjugating system, including the 
regulatory protein, can be endogenous to the cell selected to support the 
assay. Alternatively, some or all of the components can be derived from 
exogenous sources. For instance, a virally derived E3 protein, such as an 
HPV E6 protein, can be introduced into the cell by recombinant techniques 
(such as through the use of an expression vector), as well as by 
microinjecting the E3 protein itself or mRNA encoding the E3 protein. 
In any case, the cell is ultimately manipulated after incubation with a 
candidate inhibitor in order to facilitate detection of ubiquitination or 
ubiquitin-mediated degradation of the regulatory protein. As described 
above for assays performed in reconstituted protein mixtures or lysate, 
the effectiveness of a candidate inhibitor can be assessed by measuring 
direct characteristics of the regulatory protein, such as shifts in 
molecular weight by electrophoretic means or detection in a binding assay. 
For these embodiments, the cell will typically be lysed at the end of 
incubation with the candidate agent, and the lysate manipulated in a 
detection step in much the same manner as might be the reconstituted 
protein mixture or lysate. 
Indirect measurement of ubiquitination of the target protein can also be 
accomplished by detecting a biological activity associated with the 
regulatory protein that is either attenuated by ubiquitin-conjugation or 
destroyed along with the regulatory protein by ubiquitin-dependent 
proteolytic processes. As set out above, the use of fusion proteins 
comprising the regulatory protein and an enzymatic activity are 
representative embodiments of the subject assay in which the detection 
means relies on indirect measurement of ubiquitination of the regulatory 
protein by quantitating an associated enzymatic activity. 
Where the regulatory protein has a relatively short half-life due to 
ubiquitin-dependent or independent degradation in the cell, preferred 
embodiments of the assay either do not require cell lysis, or, 
alternatively, generate a longer lived detection signal that is 
independent of the regulatory protein's fate after lysis of the cell. With 
respect to the latter embodiment, the detection means can comprise, for 
example, a reporter gene construct which includes a positive 
transcriptional regulatory element that binds and is responsive to the 
regulatory protein. For instance, where the regulatory protein of interest 
is p53, p53 responsive elements can be used to construct the reporter 
gene. These include p53 binding sequences set out in Example 7 and FIG. 9, 
as well as a creatine kinase enhancer, an interleukin-6 promoter, a c-fos 
promoter, a .beta.-actin promoter, an hsc70 promoter, a c-jun promoter, a 
p53 promoter, and a CYCl hybrid promoter containing a p53-binding 
sequence. The gene product is a detectable label, such as luciferase or 
.beta.-galactosidase, and is produced in the intact cell. The label can be 
measured in a subsequent lysate of the cell. However, the lysis step is 
preferably avoided, and providing a step of lysing the cell to measure the 
label will typically only be employed where detection of the label cannot 
be accomplished in whole cells. 
Moreover, in the whole cell embodiments of the subject assay, the reporter 
gene construct can provide, upon expression, a selectable marker. For 
instance, the product of the reporter gene can be an enzyme which confers 
resistance to antibiotic or other drug, or an enzyme which complements a 
deficiency in the host cell (i.e. thymidine kinase or dihydrofolate 
reductase). To illustrate, the aminoglcycoside phosphotransferase encoded 
by the bacterial transposon gene Tn5 neo can be placed under 
transcriptional control of a promoter element responsive to the level of 
target regulatory protein present in the cell. Thus, the level of 
expression of the phenotypic marker gene is lower in the absence of an 
inhibitor of ubiquitin-mediated proteolysis of the regulatory protein, and 
such inhibitors can be detected in the assay by an ability to confer the 
measured phenotypic trait. Such embodiments of the subject assay are 
particularly amenable to high through-put analysis in that proliferation 
of the cell can provide a simple measure of inhibition of the 
ubiquitin-mediated degradation of the regulatory protein. 
In yet a further embodiment of the subject assay, the ubiquitin-conjugating 
system comprises a cell in which the biological activity of the target 
regulatory protein has been substantially impaired, the impairment being 
the result of abnormal ubiquitination of the regulatory protein. The cell, 
in the presence or absence of a candidate inhibitor, is subject to growth 
conditions that would ordinarily required the function of the regulatory 
protein for viability of the cell. Thus, an inhibitor of the 
ubiquitin-mediated degradation of the regulatory protein would restore the 
biological activity of the protein to the cell, and could easily be 
detected by the ability of the cell to proliferate. To further illustrate, 
the impairment of the regulatory protein can be the result of over 
expression of a cellular protein of the ubiquitin pathway, such as an E2 
or E3 protein, which results in hyper-ubiquitination of the regulatory 
protein. Alternatively, the impairment can result from non-cellular 
agents, such as viral proteins, which increase the ubiquitin-mediated 
degradation of the regulatory protein. For example, as described above, 
expression of the HPV E6 protein can result in decreased levels of p53 in 
the cell due to the increased ubiquitin-dependent inactivation of the 
protein. 
In embodiments of the subject method in which the target regulatory protein 
ordinarily acts as a negative regulator of mitotic events, impairment of 
the regulatory protein can result in a hyper-mitotic cell. The term 
hyper-mitotic cell denotes a cell having an impaired cell-cycle checkpoint 
which can allow the cell to proceed abherently toward subsequent mitotic 
stages and ultimately inhibits faithful proliferation of the cell. In the 
present of an agent able to inhibit the ubiquitin-mediated inactivation of 
the regulatory protein, progression of the hyper-mitotic cell through the 
cell-cycle can be reestablished under control of the regulatory protein 
and permit the cell to appropriately proliferate. 
To illustrate, a p53-impaired cell can be generated by expression of the 
HPV viral protein E6. The concomitant decrease in p53 levels brought about 
by E6 expression does not in and of itself cause abherent mitotic events 
to occur. However, exposure of the impaired cell to an agent (i.e. 
chemical or environmental) that ordinarily induces cell-cycle arrest at 
the p53 checkpoint can result in inappropriate exit of the cell from the 
chemically or environmentally induced arrest. This type of checkpoint 
override can ultimately be lethal to the cell. Such arresting agents can 
include exposure to DNA damaging radiation or DNA damaging agents; 
inhibition of DNA synthesis or repairmen using DNA polymerase inhibitors 
such as hydroxyurea or aphidicolin; topoisomerase inhibitors such as 
4'-dimethylepipodophyllotoxin (VM-26); or agents which interfere with 
microtubule assembly, such as nocadazole and taxol. 
With respect to embodiments in which the regulatory protein ordinarily acts 
as a mitotic activator, impairment of the protein's activity by 
ubiquitination can generate a hypomitotic cell in which progression of the 
cell through at least a portion of the cell-cycle is repressed. In the 
presence of an inhibitor of ubiquitin-dependent degradation of the 
regulatory protein, the activity of the mitotic activator is restored and 
the cell can proliferate at an greater rate relative to the untreated 
cell. Agents to be tested for their ability to act as inhibitor of 
ubiquitin-dependent degradation of the regulatory protein in the present 
assay can be those produced by bacteria, yeast or other organisms, or 
those produced chemically. 
With respect to sources for the proteins constituting the 
ubiquitin-conjugating system, particularly to generate the reconstituted 
protein mixture, many species of the enzymes and other proteins involved 
in ubiquitination have been identified, and in a significant number of 
instances, have been cloned so that recombinant sources exist. Isolation 
of enzymes of the ubiquitin-conjugating system has been greatly assisted 
by "covalent" ubiquitin-affinity chromatography (Crechanover et al. (1982) 
J. Biol. Chem. 257:2537-2542; and Pickart et al. (1985) J. Biol. Chem. 
260:1573-1581). This method takes advantage of the fact that the E1 enzyme 
is capable of forming a thiol ester with immobilized ubiquitin (e.g. 
ubiquitin-sepharase) in the presence of ATP. As described in Example 2, 
such a protocol can be used to purify recombinantly expressed E1. 
Moreover, E1 enzymes bound to the immobilized ubiquitin can be exchanged 
with E2 enzymes. Thus, both E1 and E2 enzymes can be specifically purified 
on such columns, and can be recovered after elution with, for example, 
dithiothreitol. Under appropriate elution conditions, ubiquitin activated 
E1 or E2 complexes can be isolated and, as described herein, used in the 
present assay to increase the selectivity of the assay for an inhibitor of 
a particular step of ubiquitin-conjugation. Moreover, with minor changes, 
this protocol can be used to isolate E1 Ub or E2:Ub conjugates (e.g. 
activated ubiquitin conjugates) for use in the reconstituted protein 
mixture. 
Identification of enzymes involved in the ubiquitin pathway from different 
sources have facilitated the cloning of corresponding genes. For instance, 
genes encoding E1 enzymes have been cloned from various organisms (see, 
for example, Adams et al. (1992) Nature 355:632-634; Handley et al. (1991) 
PNAS 88:258-262; Handley et al. (1991) PNAS 88:7456; Hatfield et al. 
(1990) J. Biol. Chem. 265:15813-15817; Kay et al. (1991) Nature 
354:486-489; McCrath eg al. (1991) EMBO J 10:227-236; Mitchell et al. 
(1991) Nature 354:483-486; and Zacksenhaus et al. (1990) EMBO J 
9:2923-2929). The sequences of various cloned E1 enzymes predict proteins 
of roughly 100 kd, and which contain the nucleotide-binding consensus 
sequence Gly-Xaa-Gly-Xaa-Xaa-Gly (McGrath et al. (1991) EMBO J 
10:227-236). For example, the gene UBA1 has been cloned from S. cerevisiae 
and shown to encode a 114 kd E1 enzyme (McGrath et al., supra). Moreover, 
more than one E1 species has been detected in the same cell-type, 
suggesting that two or more different E1 enzymes can exist. It is not yet 
known whether the different E1 enzymes are enzymatically similar, or if 
they collaborate with specific sets of ubiquitin-conjugating enzymes. In 
either case, each of the E1 species can be used to generate the 
ubiquitin-conjugating system of the subject method. 
In contrast to the ubiquitin-activating enzyme (E1), where it is generally 
believed that there are relatively few different species of the enzyme in 
a given cell, eukaryotic cells can express a large and diverse array of E2 
enzymes. This remarkable variety of E2 enzymes, along with experimental 
evidence, has implicated the E2 enzyme as the principle determinant of 
substrate selectivity in the ubiquitin system. The E2 enzyme, as set out 
above, catalyzes isopeptide bond formation between ubiquitin and substrate 
proteins, either with or without the aid of a substrate recognition factor 
(ubiquitin-ligase protein; E3 ). Accordingly, in addition to the subject 
UBC proteins, e.g., UbCE and rapUBC, the subject assays can be performed 
with other E2 enzymes. For instance, several major species of E2 enzymes 
have been identified and purified by ubiquitin-affinity chromatography of 
extracts from rabbit reticulocytes (Pickart et al. (1985) J Biol Chem 
260:1573-1581), yeast (Jentsch et al. (1987) Nature 329:131-134), and 
wheat (Sullivan et al. (1989) PNAS 86:9861-9865). Furthermore, many genes 
encoding E2 enzymes have been cloned and characterized, most notably in 
the yeast Sacchromyces cerevisiae, where the phenotypic consequences of 
their inactivation can be readily assessed. More than 10 yeast E2 genes 
have been identified to date (see Jentsch (1992) Annu Rev Genet 
26:179-207; and Jentsch (1992) Trends Cell Biol 2:98-103), and there 
evidence for over 20 E2 genes in the plant Arabipodopsis (Cook et al. 
(1992) J Biol Chem 267:15116-15121). Additionally, E2 enzymes have been 
cloned from nematode (Zhen et al. (1993) Mol Cell Biol 13:1371-1377), 
drosophila (Muralidher et al. (1993) Neuron 11:253-266; and Koken et al. 
(1991) PNAS 88:3832-3836), bovine (Chen et al. (1991) J Biol Chem 
266:15698-15704) and human cells (Koken et al. (1992) Genomics 12:447-453; 
Koken et al. (1991) PNAS 88:8865-8869; and Schneider et al. (1990) EMBO J 
9:1431-1435). Other E2 enzymes can be substituted in the subject assays in 
place of the UbCE or rapUBC proteins of the present invention, or can be 
provided in addition to a UbCE or rapUBC protein, e.g., in a differential 
screening assay. 
Some ubiquitin-conjugating enzymes require accessory factors, E3 proteins, 
for the recognition of certain protein substrates. Two E3 proteins, 
E3.alpha. and E3.beta., have been identified from rabbit reticulocytes 
(Reiss et al. (1989) J. Biol. Chem. 264:10378-10383; and Reiss et al. 
(1990) J. Biol. Chem. 265:3685-3690). A yeast gene (UBR1) encoding an E3 
functionally similar to rabbit E3.alpha. has also been cloned (Bartel et 
al. (1990) EMBO J 9:3179-3189). Rabbit E3.alpha. and yeast UBR1 bind to 
substrates with N-terminal amino acid residues that are basic or have 
bulky hydrophobic side chains, while the E3.beta. recognizes small 
unchanged residues at the N-terminus of substrates. In addition to the E3 
proteins that recognize the N-terminus of protein substrates, other E3 
proteins (collectively know as E3.gamma., capable of recognizing 
internally located signals, have been suspected. 
Proteins that facilitate ubiquitin-conjugation reactions without physically 
interacting with E2 enzymes can also be classed as E3 proteins. By this 
definition, the E6 oncoprotein of the papillomavirus is regarded as an E3 
protein, as binding of E6 triggers the ubiquitination and degradation of 
p53. For example, recombinant E6 protein from the high-risk HPV-18 (SEQ ID 
No.14), as well as the cellular factor E6-AP (SEQ ID No.15), are available 
for use in the subject assay. 
The regulatory protein provided in the subject assay can be derived by 
purification from a cell in which it is exogenously expressed, or from a 
recombinant source of the protein. For example, cDNA clones are available 
for a number of regulatory proteins, including p53 (Oren et al. (1983) 
EMBO J 2:1633-1639); p27 (Polyak et al. (1994) Cell 78:59-66; and 
Toyoshima et al. (1994) Cell 78:67-74); c-myc (Hann et al. (1988) Cell 
52:185-195); N-myc (Curran et al. (1987) Oncogene 2:79-84); MAT.alpha.2 
(Hochstrasser et al. (1990) Cell 61:697-708); and E1A (Salvicek et al. 
(1988) EMBO J 7:3171-3180). 
Additionally, the subject ubiquitin conjugating enzyme can be used to 
generate an interaction trap assay for subsequently detecting inhibitors 
of hUbCE or rapUBC biological activity (see, for example, U.S. Pat. No: 
5,283,317; PCT publication WO94/10300; Zervos et al. (1993) Cell 
72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et 
al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 
8:1693-1696) In an illustrative embodiment, Saccharomyces cerevisiae YPB2 
cells are transformed simultaneously with a plasmid encoding a 
GAL4db-hUbCE fusion and with a plasmid encoding the GAL4ad domain fused to 
p53 or E6AP. Moreover, the strain is transformed such that the 
GAL4-responsive promoter drives expression of a phenotypic marker. For 
example, the ability to grow in the absence of histidine can depends on 
the expression of the HIS3 gene if it is under control of a 
GAL4-responsive promoter and, therefore, indicates that a functional GAL4 
activator has been reconstituted through the interaction of hUbCE and p53 
or E6AP. Thus, agent able to inhibit hUbCE interaction with one of these 
proteins will result in yeast cells unable to growth in the absence of 
histidine. Alternatively, the phenotypic marker can be one which provides 
a negative selection when expressed such that agents which disrupt the 
hUbCE interactions confer positive growth selection to the cells. 
In one embodiment of the invention, the target regulatory protein is the 
tumor suppressor p53, and any one of the above assays protocols is used to 
identify inhibitors of ubiquitin-mediated destruction of p53, such as by 
disrupting interaction of hUbCE or rapUBC with p53, or interactions 
between hUbCE or rapUBC and other proteins of the ubiquitin system such as 
E6 or E6AP, or alternatively, by mechanistically inhibiting the enzymatic 
activity of the enzyme. Many lines of evidence point to the importance of 
p53 in human carcinogenesis. For instance, mutations within the p53 gene 
are the most frequent genetic aberration thus far associated with human 
cancer. Although p53 can block the progression of the cell cycle when 
artificially expressed at high levels, it appears to be dispensable for 
normal development. Thus, for mice containing homozygous deletions and 
humans harboring germline mutations of p53, development is normal and p53 
protein is expressed at very low levels in most cell types. Emerging 
evidence, however, suggests that p53 is a checkpoint protein that plays an 
important role in sensing DNA damage or regulating cellular response to 
stress. Under normal conditions, p53 is an unstable protein and is present 
at very low levels in the cell, and the level of p53 in a cell appears to 
be controlled at least in party by degradation involving the ubiquitin 
system and, based on data presented herein, is likely to be mediated by 
the subject hUbCE or rapUBC. Treating cells with UV light or X rays 
dramatically reduces the rate of p53 degradation, leading to a rapid 
increase in its concentration in the cell and presumably inducing the 
transcription of genes that block passage through the restriction point. 
However, while normal cell lines irradiated in G.sub.1 fail to enter S 
phase, many tumor lines do not. In fact, there is a perfect correlation 
between cell lines that lack this feedback control and cells that have 
mutations in the p53 gene. These mutations are of two sorts: recessive 
mutations that inactivate the gene, and dominant mutations that produce 
abnormal proteins. An inhibitor developed using the subject hUbCE or 
rapUBC in a ubiquitin-conjugating assay or by rational drug design could 
subsequently be used therapeutically to enhance the function of the p53 
checkpoint by increasing the steady state concentration of p53 in the 
treated cell. Given that elevated levels of wild-type p53 protein can lead 
to apoptosis in a variety of transformed cell types (Yonish-Rouach et al. 
(1991) Nature 352:345-347; Shaw et al. PNAS 89:4495-4499; and Caelles et 
al. (1994) Nature 370:220-223), inhibitors of hUbCE or rapUBC-mediated 
degradation of p53 may be attractive therapeutic agents not only in 
cervical cancer, but also other cancer types, by increasing the fortitude 
of the checkpoint in transformed cells which contain wild-type p53, or by 
offsetting a diminishment in p53 activity by increasing the level of 
(mutant) p53. Moreover, such agents can also be used prophylactically in 
normal cells to increase p53 levels and thereby enhance the protection 
against DNA damaging agents when it is known that exposure to damaging 
agents, such as radiation, is imminent. 
Moreover, the oncogenic activity of certain viruses, such as the simian 
virus 40 (SV40), the adenovirus type 5 (Ad5), and the high human papilloma 
virus types 16 and 18 (HPV16 and HPV18), has been correlated with the 
virus' ability to interact with and inactivate the cellular p53 protein. 
In the instance of the high-risk papilloma viruses, the association of the 
viral oncoprotein E6 with p53 leads to the specific ubiquitination and 
degradation of p53. This has suggested a model in which E6 immortalizes 
cells by deregulating cell growth control through the elimination of the 
p53 tumor suppressor protein. This models accounts for the observations 
that p53 levels are very low in HPV-immortalized cells and that the 
half-life of p53 in HPV16-immortalized keratinocytes is shorter than in 
primary keratinocytes. Thus, the present invention can be employed in the 
identification of an agent that can block the ubiquitin dependent 
degradation of p53 as mediated by E6, and thereby block proliferation of 
HPV-transformed cells. 
The subject human ubiquitin conjugating enzyme is likely to be involved in 
altering the activity of other cellular proteins, particularly proteins 
which seem to have short half-lives, and the present invention 
contemplates the use of hUbCE or rapUBC inhibitors, including antagonistic 
forms of the hUbCE or rapUBC protein, to inhibit the ubiquitination of 
other cellular proteins by hUbCE or rapUBC. For example, in another 
embodiment, the regulatory protein ubiquitinated by hUbCE or rapUBC is the 
myc oncoprotein. The myc regulatory protein is activated by translocation 
or mutation in many B-cell lymphomas or by amplification in tumor types, 
such as small cell lung cancer and breast cancer. The c-myc gene is the 
cellular homolog of the viral oncogene v-myc, which is found in a number 
of avian and feline retroviruses which induce leukemia and carcinomas. Myc 
has been implicated in the control of normal cell proliferation by many 
studies. In particular, it is one of the immediate early growth response 
genes that are rapidly induced in quiescent cells upon mitogenic 
induction, suggesting that it plays some role in mediating the transition 
from quiescence to proliferation. However, increased levels of myc itself 
is not sufficient to cause proliferation. In fact, in normal cells the 
opposite happens and the cell undergoes apoptosis. Therefore, inhibitors 
identified in the present assay can be used to effectively induce 
apoptosis in cells which do not normally overexpress myc. For example, 
specific delivery of these agents to lymphocytes can be used to inhibit 
proliferation of B- and/or T-cells in order to induce clonal deletion and 
generate tolerance to particular antigens. 
In tumor cells, on the other hand, elevated or deregulated expression of 
c-myc is so widespread as to suggest a critical role for myc gene 
activation in multi-stage carcinomas (Field et all. (1990) Anticancer Res 
10:1-22; and Spencer et al. (1991) Adv Cancer Res 56:1-48). However, such 
overexpression of myc in these cells is typically believed to be 
accompanied by expression of other cellular proteins, such as bcl-2. 
Interestingly, however, almost all tumor cells tested that overexpress myc 
readily undergo apoptosis in the presence of cytotoxic and 
growth-inhibitory drugs (Cotter et al. (1990) Anticancer Res 10:1153-1159; 
and Lennon et al. (1990) Biochem Soc Trans 18:343-345). Therefore, 
inhibitors of the ubiquitin-mediated degradation of myc can be used to 
further deregulate the expression of myc in order to render the cells even 
more sensitive to a chemotherapeutic treatment, or to possibly upset the 
careful balance of the transformed cell and cause apoptosis to occur 
evenin the absence of a second cytotoxic drug. 
The regulation of cyclin by ubiquitination is yet another therapeutic 
target which may implicate hUbCE or rapUBC inhibitors. Cyclin degradation 
is a key step governing exit from mitosis and progression into the next 
cell-cycle. For example, the transition from metaphase to anaphase which 
marks the end of mitosis in induced by the degradation of cyclin by a 
ubiquitin-mediated pathway, which in turn leads to the inactivation of 
cyclin-dependent kinases (cdk) operational at that cycle-cycle stage. As 
cells enter interphase, cyclin degradation ceases, cyclin accumulates and, 
as a result of a complex series of post-translational modifications, 
cyclin/cdk complexes are activated as kinases which drive the cell through 
mitosis. Cyclin degradation is thus one of the crucial events in exiting 
mitosis. Indeed, cyclin mutants that retain the ability to activate the 
cdk complexes, but which cannot be degraded, arrest the cell-cycle in 
mitosis. Similar cyclin-dependence exists at other points of the 
cell-cycle as well. Thus, inhibitors of ubiquitin-mediated degradation of 
a cyclin (such as where the cyclin is chosen from cyclin A, B, C, D1, D2, 
D3, E or F) can be used as antiproliterative agents. 
Yet another candidate substrate of for E2 enzymes is the cyclin-dependent 
kinase inhibitor p27.sup.kip1 (Polyak et al. (1994) Cell 78:59-66; and 
Toyoshima et al. (1994) Cell 78:67-74). This protein has been implicated 
in G.sub.1 phase arrest, such as mediated by TGF-.beta. and cell-cell 
contact. As described in the appended examples, we have found that 
ubiquitin conjugating enzymes are able to ubiquitinate p27, indicating 
that cellular turnover of that protein is dependent at least in part on 
ubiquitin-mediated destruction. Consequently, inhibition of ubiquitin 
transfer to p27 may result in accumulation of this cell-cycle inhibitor. 
An agent which inhibits the E2-mediated degradation of p27 would therefore 
be a cytostatic agent. 
Such cytostatic agents would be useful for inhibiting proliferation of both 
normal and transformed cells. For example, an inhibitor of E2-mediated 
ubquitination of p27 could be used to prevent proliferation of 
lymphocytes, much the same as rapamycin and the like, and could be used as 
an immunosuppressant. Likewise, accumulation of p27 in fibroblasts could 
be used as part of a therapy for the treatment of a connective tissue 
disorder, or for controlling would healing processes. 
P27 modulating agents may also be used for the treatment of hyperplastic 
epidermal conditions, such as psoriasis, as well as for the treatment of 
neoplastic epidermal conditions such as those characterized by a high 
proliferization rate for various skin cancers, as for example basal cell 
carcinoma and squamous cell carcinoma. 
Normal cell proliferation is generally marked by responsiveness to negative 
autocrine or paracrine growth regulators, such as members of the 
TGF-.beta. family, e.g. TGF-.beta.1, TGF-.beta.2 or TGF-.beta.3, and 
related polypeptide growth inhibitors, e.g. activins, inhibins, Muillerian 
inhibiting substance, decapentaplegic, bone morphogenic factors, and vg1 
(e.g. terminal differentiation inducers). Ordinarily, control of cellular 
proliferation by such growth regulators, particularly in epithelial and 
hemopoietic cells, is in the form of growth inhibition with p27 
accumulation being associated with at least TGF-.beta. response. This is 
generally accompanied by differentiation of the cell to a post-mitotic 
phenotype. However, it has been observed that a significant percentage of 
human cancers derived from these cells types display a reduced 
responsiveness to growth regulators such as TGF-.beta.. For instance, some 
tumors of colorectal, liver epithelial, and epidermal origin show reduced 
sensitivity and resistance to the growth-inhibitory effects of TGF-.beta. 
as compared to their normal counterparts. Treatment of such tumors with 
antagonists of ubiquitination of p27 provides an opportunity to restore 
the function of a TGF-.beta. mediated checkpoint. 
The subject E2 inhibitors can also be used in the treatment of 
hyperproliferative vascular disorders, e.g. smooth muscle hyperplasia 
(such as atherosclerosis) or restenosis, as well as other disorders 
characterized by fibrosis, e.g. rheumatoid arthritis, insulin dependent 
diabetes mellitus, glomerulonephritis, cirrhosis, and scleroderma, 
particularly proliferative disorders in which loss of TGF-.beta. autocrine 
or paracrine signaling is implicated. For example, restinosis continues to 
limit the efficacy of coronary angioplasty despite various mechanical and 
pharmaceutical interventions that have been employed. An important 
mechanism involved in normal control of intimal proliferation of smooth 
muscle cells appears to be the induction of autocrine and paracrine 
TGF-.beta. inhibitory loops in the smooth muscle cells (Scott-Burden et 
al. (1994) Tex Heart Inst J 21:91-97; Graiger et al. (1993) Cardiovasc Res 
27:2238-2247; and Grainger et al. (1993) Biochem J 294:109-112). Loss of 
sensitivity to TGF-.beta., or alternatively, the overriding of this 
inhibitory stimulus such as by PDGF autostimulation, can be a contributory 
factor to abnormal smooth muscle proliferation in restinosis. It may 
therefore be possible to treat or prevent restenosis by the use of agents 
which inhibit ubiquitination of p27, thereby causing its accumulation. 
Yet a further possible substrate of the subject hUbCE or rapUBC is the fos 
oncogene product, which can undergo ubiquitin-mediated degradation in a 
cell and has been implicated in neoplastic transformation as well as in 
mediating the action of a variety of extracellular stimuli. The control of 
gene expression by c-fos is believed to play a critical role in cellular 
proliferation and developmental responses, and alterations in the normal 
pattern of c-fos can lead to oncogenesis. Given the prominence of c-fos as 
an early response gene, apparent over-expression and prolonged lifetime of 
c-fos, as may be caused by an inhibitor of the ubiquitin-mediated 
degradation of c-fos, might sufficiently unbalance the cell-cycle and 
cause cell death. Alternatively, such inhibitors can be used to mimic the 
effects of an external stimulus on the cell, such as treatment with a 
cytokine. 
Exemplification 
The invention now being generally described, it will be more readily 
understood by reference to the following examples which are included 
merely for purposes of illustration of certain aspects and embodiments of 
the present invention, and are not intended to limit the invention. 
We have defined the biochemical roles of hUbCE and E6AP in the E6 
stimulated ubiquitination of p53 in vitro and have shown that inhibition 
of these enzymes in vivo can lead to an inhibition of E6-stimulated p53 
degradation. As described in the examples below, inhibition of hUbCE and 
E6AP enzyme function in vivo causes an inhibition of E6-stimulated p53 
degradation. The level of inhibition achieved in the micro-injection 
experiments in Example 8 was 25-30%. This may be a consequence of not 
every injected cell achieving high level expression of the injected 
construct, a phenomenon we have noted before in many different systems. It 
may also suggest that there is some redundancy in the cellular ubiquitin 
conjugation machinery, or that the intracellular concentrations of E1, 
hUbCE and E6AP are not rate-limiting for p53 degradation in the cell line 
used. All of our data suggest that E6 is absolutely required for 
ubiquitination of p53 in our in vitro and in vivo assay systems. We are 
currently investigating the possibility that hUbCE and E6AP are involved 
in the normal turnover of p53, with the possible involvement of an, as 
yet, unidentified cellular E6 homolog. 
EXAMPLE 1 
Cloning and Expression of a Novel Human Ubiquitin-conjugating Enzyme 
The cDNA encoding the human ubiquitin-conjugating enzyme of the present 
invention was cloned from HeLa cells (ATCC CCL2). Briefly, polyadenylated 
RNA was isolated from cultured HeLa cells and first strand cDNA was 
prepared following standard protocols (c.f, Chomczynski U.S. Pat. No. 
4,843,155; and Sambrook et al. Molecular Cloning: A Laboratory Manual, 
CSHL Press, Cold Spring Harbor, N.Y. (1989)). Using the nested PCR primer 
sets 5'-(GC).sub.3 AAGCTTTAYGARGGWGGWGTYTTYTT-3' (SEQ ID No. 8), 
5'-(GC).sub.3 GAATTCACNGCRTAYTTYTTNGTCCCAYTC-3' (SEQ ID No. 9) and 
5'-(GC).sub.3 AAGCTTCCNGTNGGNG-AYTTRTTYCAYTGGCA-3' (SEQ ID No. 10), 
5-(GC).sub.3 G-AATTCATNGTNARNGCNGGCGACCA-3' (SEQ ID No. 11), which also 
provided convenient restriction sites in the PCR products, the coding 
sequences for the hUbCE gene was amplified from the HeLa cDNA library, and 
a HindIII-EcoRI fragment therefrom was subsequently ligated into a 
pBluescript II KS+ phagemid (pKS+ Stratagene catalog no. 212207) for 
further manipulation. The resulting pKS-hUbCE construct was amplified in 
XL1-Blue Cells (Strategene Catalog no. 260268), and double stranded 
construct purified. The nucleic acid sequence determined for the hUbCE 
clone is represented in SEQ ID NO. 1, and the corresponding deduced amino 
acid sequence is provided in SEQ ID No. 2. 
The hUbCE gene was subsequently sub-cloned from pKS+ into other expression 
vectors to generate gene constructs for producing the recombinant hUbCE 
protein in either bacterial or insect cells. In some instances, the 
recombinant hUbCE was provided with exogenous sequences to produce fusion 
proteins, where the additional sequences of the fusion protein facilitate 
its purification. For example, after further amplification, the pKS-E2 
construct was cut with XhoI and EcoRI, and the fragment containing the 
hUbCE coding sequence sub-cloned into a pGEX vector (Pharmacia catalog no. 
PGEX-4T) previously digested with SalI and EcoRI. The resulting pGEX-hUbCE 
construct encoded a glutathione-S-transferase (GST)/hUbCE fusion (Smith et 
al. (1988) Gene 67:31-40). The pGEX construct was introduced into E. coli 
by transformation, and the transformants grown in liquid media (LB) in the 
presence of IPTG. Purification of GST/hUbCE fusion protein was by standard 
protocols (Current Protocols in Molecular Biology, eds. Ausubel et al. 
(NY:John Wiley & Sons, 1991); Pharmacia instruction booklet (for catalog 
no. 27-4570)) using a glutathione-sepharose column (Pharmacia catalog no. 
27-4570). Treatment with thrombin removed the GST domain from the fusion 
protein. 
Alternatively, the hUbCE coding sequence was excised from the pKS-hUbCE 
construct as a HindIII-EcoRI fragment and ligated into pVL1393 cut with 
Sma I and Eco I. Briefly, the hUbCE gene fragment was purified by agarose 
gel separation, and ligated into the baculorvirus vector pVL1393 
(Invitrogen catalog no. V1392-20) previously cut with Sma I and Bgl II. 
The pVL1393-hUbCE construct was then used to transfect spodoptera 
frugiperda (Sf9 cells, ATCC CRL 1711), and the cells maintained in insect 
cell culture media (Grace's Antheraea medium) supplemented with 10% FBS, 
lactal bumin hydrolysate, TC yeastolate and glutamate (Invitrogen catalog 
no. B823) following standard protocols (Invitrogen product guide; Summers 
and Smith (1987); Texas Agricultural Experiment Station Bulletin No. 1555, 
College Station, Tex.; Luckow et al. (1988) Bio/technology 6:47-55; and 
Miller et al., in Genetic Engineering, Vol. 8 ed. Setlow and Hollaender 
(Plenum Press: New York) pages 277-298). Transfected cells are grown until 
cells begin to lose their adherence to the culture plate surface, at which 
time the cells are harvested, collected by centrifugation, and lysed. The 
lysate is clarified by centrifugation to remove the cell wall debris, and 
the hUbCE can be purified from the lysate. 
For instance, the hUbCE protein was isolated on an E1:ubiquitin charged 
column. Isolation of enzymes of the ubiquitin-conjugating system has been 
greatly assisted by "covalent" ubiquitin-affinity chromatography 
(Crechanover et al. (1982) J. Biol. Chem. 257:2537-2542; and Pickart et 
al. (1985) J. Biol. Chem. 260:1573-1581). This method takes advantage of 
the fact that the E1 enzyme is capable of forming a thiol ester with 
immobilized ubiquitin (e.g. ubiquitin-Sepharose) in the presence of ATP. 
Moreover, E1 enzymes bound to the immobilized ubiquitin can be exchanged 
with the subject hUbCE protein. Thus, both E1 and the subject hUbCE 
protein can be specifically purified on such columns, and can be recovered 
after elution with, for example, dithiothreitol. Moreover, with minor 
changes, this protocol can be used to isolate hUbCE:Ub conjugates (e.g. 
activated ubiquitin conjugates) for use in therapeutic target assays. 
As described in U.S. patent application Ser. No. 08/176,937, the an 
E1-containing lysate was applied to a sepharose-ubiquitin column (Hershko 
et al. (1983) J. Biol. Chem. 257:2537-2542) in the presence of ATP (e.g. 5 
mM ATP, 10 mM MgCl.sub.2, and 0.2 mM dithiothreitol, 50 mM Tris-HCl (pH 
7.2)). The column was washed several times with this buffer. A clarified 
lysate of the hUbCE-producing insect cells, adjusted to 50 mM Tris-HCl (pH 
7.2), 5 mM ATP, 10 mM MgCl.sub.2, and 0.2 mM dithiothreitol, was then 
applied to the Ub:E1 column, washed, then eluted to remove any remaining 
ub:E1 (e.g. hUbCE will be exchanged for E1 on the column). The subject 
hUbCE protein was then eluted from the column by washing with 50 mM 
Tris-HCl (pH 9.0) containing 2 mM dithiothreitol. 
In another exemplary embodiment, the recombinant hUbCE protein is generated 
as a poly(His) fusion protein for purification on a Ni.sup.2+ metal 
column. An XhoI to EcoRI fragment of the pKS construct is cloned into the 
pBlueBac A baculovirus (Intvitrogen catalog no. V360-20) previously 
digested with XhoI and EcoRI. Following the manufacturer's protocols, the 
His.sub.6 -hUbCE fusion protein is then expressed in Sf9 insect cells, and 
purified on a Ni.sup.2+ charged sepharose resin (Invitrogen catalog no. 
R801; see also Hochuli et al. (1987) J. Chromatography 411:177-184; and 
Janknecht et al. (1991) PNAS 88:8972-8976). Following purification of the 
fusion protein, the His.sub.6 tag can be removed by treatment with 
entrokinase. 
EXAMPLE 2 
Isolation of components of an in vitro ubiquitin conjugating system 
Ubiquitin was obtained from commercial sources, and the remaining protein 
components of the reconstituted protein system were cloned from HeLa cells 
(ATCC CCL2). Briefly, polyadenylated RNA was isolated from cultured HeLa 
cells and first strand cDNA was prepared following standard protocols 
(c.f., Chomczynski U.S. Pat. No. 4,843,155; and Sambrook et al. Molecular 
Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y. 
(1989)). PCR primers, designed to amplify DNA sequences encoding each of 
the component proteins, as well as provide convenient restriction sites to 
the PCR products, were used to isolate coding sequences for a human E1, 
human p53, HPV-18 E6, human E6-AP, and various human E2's, which were 
subsequently ligated into a pBluescript II KS+ phagemid (pKS+ Stratagene 
catalog no. 212207) for further manipulation. As described below, each of 
the component proteins genes were subsequently sub-cloned from pKS+ into 
other expression vectors to generate gene constructs for producing the 
recombinant proteins in either bacterial or insect cells. In some 
instances, the recombinant proteins have been provided with exogenous 
sequences to produce fusion proteins, where the additional sequences of 
the fusion protein facilitate its purification. 
i) Human E1 
Utilizing the primers 5'-(GC).sub.3 AAGCTTATGTCCAGCTCGCCGCTGTCCAAG-3' and 
5'-(GC).sub.3 GGATCCTCAGCGGATGGTGTATCGGACATA-3'. The coding sequence for a 
human E1 (SEQ ID No. 14) was amplified from a HeLa cell cDNA library. The 
PCR amplification product containing the E1 coding sequences was purified 
and cut with Hind III and Bam HI (restriction sites provided by the PCR 
primers), and ligated into the pKS+ phagemid. The resulting pKS-E1 
construct was amplified in XL1-Blue Cells (Strategen catalog no. 260268), 
and double stranded construct purified. 
A Hind III/fill to BamHI fragments containing the E1 coding sequence was 
isolated from the pKS-E1 construct, where "Hind III/fill" indicates that a 
Hind III overhand generated in the fragment has been filled to form a 
blunt-end using Klenow and dNTPs. The E1 gene fragment was purified by 
agarose gel separation, and ligated into the baculorvirus vector pVL1393 
(Invitrogen catalog no. V1392-20) previously cut with Sma I and Bgl II. 
The pVL1393-E1 construct was used to transfect spodoptera frugiperda (Sf9) 
cells) (ATCC CRL 1711), and the cells maintained in insect cell culture 
media (Grace's Antheraea medium) supplemented with 10% FBS, lactal bumin 
hydrolysate, TC yeastolate and glutamate (Invitrogen catalog no. B823) 
following standard protocols (Invitrogen product guide; Summers and Smith 
(1987); Texas Agricultural Experiment Station Bulletin No. 1555, College 
Station, Tex.; Luckow et al. (1988) Bio/technology 6:47-55; and Miller et 
al., in Genetic Engineering, Vol. 8 (Setlow and Hollaender, eds) pp. 
277-298, Plenum, N.Y.). Transfected cells are grown until cells begin to 
lose their adherence to the culture plate surface, at which time the cells 
are harvested, collected by centrifugation, and lysed. The lysate is 
clarified by centrifugation to remove the cell wall debris, and the E1 
containing lysate is applied to a sepharose-ubiquitin column (Hershko et 
al. (1983) J. Biol. Chem. 257:2537-2542) in the presence of ATP (e.g. 5 m 
MATP, 10 mM MgCl.sub.2, and 0.2 mM clithiothreitol, 50 mM Tris-HCl (pH 
7.2)). The column is washed several times with this buffer, and the E1 
protein eluted with the following solutions: 1M KCl containing 50 mM 
Tris-HCl, pH7.2 (KCl eluate); the above Tris buffer, to remove salt; and 
finally 2 mM ATP and 0.04 mM sodium pyrophosphate in the above Tri buffer. 
The E1-containing eluate can be concentrated, as well as placed in new 
buffer solution, by centrifuge ultrafiltration with CentriPrep or 
Centricon membranes (Amicon Corp., Mass.). Alternatively, the 
ubiquitin-immobilized E1 can be used, as described below, in the 
purification of E2 enzymes. 
ii) Human E2 
A human rad6 homolog (SEG ID No. 15) was amplified from the HeLa cel cDNA 
using the primers 5'-(GC).sub.3 AAGCTTATGTCGACCCCGGCCCGGAGGAGG-3' and 
5'-(GC).sub.3 GAATTCTTATGAATCATTCCAGCTTTGTTC-3' and cloned into 
pBluescript II pKS+ as a Hind III-EcoRI fragment. After further 
amplification, the pKS-E2 construct was cut with XhoI and NotI, and the 
fragment containing E2 coding sequence sub-cloned into a pGEX vector 
(Pharmacia catalog no. PGEX-4T-3) previously digested with SalI and NotI. 
The resulting pGEX-E2 construct encoded a glutathione-S-transferase 
(GST)/E2 fusion (Smith et al. (1988) Gene 67:31-40). The pGEX construct 
was introduced into E. coli by transformation, and the transformants grown 
in liquid media (LB) in the presence of IPTG. Purification of GST/E2 
fusion protein was by standard protocols (Current Protocols in Molecular 
Biology, eds. Ausubel et al. (NY:John Wiley & Sons, 1991); Pharmacia 
instruction booklet (for catalog no. 27-4570)) using a 
glutathione-sepharose column (Pharmacia catalog no. 27-4570). Treatment 
with thrombin removed the GST domain from the fusion protein. 
Alternatively, the rad6 coding sequence was excised from the pKS-rad6 
construct as a HindIII-EcoRI fragment and ligated into pVL1393 cut with 
Sma I and Eco I. The E2 protein is produced in Sf9 cells, as described 
above, and purified on a sepharose-uibiquitin:E1 column. As above, a 
clarified lysate of the E2-producing insect cells, adjusted to 50 mM 
Tris-HCl (pH 7.2), 5 mM ATP, 10 mM MgCl.sub.2, and 0.2 mM dithiothreitol, 
is applied to the ub:E1 column, washed, then eluted to remove any 
remaining ub:E1 (e.g. E2 will be exchanged for E1 on the column). Rad6 is 
then eluted from the column by washing with 50 mM Tris-HCl (pH 9.0) 
containing 2 mM dithiothreitol. 
In similar fashion, recombinant forms of human UBC3 /CDC34 (SEQ ID No. 19) 
were produced. 
iii) HPV-18 E6 
The coding-sequence for HPV-18 E6 (SEQ. ID No. 16) was amplified from the 
HeLa cell cDNA library using the primers 5'-(GC).sub.3 
AAGCTTATGGCGCGCTTTGAGGATCCAACA-3' and 5'-(GC).sub.3 
GAATTCTTATACTTGTGTTTCTCTGCGTCG-3', the PCR products purified, and the 
amplified E6 sequences digested with Hind III and EcoRI and ligated into a 
pBlueScript II pKS+ phagemid. Several different expression vectors were 
generated by subcloning the E6 sequences from the pKS-E6 construct. For 
example, a Hind III to EcoRI fragment containing E6 coding sequences was 
ligated into pVL1393 cut with SmaI and EcoRI to produce baculovirus 
expression system as described above. 
Alternatively, E6 has been generated as His.sub.6 fusion protein for 
purification on a Ni.sup.2+ metal column. An XhoI to EcoRI fragment of 
the pKS construct was cloned into the pBlueBac A baculovirus (intvitrogen 
catalog no. V360-20) previously digested with XhoI and EcoRI. Following 
the manufacturer's protocols, the His.sub.6 -E6 fusion protein was 
expressed in Sf9 insect cells, and purified on a Ni.sup.2+ charged 
sepharose resin (Invitrogen catalog no. R801; sell also Hochuli et al. 
(1987) J. Chromatography 411:177-184; and Janknecht et al. (1991) PNAS 
88:8972-8976). Following purification of the fusion protein, the His.sub.6 
tag can be removed by treatment with entrokinase. 
iv) Human E6-AP 
E6-AP (SEQ ID No. 17) was cloned from the HeLa cell cDNA library using the 
PCR primers 5'-(GC).sub.3 AAGCTTTCAGGACCTCAGTCTGACGAC-3' and 5'(GC).sub.3 
GGATCCTTACAGCATGCCAAATCCTTTGGC-3', wherein the amplified E6-AP sequences 
were digested with Hind III and Bam HI and ligated into pBluescript II 
pkst. Constructs for expressing both HIS.sub.6 tagged and GST tagged 
versions of E6-AP were generated. In one instance, an NheI to BamHI E6-AP 
containing fragment was cloned into pBlueBacA (cut with NheI and BamHI), 
and the construct expressed in insect cells. As above, the His-tagged 
E6-AP protein was purified by Ni.sup.+2 affinity, and the his-tag 
subsequently removed by treatment with enterokinase. 
Alternatively, a HindIII (fill) to NotI fragment has been isolated from the 
pKS-E6AP construct and subsequently ligated into the SmaI-Not I sites of 
pGEX-4T-3, to produce a GST fusion protein in E. coli which was purified 
using a gluathione-sepharose resin. 
v) Human p53 
Human p53 (SEQ ID No. 18) was cloned into pBluescript II pKS+ from the HeLa 
cell cDNA library using the primers 5' (GC).sub.3 
GAATTCGCCATGGAGGAGCCGCAGTCAGATCCT-3' and 5'-(GC).sub.3 
AAGCTT-TCAGTCTGAGTCAGGCCCTTCTGT-3'. In similar fashion to the other 
component proteins above, several different expression constructs were 
generated for p53, some of which included extra polypeptide sequence to 
facilitate purification. For expression in insect cells, two baculoviral 
constructs were made. For native p53, a BamHI fragment of the pKS-p53 
vector was ligated into BamHI digested pVL1393. For His.sub.6 -tagged p53, 
the BamHI fragment was ligated into pBlueBacA previously cut with BamHI. 
Likewise, a GST-p53 was generated in E. coli by expression of a pGEX 
construct made by ligating a p53-containing EcoRI to NotI fragment of the 
pKS-p53 construct into pGEX-4T-1. 
In the instance of each of the two fusion proteins, standard protocols were 
used to purify p53 from lysed transformants. For the native p53 produced 
by the pVL1393-p53 construct, the method of Hupp et al. was used to purify 
the p53 on a heparin-sepharose column (Hupp et al. (192) Cell 71:875-886). 
vi) Ubiquitin 
Ubiquitin is available from commercial sources (Bovine ubiquitin, Sigma 
catalog no. 6253; yeast ubiquitin, Sigma catalog no. 2129). Various 
modified forms of ubiquitin are also available as for example, 
fluorescein-labeled ubiquitin (Sigma catalog no. U5504), and 
horseradish-peroxidase labeled ubiquitin (Sigma catalog no. U9879). 
Biotinylated ubiquitin can be prepared from biotin-NHS 
(N-hydroxy-succinimide ester) using well-known techniques (biotinylation 
kit; Pierce catalog no. 214206, 203188 (6 atom spacer), or 203114 (14 atom 
spacer)). 
vii) Additional Reagents 
For generating certain of the detection means as described herein, some of 
the following reagents can be employed: polyclonal sera to ubiquitin 
(Sigma catalog no. U5379); labeled antibodies to biotin (Sigma catalog 
nos. A4541 (peroxidase conjugated) and F6762 (FITC conjugated)); labeled 
avidin (Sigma catalog nos. A7294, E2636 (peroxidase conjugated) and A2050, 
E2761 (FITC conjugated)); streptavidin (Sigma catalog no. S3762 (FITC 
conjugated) and S5512 (peroxidase conjugated)); Streptavidin-coated beads 
(Sigma catalog no. 400996; Pierce catalog no. 20347G); Streptavidin-coated 
96 well microtrite plates (Pierce catalog no. 15124); Maleic 
anhydride-activated polystyrene 96 well plates (Pierce catalog no. 15110); 
and antibody to human p53 (PharMingen catalog Nos. 14091A and 14211A). 
EXAMPLE 3 
In Vitro Ubiquitination of p53 
We describe the cloning of a new human ubiquitin-conjugating enzyme hUbCE 
in Example 1. In Examples 4 and 5, we show that hUbCE specifically 
ubiquitinylates E6AP and is involved in the turnover of p53 in vivo. We 
have defined several discrete biochemical steps in the activation and 
transfer of ubiquitin onto p53. These biochemical reactions provide two 
levels of specificity in the ubiquitination of p53; the hUbCE dependent 
ubiquitination of E6AP, and the E6-dependent transfer of ubiquitin from 
ubiquitinylated E6AP to p53. 
Proteins 
To perform an in vitro ubiquitination reaction, native hUbCE and UBC2, the 
human homolog of the S.cerevisiae DNA repair gene, Rad6 (Koken et al. 
(1991) PNAS 88:8865-8869) were expressed and purified from E.coli BL21(DE3 
). Both proteins are readily soluble and easily purified using standard 
procedures. The cloning and purification of each of the proteins hUbCE, 
UBC2, p53, human E1, E6, and E6AP are described in Example 2 above. 
Briefly, native p53 was expressed from the baculoviral vector pVL1392 in 
Sf9 insect cells according to the manufacturer's instructions (Pharmingen) 
and purified on a p53 affinity column. HPV18 E6 was expressed E. coli BL21 
as a GST fusion protein and purified on GSH-sepharose. Human E1 was cloned 
by PCR from the published cDNA sequence (Handley et al. (1991) PNAS 
88:258-262), and native protein was expressed and purified from 
baculoviral infected cells. E6AP was expressed in E.coli JM109 as a GST 
fusion protein and purified on GSH-sepharose. 
Ubiguitination reactions 
Ubiquitination reactions contained 50-200 ng of the indicated proteins in 
50 mM Tris pH 7.5, 5 mM MgCl.sub.2, 2 mM ATP-.gamma.-S, 0.1 mM DTT and 5 
.mu.M ubiquitin. Total reactions (30 .mu.l) were incubated at 25.degree. 
C. for 3 hrs and then loaded on an 8% SDS gel for analysis of p53 
ubiquitination or a 4-20% gradient gel for analysis of ubiquitination of 
the ubiquitin-conjugating enzymes and E6AP. The gels were run and proteins 
were electrophoretically transferred to nitrocellulose. p53 proteins were 
revealed with the monoclonal antibody DO-1 (Oncogene Science) and the ECL 
system from NEN. Ubiquitinylated proteins were visualized using 
Extravidin-HRP from Sigma and the ECL system from NEN. 
As demonstrated in FIG. 2, the appearance of specific p53-ubiquitin 
conjugates requires hUbCE, HPV18-E6, E6AP, ubiquitin and E1, the ubiquitin 
activating enzyme. In contrast, UBC2 was active in a minimal conjugation 
reaction containing E1, ATP and ubiquitin, in that E1 could activate 
ubiquitin and transfer it onto UBC2. However, UBC2 could not substitute 
for hUbCE in the p53 conjugation reaction (FIG. 2, lane 3). In addition, 
we made an active site cysteine-to-serine mutation in hUbCE. Such active 
site E2 mutants should accept activated ubiquitin from E1 but should not 
ubiquitinylate their downstream substrates owing to the high stability of 
the esther linkage formed between the active site serine and the 
carboxy-terminus of ubiquitin. This mutant was inactive in the p53 
conjugation reaction (FIG. 6, lane 7). These results demonstrate that a 
catalytically active hUbCE is absolutely required for generation of 
ubiquitinylated p53 in this in vitro system. 
In FIG. 3A we show that ubiquitinated E1 could transfer ubiquitin 
efficiently to hUbCE but not directly to E6AP and that ubiquitinated hUbCE 
transferred ubiquitin to E6AP in a reaction that was not further 
stimulated by E6. All of these ubiquitination reactions required the 
presence of the ubiquitin-activating enzyme, E1, and ubiquitin. 
To address the issue of the specificity of hUbCE-mediated ubiquitination of 
E6AP we performed ubiquitination reactions with purified recombinant 
hUbCE, GST-UBC2, GST-UBC8 (Kaiser et al. (1994) J. Biol. Chem. 
269:8797-8802) and a GST-fusion of the so-called epidermal ubiquitin 
conjugating enzyme (Liu et al. (1992) J Biol Chem 267:15829-15835). Each 
of these recombinant proteins could accept activated ubiquitin from E1, 
but only hUbCE could donate ubiquitin to E6AP (FIG. 3B). We also confirmed 
that native UBC2 could accept ubiquitin from E1 but could not donate 
ubiquitin to E6AP (data not shown). 
We then purified the ubiquitinated E6AP by affinity chromatography on 
glutathione-Sepharose and demonstrated that it did not contain appreciable 
amounts of ubiquitinated E1, ubiquitinylated hUbCE or free ubiquitin. We 
found that this purified, ubiquitinated E6AP could donate ubiquitin to p53 
in an E6-dependent reaction. 
EXAMPLE 4 
Radiolabel-Detection Assay 
.sup.35 S-labeled p53, prepared by cell culture technique utilizing .sup.35 
S-methionine, is incubated with combined purified components of a 
ubiquitin conjugating system, including biotinylated ubiquitin. The 
reaction is conducted in a 96 well microtitre plate and stopped with 
iodoacetate. The reaction mixture is transferred to the wells of a 
streptavidin-coated microtitre plate and incubated to capture the complex 
of biotinylated ubiquitin and p53 (free biotinylated ubiquitin will also 
compete for binding sites on the well). The wells are washed with buffer 
(e.g. phosphate-buffered saline, or conjugation buffer lacking ubiquitin 
and ATP) to remove uncomplexed p53. Ubiquinated p53 is detected by 
addition of scintillant to the well and counting in a scintillation 
instrument. Inhibition of the ubiquitin conjugation system by an added 
candidate agent is indicated by a reduced radioactive count 
EXAMPLE 5 
Immunodetection Assay 
p53 is incubated with combined purified components of a ubiquitin 
conjugating system as described above, including biotinylated ubiquitin. 
The reaction is conducted in a 96 well microtitre plate and stopped with 
iodoacetate. The reaction mixture is transferred to the wells of a 
streptavidin coated microtitre plate and incubated to capture the complex 
of biotinylated ubiquitin and p53 (free biotinylated ubiquitin will also 
compete for binding sites on the well). The wells are washed with buffer 
to remove uncomplexed p53. Next, the ub:p53 complexes capatured on the 
plate are decorated with a murine monoclonal antibody to p53. The wells 
are washed and binding of monoclonal antibody is detected by addition of 
peroxidase-conjugated antibody to mouse IgG (H+L) (Pierce catalog nos. 
91430G and 91450G) and contacting with an appropriate substrate system, 
such as o-phenylenediamine dihydrochloride (Sigma catalog no. P9187). 
EXAMPLE 6 
GST Detection Assay 
The GST-p53 fusion product is incubated with combined purified components 
of a ubiquitin conjugating system, including biotinylated ubiquitin. The 
reaction is conducted in a 96 well microtitre plate and stopped with 
iodoacetate. The reaction mixture is transferred to the wells of a 
streptavidin coated microtitre plate and incubated to capture the complex 
of biotinylated ubiquitin and GST-p53 (free biotinylated ubiquitin will 
also compete for binding sites on the well). The wells are washed with 
buffer to remove uncomplexed GST-p53. Binding of ubiquitinated GST-p53 is 
monitored with a detection system, based either on a biochemical assay for 
GST (e.g., 1-chloro-2,4-dinitrobenzene, Pharmacia catalog no. 27-4590-01) 
or an immunological assay using goat anti-GST antibody (Pharmacia catalog 
no. 27-4590-01). 
EXAMPLE 7 
Reporter Construct Detection Assay 
The plasmid pTKluc, shown in FIG. 5, comprises a luciferase gene whose 
expression is driven by the core Herpes simplex virus thymidine-kinase 
(TK) promoter which has been modified with either p53 (p53RE/TK), myc 
(mycRE/TK), or Sp1 (Sp1 RE/TK) binding sites. When the construct lacking 
any of the modifications to the TK promoter is transfected into mammalian 
cells, the detectable luciferase activity is low because this core TK 
promoter fragment does not contain the upstream activating sequences 
necessary for efficient transcriptional activation of the luciferase gene. 
However transfection with the constructs in which TK is further modified 
to contain either 3 or 6 response-elements (RE) for one of p53, myc or 
Sp1, the detectable luciferase activity increases in cells which express 
the appropriate protein. For example, the level of luciferase expression 
is significantly higher in p53-producing cells (e.g. ML1 cells) 
transfected with the p53 RETK-containing construct than with the TK 
construct. Likewise, endogenous myc and Sp1 proteins can drive expression 
of the mycRE/TK and Sp1RE/TK constructs. As set out above, both p53 and 
myc can be degraded by the ubiquitin pathway. However, Sp1 is not known to 
be degraded by any ubiquitin-mediated pathway, and the SP1RE/TK construct 
can therefore be used as a control in the present assays. Thus, in the 
presence of an agent which inhibits ubiquitin-mediated degradation of p53 
in a cell harboring the p53 RE/TK construct, the level of luciferase 
activity would increase relative to that in the cell not treated with the 
candidate agent. 
To construct the luciferase reporter constructs shown in FIG. 5, the 
pGL2-Basic vector (Promega catalog no. E1641) was modified by addition, in 
the multiple cloning region, of a SalI to BamHI fragment containing the TK 
promoter sequence with either 3 or 6 tandemly arranged binding sites 
placed upstream of the TK promoter. Prior to addition of the RE/TK 
promoter sequences, a SalI restriction site at 2744 of pGL2-Basic was 
destroyed by oligonucleotide site-directed mutagenesis. The resulting 
constructs, designated p53RE/TK, mycRE/TK, and Sp1RE/TK, were each 
subsequently used to transfect mammalian cells following the 
manufacturer's suggests (Technical notes, Part #TM003 of Promega Catalog 
no. E164). 
In an alternative embodiment, a SalI to BamHI fragment of p53/RE/TK 
containing the luciferase reporter gene was isolated and sub-cloned into 
another eukaryotic expression vector pcDNAIII (Invitrogen, San Diego, 
Calif.) previously digested with BglII and XhoI. 
The vector p53RE/TK is transfected into the human chronic leukemia cell 
line MLI that expresses wild-type p53. In this in vivo situation, 
luciferase expression is upregulated by the presence of p53, which 
functions as a transcriptional activating factor by binding to the p53 
response element upstream of the TK promoter. The ubiquitin conjugating 
system participates in the degradation of p53 and, when functional, down 
regulates the expression of luciferase in this system. Measurement of 
luciferase activity are carried out by standard protocols (see, for 
example, Promega Technical Bulletin #TB161). Cells are grown and 
transfected in a tissue culture grade 96 well microtitre plate. The 
cultured cells are incubated in the presence and absence of a candidate 
agent, then harvested and centrifuged. The harvested cells are then lysed 
with lysis buffer. The lysates clarified by centrifugation, and the 
supernatants transferred to luminescent grade microtitre plates. 
Luciferase assay sustrate (Beetle luciferin, Promega catalog no. E1603) is 
added, and the reaction in each well monitored in a luminometer or 
scintillation counter. Inhibition of the ubiquitin conjugating system 
results in a greater luminescence signal than the uninhibited system. 
Although an in vivo assay, this screen will ignore general cytotoxic 
compounds. 
EXAMPLE 8 
Microinjection of Sense and Anti-Sense Constructs of the hUbCE Gene 
To investige the consequences of interfering with hUbCE and E6AP function 
in p53 degradation, we performed microinjection experiments using sense 
and anti-sense constructs of the hUbCE gene. To facilitate the detection 
of p53 by indirect immunofluorescence, the experiments were performed in 
the human tumor cell line MDA-MB-468 which contain high levels of mutant 
p53 (Arg273His). In this line, the degradation of p53 could be stimulated 
by microinjection of an HPV-18 E6 expression plasmid. 
In order to determine whether hUbCE and E6AP mediate the E6-dependent 
ubiquitination and degradation of p53 in vivo co-injection experiments 
were performed. To briefly describe the experiments, the CMV expression 
vectors were obtained by inserting the entire open-reading frame of one of 
HPV-18 E6, human E1, human E6-AP, hUbCE, or a Cys-85 mutant of hUbCE, in 
either a sense or anti-sense orientation (as indicated in FIG. 4) in the 
pX-plasmid (Baldin et al. (1993) Genes & Devel., 7:812-821). Plasmids were 
purified with a Promega Wizard Maxi-prep kit and injected at a 
concentration of 50 to 100 .mu.g/.mu.l in the presence of normal 
affinity-purified rabbit or mouse antibody (5 mg/ml in PBS) used as 
microinjection marker. 
Cell monolayers of asynchronous MDA-MB-468 cells were injected with the 
indicated DNAs (FIG. 4) along with rabbit IgG to allow identification of 
injected cells with an automated microinjection system (AIS, Zeiss; 
Ansorge et al. (1988) J. Biochem. Biophys. Meth., 16:283-292). All 
microinjection experiments were carried out in 3.5 cm Petri dishes 
containing 3 ml of DMEM medium carbonate free, in order to avoid the 
decrease in pH of the medium during the injection. Each cell was injected 
at a pressure between 50 and 150 hPa. After 24 hrs the cells were fixed 
and stained with a p53 specific monoclonal antibody (DO-1; Oncogene 
Sciences) followed by a biotinylated horse anti-rabbit antibody and Texas 
red conjugated streptavidin. Injected cells were identified by staining 
with an FITC conjugated goat anti-rabbit antibody (Baldin et al. (1993) 
Genes and Dev 7:812-821). 
When either an anti-sense or mutant hUbCE expression plasmid or an 
expression plasmid encoding anti-sense E6AP was co-injected with the E6 
expression plasmid, the E6 stimulated degradation of p53 was inhibited 
(FIG. 4). Similar results were obtained when polyclonal antibodies 
generated against human hUbCE or an expression plasmid encoding a mutant 
form of E6AP were microinjected (not shown). 
Co-injection of an E6 expression plasmid with an expression plasmid 
encoding anti-sense E1 also inhibited the E6 stimulated degradation of 
p53. Co-injection of anti-sense or mutant UBC2 expression plasmids had a 
negligible effect on the E6 stimulated degradation of p53 (FIG. 4). 
Moreover, the data that an hUbCE mutant, Cys-85.fwdarw.Ser, which produces 
an inactive form of the enzyme, is possibly a dominant negative mutant 
able to at least partially rescue p53. 
EXAMPLE 9 
Cloning of Yeast UbCE Genes 
In order to clone homologs of the hUbCE gene, degenerate oligonucleotides 
based on the conserved regions PVGDDLFHWH/Q and ITLAPSW (see SEQ ID No. 1) 
were designed and used to amplify S. pombe genomic DNA and cDNA in 
.lambda.ZAP (strain h+.sup.N his3-) and C. albicans genomic and cDNA in 
.lambda.ZAP (strain 3153A). The amplification consisted of 30 cycles of 
94.degree. C. for 1 minute, 55.degree. C. for 1 minute and 72.degree. C. 
for 1 minute. The PCR reactions were separated on a 2.5% low melting 
agarose gel, that identified a 250 bp fragment for both genomic and 
complementary DNA from C. albicans. From S. pombe 250 and 650 bp fragments 
were detected for complementary and genomic DNA respectively. The size 
discrepancy between complementary DNA and genomic S. pombe DNA fragments 
probably reflects the presence of an intron. The fragments of 250 bps were 
eluted and cloned into pCRII (TA cloning system, Invitrogen corporation). 
The S. pombe and C. albicans DNA probes were .sup.32 P labeled by nick 
translation and used on Southern blots to confirm the species identity of 
the fragments and to screen S. pombe and C. albicans cDNA libraries. 
Sequencing of the full length cDNAs confirmed the identity of the clones. 
The C. albicans and S. pombe UbCE open-reading frames are both 147 aa 
residues long (SEQ ID Nos: 3 and 5, respectively). The newly isolated 
genes are named caUbCE and spUbCE for C. albicans and S. pombe 
respectively. 
EXAMPLE 10 
Cloning of the Human rapUBC Enzyme 
Utilizing a two hybrid assay comprising an FKBP12-bait protein, a 
drug-dependent interaction trap assay was used to screen a WI38 (mixed 
G.sub.0 and dividing fibroblast) cDNA library (Clonetech, Palo Alto 
Calif.) in pGADGH (XhoI insert, Clonetech). Briefly, the two hybrid assay 
was carried out in an HF7C yeast cell (Clonetech) in which FKB1 gene was 
disrupted. Of the clones isolated, a novel human ubiquitin-conjugating 
enzyme (rap-UBC, SEQ ID Nos. 12 and 13) has been identified. The original 
clones contained 5' end of the gene which included substantial portion of 
the coding region for rapUBC, including the active site cysteine and the 
3' end of the gene. In order to obtain full length sequence of the rapUBC 
gene, the 5' end was cloned using a library vector (MTXP37) and oligos 
corresponding to sequences near the 5' end of the original cDNA clone 
SMR4-15. The oligos used were: VB1040: CTACTAATAGGTAGAAGCGGTGG (SEQ ID 
No:20) and VB1041: GGTAAACCAAAGCCCGACAGGG (SEQ ID No:21). PCR products 
were obtained from a cDNA library made from normal human fibroblasts 
(dividing WI38 cells). 
EXAMPLE 11 
Ubiquitination of p27 by UBC3 
The protein p27 is a potent inhibitor of cyclin-dependent kinases and its 
overexpression in mammalian cells causes a G1 arrest. In serum deprived 
cells p27 accumulates without an increase in mRNA or protein synthesis 
indicating that the regulation of its abundance occurs at the level of 
protein stability. We demonstrate here that p27 is degraded in vivo and in 
vitro through the ubiquitin-proteasome pathway. In human MG-63 cells, 
inhibition of the proteasome using the peptide-aldehyde, LLnL, induced an 
accumulation of p27 protein, but not p21, a distinct cdk inhibitor. 
Because of lack of proteasome activity, accumulation of ubiquitinated 
forms of p27 was observed. We also found that p27 was ubiquitinated and 
degraded in an ATP dependent manner in a rabbit reticulocyte lysate (RRL) 
system. Inhibition or depletion of the proteasome blocked p27 degradation 
in vitro. Addition of purified human Ubc2 or Ubc3 enzymes, but not of four 
other different human Ubcs to RRL, induced an increase in p27 turnover. 
Consistent with these results, inhibition of Ubc2 or Ubc3, using inactive 
mutant proteins, specifically slowed the kinetics of p27 proteolysis. 
These results represent the first demonstration that the 
ubiquitin-proteasome pathway plays a role in the regulation of a cell 
cycle protein in human cells, namely the cdk inhibitor p27. This specific 
proteolysis of p27 may, therefore, represent a novel mechanism for 
regulating cyclin dependent kinases. 
Immunoreagents 
Anti-p27 polyclonal antibody (F-L) was generated against mouse bacterial 
expressed purified p27 -his6. Characterization of this antibody is 
reported elsewhere (S.W.T. and M.P., manuscript in preparation). The 
monoclonal antibody to p27 was from Transduction Laboratories (#K25020) 
and the C-T polyclonal antiserum generated against a synthetic peptide (19 
amino acids) carboxyl terminus was from Santa Cruz Biotechnology 
(#sc-528). The preparation and characterization of the rabbit polyclonal 
antiserum against human p21 and of 4F3 monoclonal antibody to ubiquitin 
have been described previously in the art. 
Cell culture and cell synchronization 
The human osteosarcoma cell line MG-63 was obtained from the American Type 
Culture Collection (ATCC) and cultured in Dulbecco's-Modified Eagle's 
Medium (DMEM). Cells were synchronized in G1. In brief, cells were 
incubated 2-3 days in DMEM containing 0.2% fetal calf serum (FCS). After 
these periods of incubation, more than 95% of the cells presented a 2N DNA 
content. Cell cycle phases were monitored by flow cytometry (FACSCAN, 
Becton Dickinson) and by BrdU incorporation (see Immunofluorescence 
paragraph). 
Electroporation 
Cells were electroporated. In brief, cell monolayers growing on glass 
coverslips (at ca. 60% density) were trypsinized and about 
3.times.10.sup.6 cells were incubated at 37.degree. C. in suspension with 
1 ml DMEM supplemented with 10% FCS and LLnL or E64 peptide at a 
concentration of 50 .mu.M. After one hour, cells were washed twice with 
PBS, resuspended in 100 .mu.l of cold PBS containing 1 .mu.g/ml of either 
peptide and left on ice for 10 minutes. After this time, cells were 
transferred to a precooled cuvette (Bio-Rad, 0.4 cm electrode distance) 
and electroporated using a Bio-Rad GenePulser electroporator (200v,125 
.mu.F, infinite resistance). After electroporation, cells were incubated 
again on ice for five minutes and then immediately reincubated at 
37.degree. C. in prewarmed DMEM supplemented with 10% FCS and LLnL or E64 
peptide for 1-2 additional hours. 
Extract preparation, Immunoblotting and Immunoprecipitation 
Cell extracts were prepared as previously described in the literature, with 
the following modifications. Three to 5 volumes of lysis buffer (50 mM 
Tris-HCl, pH 7.4, 0.25 M NaCl, 1% Triton-X100, 0.1% SDS, 0.5% 
deoxycholate, 1 mM EDTA, 50 mM NaF, 0.1 mM Na.sub.3 VO.sub.4) were added 
to pelletted cells. The following protease inhibitors were added: 0.1 mM 
phenyl-methyl sulfonyl fluoride (PMSF), 1 .mu.g/ml of leupeptin, 10 
.mu.g/ml of soybean trypsin inhibitor, 10 .mu.g/ml of L-1 
Chlor-3-(4-tosylamido)-4 Phenyl-2-butanon (TPCK), 10 .mu.g/ml of L-1 
Chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK), 1 .mu.g/ml 
of aprotinin, 10 mM N-ethylmaleimide (NEM). After incubation on ice for 30 
minutes, the samples were centrifuged at 14,000 rpm in an Eppendorf 
microfage for 5 minutes at 4.degree. C. to recover the supernatant. 
Proteins were transferred from gel to a nitrocellulose membrane (Novex) by 
wet blotting. Filters were subjected to immunoblotting using the ECL (NEN) 
detection system according to the manufacturer's instructions. For 
immunodetection with anti-Ubiquitin antibody, in order to completely 
denature the ubiquitin-containing proteins, filters were immersed in 
distilled water and autoclaved using a sterilization program for 15 
minutes before processing. Immunoprecipitation were performed as described 
in [Tam, 1994 #2163]. 
Northern blot analysis 
RNA was isolated from exponentially growing cells using standard 
Chomczynski extraction methods. Northern analysis was performed. Briefly, 
total RNA was separated by 1% agarose denaturing gel electrophoresis, 
transferred to Nytran-Plus nylon membranes, and hybridized with probes 
which were radiolabeled with [.alpha..sup.32 P]dCTP (Amersham, Inc.) using 
a random primer DNA labeling kit (Boehringer, Inc.) and used for 
hybridization at 1.times.10.sup.6 cpm/ml. Washing conditions were done to 
a final stringency of 0.1.times.SSC, 0.1% SDS, at 65.degree. C. 
Immunofluorescence 
Indirect immunofluorescence was performed as generally described in the 
art, with the following modifications. Cell monolayers growing on glass 
coverslips were rinsed in PBS and fixed for 10 minutes in 4% formaldehyde 
(Sigma, HT50-1-1). Fixed cells were permeabilized with 0.25% Triton X-100 
and processed for cell staining. Incubation with primary antibodies 
(anti-p27 monoclonal antibody; 10 .mu.g/ml) was carried out for one hour 
in a humidified chamber. After three washes in PBS the coverslips were 
incubated for 30 minutes with biotinylated horse anti-mouse secondary 
antibody (Vector Laboratories, dilution 1:50). Cells were washed again 
three times with PBS and incubated with Texas red-conjugated streptavidin 
(Vector Laboratories, dilution 1:100) or FITC-conjugated streptavidin 
(Vector Laboratories, dilution 1:50). All reactions were carried out at 
room-temperature and antibody dilutions were made in DMEM containing 10% 
FCS. Counterstaining for DNA was performed by adding 1 .mu.g/ml 
bisbenzimide (Hoechst 33258) into the final PBS wash. Immunofluorescence 
samples were directly mounted in Crystal/mount medium (Biomeda Corp.). 
Photographs were taken using a Plan-Neofluar 100.times. or a Plan-Neofluar 
40.times. lens mounted on a Zeiss Axiophot Photomicroscope and a Color 
Video Printer Mavigraph, on Sony UPC-3010 print paper. 
Protein expression and purification 
p27-his6, Ubc3-his6 and mutant Ubc3-his6 were bacterially expressed and 
purified by Ni-NTA-agarose (Invitrogen) according to the manufacturer's 
instructions. Native Ubc2 and native hUbCE (bacterially expressed) and E1 
proteins (from baculovirus infected cells) were purified as described 
above. GST-Ubc2 mutant, GST-hUbCE mutant, GST-Ubc8, GST-rapUBC and 
GST-Ubcepi were bacterially expressed and purified by GH-Sepharose 
(Pharmacia) according to the manufacturer's instructions. 
In vitro Ubiquitination and degradation of p27 
p27-his.sub.6 was incubated at 37.degree. C. for different times in 30 
.mu.l of degradation mix [final concentration 33% (v/v) untreated 
reticulocyte lysate (Promega), 50 mM Tris-HCl (pH 8.3), 5 mM MgCl.sub.2, 5 
mM CaCl.sub.2, 2 mM DTT], in presence or in absence of 3.5 mM 
ATP-.gamma.-S and in presence or in absence of purified bacterially 
expressed Ubc enzymes. All purified protein were used in the degradation 
mix at .about.1 .mu.M concentration. The reaction was stopped by addition 
of Laemmli sample buffer followed by immediate freezing of the samples in 
liquid nitrogen. Ubiquitinated p27 and p27 degradation was analyzed by 
electrophoresis and immunoblot with monoclonal anti-p27 antibody. In some 
cases, ubiquitinated p27 was detected by adding 5 .mu.M biotinylated 
ubiquitin to the degradation mix. After the reaction was stopped, p27 was 
purified by either Ni-NTA-agarose or F-L anti-p27 antibody, 
electrophoresed, transferred on a nitrocellulose membrane (Novex) and 
visualized using HRP-conjugated streptavidin (Extravidin, Sigma) and the 
ECL (NEN) detection system according to the manufacturer's instructions. 
In some experiments, to remove the proteasome, the RRL was 
ultracentrifuged at 100,000 g for six hours in the presence of 5 mM 
MgCl.sub.2. In others, ATP was depleted from RRL by treatment (20 minutes 
at 30.degree. C.) with apyrase [5 units/ml in 50 mM Tris-HCl (pH 8.0), 4 
mM CaCl.sub.2, 0.05% BSA)]. 
Ubiquitination in purified in vitro system 
Purified protein were incubated at 37.degree. C. for 30 minutes in 30 .mu.l 
of ubiquitination mix [50 mM Tris-HCl (pH 8.3), 5 mM MgCl.sub.2, 5 mM 
CaCl.sub.2, 1 mM DTT, 2 mM ATP-.gamma.-S, 5 .mu.M biotinylated ubiquitin]. 
All purified protein were used in the ubiquitination mix at .about.1 .mu.M 
concentration. The reaction was stopped by addition of Laemmli sample 
buffer followed by immediate freezing of the samples in liquid nitrogen. 
Samples were electrophoresed, transferred on a nitrocellulose membrane 
(Novex) and visualized using either HRP-conjugated streptavidin 
(Extravidin, Sigma) or anti-p27 monoclonal antibody and the ECL (NEN) 
detection system according to the manufacturer's instructions. 
p27 protein accumulation in serum starved human osteosarcoma cells, without 
increases in mRNA and protein synthesis 
We analyzed the levels of p27 and p21 protein and mRNA in serum deprived 
human MG-63 cells. We found that 48-72 hours after serum starvation, when 
95% of the cells showed a 2n DNA content by flow cytometry, the p27 
protein level was strongly induced compared to proliferating cells. In 
contrast, the p21 levels remained constant. Interestingly, the mRNA levels 
for both p27 and p21 were found to be comparable in asynchronously growing 
cells and in G1 cells. Similar results were obtained with normal human 
fibroblasts. We also analyzed whether the cellular abundance of p27 and 
p21 varied after serum readdition. Arrested cells were stimulated to 
reenter the cell cycle and monitored for a period of 18 hours. Cells 
started to synthesize DNA, as monitored by flow cytometry, approximately 
12 hours after serum addition. By 18 hours about 80% of the cells had 
incorporated 5-bromo deoxyuridine (BrdU) (data not shown) and show a DNA 
content higher than 2N. At different time points cell lysates were 
analyzed by immunoblotting with antibodies to p27 and p21. The overall 
abundance of p27 protein gradually decreased after serum readdition, and 
by 18 hours reached a level similar to that found in asynchronous cells. 
In contrast, the p21 level initially increased after serum stimulation, 
then decreased by 9 hours, and by 18 hours reached a level comparable to 
that present in asynchronous cells. 
The increase in p27 abundance in quiescent cells could be regulated by 
either an increase in the rate of protein synthesis or by a decrease in 
the rate of protein turnover, or by both. Metabolic labeling revealed that 
the rate of p27 synthesis was similar in both proliferating and serum 
deprived cells. These results suggest that levels of p27 protein are 
regulated at the level of protein turnover. 
Accumulation of p27 and ubiquitinated forms of p27, but not of p21, upon 
proteasome inhibition in vivo 
The above results prompted us to test whether the intracellular regulation 
of p27 abundance involved the ubiquitin-proteasome pathway. We examined 
the effect of a peptide-aldehyde, LLnL 
(N-acetyl-leucinyl-leucinyl-norleucinal-H), a potent inhibitor of the 
chymotryptic site on the proteasome, on p27 levels. As a control, we used 
the cysteine protease inhibitor E64 (L-transepoxysuccinic acid) or vehicle 
(dimethyl sulfoxyde, DMSO) alone. MG-63 cells were treated for the various 
times with these compounds, then collected by trypsinization, washed with 
PBS and lysed as described in above. Cell lysates were electrophoresed, 
transferred to a nitrocellulose membrane and immunoblotted with either an 
anti-p27 monoclonal antibody or an anti-p27 carboxy terminus antiserum 
(C-T) or with a polyclonal antibody to p21. Addition of LLnL, but not of 
E64 or DMSO, induced an accumulation of p27 protein after 60 minutes of 
treatment. In contrast, p21 was not found to accumulate in LLnL treated 
cells. Interestingly, at later points, we noticed that the two anti-p27 
antibodies, which do not recognize the same epitope, both recognized a 
doublet of approximately Mr 70,000. The monoclonal antibody also 
recognized an approximately Mr 100,000 band in the extract from the 
24-hour LLnL time point. We reasoned that, since the proteasome is 
essential for the degradation of proteins covalently conjugated to 
ubiquitin, presumably these slower migrating bands represented 
ubiquitinated forms of p27 that accumulated in LLnL treated cells. To 
determine whether these bands contained ubiquitinated p27, lysates from 
cells treated for 24 hours with LLnL were subjected to immunoprecipitation 
with either an anti-p27 full-length antiserum (F-L) or with normal rabbit 
immunoglobulin and then immunoblotted with a monoclonal antibody to 
ubiquitin. The 70K doublet and a group of bands migrating as a high 
molecular weight smear were detected by anti-ubiquitin antibody 
exclusively in the anti-p27 immunoprecipitates. Immunoblot with a control 
antibody of similar immunoprecipitates did not visualize any band. When 
LLnL was introduced into cells by electroporation, the accumulation of p27 
and the ubiquitinated forms of p27 was evident after two hours treatment 
(one hour before and one hour after electroporation). In contrast, 
introduction of E64 or DMSO alone by electroporation did not lead to an 
increase in p27 abundance. Again, p21 levels were unchanged by 
electroporation of either LLnL or E64. 
We also used indirect immunofluorescence to analyze the subcellular 
localization of p27 after LLnL treatment. p27 was detectable in the 
nucleus of about 50-60% of serum starved cells. Twelve hours after serum 
readdition, nuclear p27 staining was reduced to less than 2%. E64 or DMSO 
did not affect the serum-stimulated p27 turnover. In contrast, after 6 
hours or 12 hours in the presence of serum and LLnL, a bright nuclear p27 
staining was detected in approximately 78% of the cells. 
These results show that inhibition of the proteasome in intact cells leads 
to p27 accumulation and to the appearance of ubiquitinated forms of p27. 
p27 is ubiquitinated and degraded in rabbit reticulocyte lysate 
We tested whether purified bacterial expressed hexahistidine-tagged p27 
(p27 -his6) was an in vitro substrate for ubiquitination in a rabbit 
reticulocyte lysate (RRL) system, an established source of ubiquitinating 
enzymes and proteasome complexes. Incubation of p.sup.27 -his6 with RRL 
for one minute, produced a ladder of bands higher than 27,000 as 
visualized by immunoblotting with a monoclonal antibody anti-p27. The 
ladder of bands was not detected if RRL was omitted from the reaction. 
Furthermore, no bands were recognized by the anti-p27 antibody when only 
RRL was present in the reaction, thus demonstrating that the bands 
recognized by the anti-p27 antibody are due to the presence of p27 -his6 
and not to a crossreactivity with proteins present in the RRL. Time course 
experiments showed that after three hours, the overall intensity of the 
bands produced by the incubation of p27 -his6 with RRL decreases. After 
six hours of incubation, the p27 band was dramatically reduced and the 
ladder of bands had almost totally disappeared, suggesting that the RRL 
contained an activity able to degrade p27. 
The degradation of p27 appeared to require ATP hydrolysis. Since RRL 
contains ATP, the addition of exogenous ATP did not change the kinetics of 
the reaction. In contrast, preincubation of RRL with apyrase, which 
hydrolyzes ATP, prevented the appearance of the slower migrating bands and 
inhibited the proteolysis of p27. ATP-.gamma.-S, a non-hydrolysable ATP 
analog, when added to the degradation mix, led to a substantial reduction 
in the kinetics of proteolysis of p27. 
To demonstrate that the ladder of bands obtained in these reactions was due 
to ubiquitination of p27, we added biotinylated-ubiquitin to the 
degradation mix and, after the reaction was terminated, we re-purified p27 
-his6 with either an antibody to p27 or with Nickel-chromatography on 
nitriolotriacetic acid-agarose (Ni-NTA-agarose). The purified material was 
analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by 
transfer to a nitrocellulose membrane. Ubiquitinated proteins were 
visualized using streptavidin-HRP. Two major ubiquitin cross-reactive 
groups of bands co-migrated with the two groups of higher molecular weight 
bands identified by the anti-p27 antibody and were not detected in 
identical samples lacking biotinylated-ubiquitin or p27 in the degradation 
mix. This result demonstrates that the higher molecular weight bands 
obtained in the reaction are ubiquitinated forms of p27. 
To demonstrate that the in vitro degradation of p27 required the 
proteasome, we added either 50 .mu.M LLnL or 50 .mu.M E64 or DMSO to the 
degradation mix. LLnL, but not E64 or DMSO, strongly inhibited p27 
degradation). As described in other systems, LLnL had a lesser inhibitory 
effect in vitro compared to the effect observed in vivo. Two other 
protease inhibitors, the serine protease inhibitor, L-1 
Chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK), used at 
150 .mu.M, or the cystein protease inhibitor, N-ethylmaleimide (NEM), used 
at 50 .mu.M, did not inhibit p27 degradation. It has been previously shown 
that ultracentrifugation can deplete an extract of proteasome particles. 
We subjected the RRL to centrifugation at 100,000 g for 6 hours. 
Incubation of p27 with proteasome-depleted supernatant did not result in 
p27 degradation. Interestingly, when the proteasome-rich pellet was added 
back to the supernatant, p27 degradation was completely restored. 
Ubc3 and Ubc2 specifically accelerate the turnover of p27 in rabbit 
reticulocyte lysate 
We tested whether the addition of human purified bacterially expressed 
ubiquitin-conjugating enzymes (UBCs) to the RRL altered the kinetics of 
the reaction. We tested Ubc2 (Rad6), Ubc3 (Cdc34), hUbCE, Ubc8, an 
epidermal Ubc (Ubc-epi) and rapUBC. We also tested the addition of HPV-18 
E6 which increases the rate of p53 degradation in a similar assay. In all 
reactions ATP-.gamma.-S was used to slow down the kinetics of reaction in 
order to highlight any potential difference. While Ubc2 and Ubc3 increased 
the rate of p27 turnover, the other proteins had no effect compared to the 
control. This difference was not due to a difference in their ability to 
accept ubiquitin from E1, because all of the Ubcs were efficiently charged 
in reactions containing purified recombinant human E1, the 
ubiquitin-activating enzyme. Interestingly, incubation with Ubc3 
specifically produced a smear of bands of high molecular weight. 
Incubation of purified p27 with purified Ubc2 or Ubc3 generated a 
mono-ubiquitinated form of p27. This reaction was ATP, Ubiquitin and E1 
dependent. The fact that the efficiency of this reaction was not very high 
and that only a single ubiquitin molecule was added to p27, strongly 
suggests that efficient multi-ubiquitination of p27 requires one or more 
factors which can be provided by the RRL. 
Inhibition of Ubc2 and Ubc3 slows down the turnover of p27 in rabbit 
reticulocyte lysate 
The result that Ubc2 and Ubc3 accelerate the turnover of p27 in vitro, 
prompted us to test the effect of inhibition of these Ubc functions in 
vitro. We made active site cysteine-to-serine mutations in human Ubc3, 
Ubc2 and hUbCE (on cysteine 93, 88 and 85, respectively). The Ubc3 mutant 
also contained a leucine97-to-serine mutation which has been shown to 
increase the dominant negative effect of yeast Cdc34. Such active site E2 
mutants are unable to accept activated ubiquitin from E1 and therefore 
should not ubiquitinate their downstream substrates. In addition, they 
efficiently inhibit the ubiquitination of their respective wild type in an 
in vitro reaction. In all reactions ATP-.gamma.-S was used to slow down 
the kinetics of the reaction. Compared to a control reaction, addition of 
the Ubc3 mutant considerably slowed down the in vitro turnover of p27, 
while the Ubc2 mutant had a less pronounced effect. Addition of both Ubc2 
and Ubc3 mutant proteins had the same effect as the Ubc3 mutant protein 
alone. Finally, the hUbCE mutant had no effect on p27 turnover. 
All of the above-cited references and publications are hereby incorporated 
by reference. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 45 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 444 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..441 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - ATG GCG CTG AAA CGG ATC CAC AAG GAA TTG AA - #T GAT CTG GCA CGG 
GAC 48 
Met Ala Leu Lys Arg Ile His Lys Glu Leu As - #n Asp Leu Ala Arg Asp 
1 5 - # 10 - # 15 
- - CCT CCA GCA CAG TGT TCA GCA GGT CCT GTT GG - #A GAT GAT ATG TTC CAT 
96 
Pro Pro Ala Gln Cys Ser Ala Gly Pro Val Gl - #y Asp Asp Met Phe His 
20 - # 25 - # 30 
- - TGG CAA GCT ACA ATA ATG GGG CCA AAT GAC AG - #T CCC TAT CAG GGT GGA 
144 
Trp Gln Ala Thr Ile Met Gly Pro Asn Asp Se - #r Pro Tyr Gln Gly Gly 
35 - # 40 - # 45 
- - GTA TTT TTC TTG ACA ATT CAT TTC CCA ACA GA - #T TAC CCC TTC AAA CCA 
192 
Val Phe Phe Leu Thr Ile His Phe Pro Thr As - #p Tyr Pro Phe Lys Pro 
50 - # 55 - # 60 
- - CCT AAG GTT GCA TTT ACC ACA AGA ATT TAT CA - #T CCA AAT ATT AAC AGT 
240 
Pro Lys Val Ala Phe Thr Thr Arg Ile Tyr Hi - #s Pro Asn Ile Asn Ser 
65 - # 70 - # 75 - # 80 
- - AAT GGC AGC ATT TGT CTT GAT ATT CTA CGA TC - #A CAG TGG TCT CCA GCA 
288 
Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg Se - #r Gln Trp Ser Pro Ala 
85 - # 90 - # 95 
- - CTA ACT ATT TCA AAA GTA CTC TTG TCC ATC TG - #T TCT CTG TTG TGT GAT 
336 
Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cy - #s Ser Leu Leu Cys Asp 
100 - # 105 - # 110 
- - CCC AAT CCA GAT GAT CCT TTA GTG CCT GAG AT - #T GCT CGG ATC TAC CAA 
384 
Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Il - #e Ala Arg Ile Tyr Gln 
115 - # 120 - # 125 
- - ACA GAT AGA GAA AAG TAC AAC AGA ATA GCT CG - #G GAA TGG ACT CAG AAG 
432 
Thr Asp Arg Glu Lys Tyr Asn Arg Ile Ala Ar - #g Glu Trp Thr Gln Lys 
130 - # 135 - # 140 
- - TAT GCG ATG TAA - # - # 
- # 444 
Tyr Ala Met 
145 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 147 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Ala Leu Lys Arg Ile His Lys Glu Leu As - #n Asp Leu Ala Arg Asp 
1 5 - # 10 - # 15 
- - Pro Pro Ala Gln Cys Ser Ala Gly Pro Val Gl - #y Asp Asp Met Phe His 
20 - # 25 - # 30 
- - Trp Gln Ala Thr Ile Met Gly Pro Asn Asp Se - #r Pro Tyr Gln Gly Gly 
35 - # 40 - # 45 
- - Val Phe Phe Leu Thr Ile His Phe Pro Thr As - #p Tyr Pro Phe Lys Pro 
50 - # 55 - # 60 
- - Pro Lys Val Ala Phe Thr Thr Arg Ile Tyr Hi - #s Pro Asn Ile Asn Ser 
65 - # 70 - # 75 - # 80 
- - Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg Se - #r Gln Trp Ser Pro Ala 
85 - # 90 - # 95 
- - Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cy - #s Ser Leu Leu Cys Asp 
100 - # 105 - # 110 
- - Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Il - #e Ala Arg Ile Tyr Gln 
115 - # 120 - # 125 
- - Thr Asp Arg Glu Lys Tyr Asn Arg Ile Ala Ar - #g Glu Trp Thr Gln Lys 
130 - # 135 - # 140 
- - Tyr Ala Met 
145 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 582 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 25..465 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - CACGAGTAAC TATTGCTTTA AATC ATG TCA TTA AAA CGT A - #TT AAC AAA GAA 
51 
- # Met Ser Leu Lys Arg - #Ile Asn Lys Glu 
- # 1 - # 5 
- - TTA TCT GAC TTA GGA AGA GAT CCA CCA TCA TC - #A TGT TCA GCC GGT CCA 
99 
Leu Ser Asp Leu Gly Arg Asp Pro Pro Ser Se - #r Cys Ser Ala Gly Pro 
10 - # 15 - # 20 - # 25 
- - GTT GGA GAT GAC TTA TAC CAC TGG CAA GCA TC - #T ATC ATG GGA CCA CCA 
147 
Val Gly Asp Asp Leu Tyr His Trp Gln Ala Se - #r Ile Met Gly Pro Pro 
30 - # 35 - # 40 
- - GAC TCT CCA TAC GCT GGT GGG GTA TTT TTC TT - #G AGT ATC CAT TTC CCA 
195 
Asp Ser Pro Tyr Ala Gly Gly Val Phe Phe Le - #u Ser Ile His Phe Pro 
45 - # 50 - # 55 
- - ACA GAT TAT CCT TTA AAA CCA CCA AAG ATT GC - #T TTA ACA ACA AAA ATC 
243 
Thr Asp Tyr Pro Leu Lys Pro Pro Lys Ile Al - #a Leu Thr Thr Lys Ile 
60 - # 65 - # 70 
- - TAT CAT CCA AAT ATT AAT AGT AAT GGT AAC AT - #C TGT TTA GAT ATC TTA 
291 
Tyr His Pro Asn Ile Asn Ser Asn Gly Asn Il - #e Cys Leu Asp Ile Leu 
75 - # 80 - # 85 
- - AAG GAT CAA TGG TCA CCT GCA TTA ACA ATT TC - #C AAA GTG TTA TTG TCT 
339 
Lys Asp Gln Trp Ser Pro Ala Leu Thr Ile Se - #r Lys Val Leu Leu Ser 
90 - # 95 - #100 - #105 
- - ATT TGT TCA TTA TTA ACT GAT GCC AAC CCA GA - #C GAT CCA TTA GTG CCA 
387 
Ile Cys Ser Leu Leu Thr Asp Ala Asn Pro As - #p Asp Pro Leu Val Pro 
110 - # 115 - # 120 
- - GAA ATC GCT CAC ATT TAT AAA CAA GAT AGA AA - #G AAG TAT GAA GCT ACT 
435 
Glu Ile Ala His Ile Tyr Lys Gln Asp Arg Ly - #s Lys Tyr Glu Ala Thr 
125 - # 130 - # 135 
- - GCC AAA GAA TGG ACT AAG AAA TAT GCT GTG TG - #ATTTTAGA GAAAAACAAA 
485 
Ala Lys Glu Trp Thr Lys Lys Tyr Ala Val 
140 - # 145 
- - AACATCTAAT TTCTACATGT ATTATGTCGT AATGCTTTCA CACAATACAA AA - 
#ACATCTAA 545 
- - TTTCTACATG TATTATGTCG TAATGCTTTC ACACAAT - # 
- # 582 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 147 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Met Ser Leu Lys Arg Ile Asn Lys Glu Leu Se - #r Asp Leu Gly Arg Asp 
1 5 - # 10 - # 15 
- - Pro Pro Ser Ser Cys Ser Ala Gly Pro Val Gl - #y Asp Asp Leu Tyr His 
20 - # 25 - # 30 
- - Trp Gln Ala Ser Ile Met Gly Pro Pro Asp Se - #r Pro Tyr Ala Gly Gly 
35 - # 40 - # 45 
- - Val Phe Phe Leu Ser Ile His Phe Pro Thr As - #p Tyr Pro Leu Lys Pro 
50 - # 55 - # 60 
- - Pro Lys Ile Ala Leu Thr Thr Lys Ile Tyr Hi - #s Pro Asn Ile Asn Ser 
65 - # 70 - # 75 - # 80 
- - Asn Gly Asn Ile Cys Leu Asp Ile Leu Lys As - #p Gln Trp Ser Pro Ala 
85 - # 90 - # 95 
- - Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cy - #s Ser Leu Leu Thr Asp 
100 - # 105 - # 110 
- - Ala Asn Pro Asp Asp Pro Leu Val Pro Glu Il - #e Ala His Ile Tyr Lys 
115 - # 120 - # 125 
- - Gln Asp Arg Lys Lys Tyr Glu Ala Thr Ala Ly - #s Glu Trp Thr Lys Lys 
130 - # 135 - # 140 
- - Tyr Ala Val 
145 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 522 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 22..462 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - CGCAAAAGCA AACCAGTAAC G ATG GCT TTG AAA AGA ATT - #AAC CGT GAA TTA 
51 
- # Met Ala Leu Lys Arg Ile Asn - #Arg Glu Leu 
- # 1 - # 5 - # 10 
- - GCT GAT CTT GGA AAA GAC CCA CCG TCT TCT TG - #T TCC GCC GGC CCT GTT 
99 
Ala Asp Leu Gly Lys Asp Pro Pro Ser Ser Cy - #s Ser Ala Gly Pro Val 
15 - # 20 - # 25 
- - GGC GAT GAT TTA TTC CAT TGG CAA GCT ACA AT - #C ATG GGT CCT GCT GAC 
147 
Gly Asp Asp Leu Phe His Trp Gln Ala Thr Il - #e Met Gly Pro Ala Asp 
30 - # 35 - # 40 
- - AGC CCT TAT GCG GGT GGT GTC TTC TTC TTG TC - #C ATT CAT TTC CCT ACG 
195 
Ser Pro Tyr Ala Gly Gly Val Phe Phe Leu Se - #r Ile His Phe Pro Thr 
45 - # 50 - # 55 
- - GAC TAC CCA TTC AAG CCA CCA AAG GTA AAC TT - #T ACA ACC AGA ATC TAT 
243 
Asp Tyr Pro Phe Lys Pro Pro Lys Val Asn Ph - #e Thr Thr Arg Ile Tyr 
60 - # 65 - # 70 
- - CAT CCC AAC ATC AAT TCA AAC GGT AGC ATT TG - #T TTG GAT ATC CTT CGT 
291 
His Pro Asn Ile Asn Ser Asn Gly Ser Ile Cy - #s Leu Asp Ile Leu Arg 
75 - # 80 - # 85 - # 90 
- - GAC CAA TGG TCT CCA GCG TTG ACT ATA TCA AA - #G GTA TTA CTG TCT ATC 
339 
Asp Gln Trp Ser Pro Ala Leu Thr Ile Ser Ly - #s Val Leu Leu Ser Ile 
95 - # 100 - # 105 
- - TGC TCA TTG TTG ACA GAT CCT AAT CCT GAT GA - #T CCG CTT GTG CCT GAA 
387 
Cys Ser Leu Leu Thr Asp Pro Asn Pro Asp As - #p Pro Leu Val Pro Glu 
110 - # 115 - # 120 
- - ATT GCG CAC GTC TAC AAA ACT GAC AGA TCC CG - #T TAT GAA TTA AGT GCT 
435 
Ile Ala His Val Tyr Lys Thr Asp Arg Ser Ar - #g Tyr Glu Leu Ser Ala 
125 - # 130 - # 135 
- - CGT GAA TGG ACT AGA AAA TAC GCA ATC TAGAGTTTG - #T TTCTGTGTTG 
482 
Arg Glu Trp Thr Arg Lys Tyr Ala Ile 
140 - # 145 
- - ATATTAAATA TTCATCTCTT AAAAAAAAAA AAAAAACTCG - # 
- # 522 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 147 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - Met Ala Leu Lys Arg Ile Asn Arg Glu Leu Al - #a Asp Leu Gly Lys Asp 
1 5 - # 10 - # 15 
- - Pro Pro Ser Ser Cys Ser Ala Gly Pro Val Gl - #y Asp Asp Leu Phe His 
20 - # 25 - # 30 
- - Trp Gln Ala Thr Ile Met Gly Pro Ala Asp Se - #r Pro Tyr Ala Gly Gly 
35 - # 40 - # 45 
- - Val Phe Phe Leu Ser Ile His Phe Pro Thr As - #p Tyr Pro Phe Lys Pro 
50 - # 55 - # 60 
- - Pro Lys Val Asn Phe Thr Thr Arg Ile Tyr Hi - #s Pro Asn Ile Asn Ser 
65 - # 70 - # 75 - # 80 
- - Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg As - #p Gln Trp Ser Pro Ala 
85 - # 90 - # 95 
- - Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cy - #s Ser Leu Leu Thr Asp 
100 - # 105 - # 110 
- - Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Il - #e Ala His Val Tyr Lys 
115 - # 120 - # 125 
- - Thr Asp Arg Ser Arg Tyr Glu Leu Ser Ala Ar - #g Glu Trp Thr Arg Lys 
130 - # 135 - # 140 
- - Tyr Ala Ile 
145 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 147 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (v) FRAGMENT TYPE: internal 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - Met Xaa Leu Lys Arg Ile Xaa Xaa Glu Leu Xa - #a Asp Leu Xaa Xaa Asp 
1 5 - # 10 - # 15 
- - Pro Pro Xaa Xaa Cys Ser Ala Gly Pro Val Gl - #y Asp Asp Xaa Xaa His 
20 - # 25 - # 30 
- - Trp Gln Ala Xaa Ile Met Gly Pro Asn Asp Se - #r Pro Tyr Xaa Gly Gly 
35 - # 40 - # 45 
- - Val Phe Phe Leu Xaa Ile His Phe Pro Thr As - #p Tyr Pro Xaa Lys Pro 
50 - # 55 - # 60 
- - Pro Lys Xaa Xaa Xaa Thr Thr Xaa Ile Tyr Hi - #s Pro Asn Ile Asn Ser 
65 - #70 - #75 - #80 
- - Asn Gly Xaa Ile Cys Leu Asp Ile Leu Xaa Xa - #a Gln Trp Ser Pro Ala 
85 - # 90 - # 95 
- - Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cy - #s Ser Leu Leu Xaa Asp 
100 - # 105 - # 110 
- - Xaa Asn Pro Asp Asp Pro Leu Val Pro Glu Il - #e Ala Xaa Xaa Tyr Xaa 
115 - # 120 - # 125 
- - Xaa Asp Arg Xaa Xaa Tyr Xaa Xaa Xaa Ala Xa - #a Glu Trp Thr Xaa Lys 
130 - # 135 - # 140 
- - Tyr Ala Xaa 
145 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - GCGCGCAAGC TTTAYGARGG WGGWGTYTTY TT - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - GCGCGCGAAT TCACNGCRTA YTTYTTNGTC CCAYTC - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - GCGCGCAAGC TTCCNGTNGG NGAYTTRTTY CAYTGGCA - # 
- # 38 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - GCGCGCGAAT TCATNGTNAR NGCNGGCGAC CA - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 907 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 34..507 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - GCCGGGGCTG CGGCCGCCCG AGGGACTTTG AAC ATG TCG GGG AT - #C GCC CTC 
AGC 54 
- # - # Met Ser Gly Ile Ala Leu Ser 
- # - # 1 - #5 
- - AGA CTC GCC CAG GAG AGG AAA GCA TGG AGG AA - #A GAC CAC CCA TTT GGT 
102 
Arg Leu Ala Gln Glu Arg Lys Ala Trp Arg Ly - #s Asp His Pro Phe Gly 
10 - # 15 - # 20 
- - TTC GTG GCT GTC CCA ACA AAA AAT CCC GAT GG - #C ACG ATG AAC CTC ATG 
150 
Phe Val Ala Val Pro Thr Lys Asn Pro Asp Gl - #y Thr Met Asn Leu Met 
25 - # 30 - # 35 
- - AAC TGG GAG TGC GCC ATT CCA GGA AAG AAA GG - #G ACT CCG TGG GAA GGA 
198 
Asn Trp Glu Cys Ala Ile Pro Gly Lys Lys Gl - #y Thr Pro Trp Glu Gly 
40 - # 45 - # 50 - # 55 
- - GGC TTG TTT AAA CTA CGG ATG CTT TTC AAA GA - #T GAT TAT CCA TCT TCG 
246 
Gly Leu Phe Lys Leu Arg Met Leu Phe Lys As - #p Asp Tyr Pro Ser Ser 
60 - # 65 - # 70 
- - CCA CCA AAA TGT AAA TTC GAA CCA CCA TTA TT - #T CAC CCG AAT GTG TAC 
294 
Pro Pro Lys Cys Lys Phe Glu Pro Pro Leu Ph - #e His Pro Asn Val Tyr 
75 - # 80 - # 85 
- - CCT TCG GGG ACA GTG TGC CTG TCC ATC TTA GA - #G GAG GAC AAG GAC TGG 
342 
Pro Ser Gly Thr Val Cys Leu Ser Ile Leu Gl - #u Glu Asp Lys Asp Trp 
90 - # 95 - # 100 
- - AGG CCA GCC ATC ACA ATC AAA CAG ATC CTA TT - #A GGA ATA CAG GAA CTT 
390 
Arg Pro Ala Ile Thr Ile Lys Gln Ile Leu Le - #u Gly Ile Gln Glu Leu 
105 - # 110 - # 115 
- - CTA AAT GAA CCA AAT ATC CAA GAC CCA GCT CA - #A GCA GAG GCC TAC ACG 
438 
Leu Asn Glu Pro Asn Ile Gln Asp Pro Ala Gl - #n Ala Glu Ala Tyr Thr 
120 1 - #25 1 - #30 1 - 
#35 
- - ATT TAC TGC CAA AAC AGA GTG GAG TAC GAG AA - #A AGG GTC CGA GCA 
CAA 486 
Ile Tyr Cys Gln Asn Arg Val Glu Tyr Glu Ly - #s Arg Val Arg Ala Gln 
140 - # 145 - # 150 
- - GCC AAG AAG TTT GCG CCC TCA TAAGCAGCGA CCTTGTGGC - #A TCGTCAAAAG 
537 
Ala Lys Lys Phe Ala Pro Ser 
155 
- - GAAGGGATTG GTTTGGCAAG AACTTGTTTA CAACATTTTT GGCAAATCTA AA - 
#GTTGCTCC 597 
- - ATACAATGAC TAGTCACCTG GGGGGGTTGG GCGGGCGCCA TCTTCCATTG CC - 
#GCCGCGGG 657 
- - TGTGCGGTCT CGATTCGCTG AATTGCCCGT TTCCATACAG GGTCTCTTCC TT - 
#CGGTCTTT 717 
- - TGGTATTTTT GGATTGTTAT GTAAAACTCG CTTTTATTTT AATATTGATG TC - 
#AGTATTTC 777 
- - AACTGCTGTA AAATTATAAA CTTTTATACT GGGTAAGTCC CCCAGGGGCG AG - 
#TTNCCTCG 837 
- - CTCTGGGATG CAGGCATGCT TCTCACCGTG CAGAGCTGCA CTTGNCCTCA GC - 
#TGNCTGNA 897 
- - TGGAAATGCA - # - # 
- # 907 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 158 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - Met Ser Gly Ile Ala Leu Ser Arg Leu Ala Gl - #n Glu Arg Lys Ala Trp 
1 5 - # 10 - # 15 
- - Arg Lys Asp His Pro Phe Gly Phe Val Ala Va - #l Pro Thr Lys Asn Pro 
20 - # 25 - # 30 
- - Asp Gly Thr Met Asn Leu Met Asn Trp Glu Cy - #s Ala Ile Pro Gly Lys 
35 - # 40 - # 45 
- - Lys Gly Thr Pro Trp Glu Gly Gly Leu Phe Ly - #s Leu Arg Met Leu Phe 
50 - # 55 - # 60 
- - Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cy - #s Lys Phe Glu Pro Pro 
65 - # 70 - # 75 - # 80 
- - Leu Phe His Pro Asn Val Tyr Pro Ser Gly Th - #r Val Cys Leu Ser Ile 
85 - # 90 - # 95 
- - Leu Glu Glu Asp Lys Asp Trp Arg Pro Ala Il - #e Thr Ile Lys Gln Ile 
100 - # 105 - # 110 
- - Leu Leu Gly Ile Gln Glu Leu Leu Asn Glu Pr - #o Asn Ile Gln Asp Pro 
115 - # 120 - # 125 
- - Ala Gln Ala Glu Ala Tyr Thr Ile Tyr Cys Gl - #n Asn Arg Val Glu Tyr 
130 - # 135 - # 140 
- - Glu Lys Arg Val Arg Ala Gln Ala Lys Lys Ph - #e Ala Pro Ser 
145 1 - #50 1 - #55 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3176 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3174 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - ATG TCC AGC TCG CCG CTG TCC AAG AAA CGT CG - #C GTG TCC GGG CCT GAT 
48 
Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Ar - #g Val Ser Gly Pro Asp 
1 5 - # 10 - # 15 
- - CCA AAG CCG GGT TCT AAC TGC TCC CCT GCC CA - #G TCC GTG TTG TCC GAA 
96 
Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala Gl - #n Ser Val Leu Ser Glu 
20 - # 25 - # 30 
- - GTG CCC TCG GTG CCA ACC AAC GGA ATG GCC AA - #G AAC GGC AGT GAA GCA 
144 
Val Pro Ser Val Pro Thr Asn Gly Met Ala Ly - #s Asn Gly Ser Glu Ala 
35 - # 40 - # 45 
- - GAC ATA GAC GAG GGC CTT TAC TCC CGG CAG CT - #G TAT GTG TTG GGC CAT 
192 
Asp Ile Asp Glu Gly Leu Tyr Ser Arg Gln Le - #u Tyr Val Leu Gly His 
50 - # 55 - # 60 
- - GAG GCA ATG AAG CGG CTC CAG ACA TCC AGT GT - #C CTG GTA TCA GGC CTG 
240 
Glu Ala Met Lys Arg Leu Gln Thr Ser Ser Va - #l Leu Val Ser Gly Leu 
65 - # 70 - # 75 - # 80 
- - CGG GGC CTG GGC GTG GAG ATC GCT AAG AAC AT - #C ATC CTT GGT GGG GTC 
288 
Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Il - #e Ile Leu Gly Gly Val 
85 - # 90 - # 95 
- - AAG GCT GTT ACC CTA CAT GAC CAG GGC ACT GC - #C CAG TGG GCT GAT CTT 
336 
Lys Ala Val Thr Leu His Asp Gln Gly Thr Al - #a Gln Trp Ala Asp Leu 
100 - # 105 - # 110 
- - TCC TCC CAG TTC TAC CTG CGG GAG GAG GAC AT - #C GGT AAA AAC CGG GCC 
384 
Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Il - #e Gly Lys Asn Arg Ala 
115 - # 120 - # 125 
- - GAG GTA TCA CAG CCC CGC CTC GCT GAG CTC AA - #C AGC TAT GTG CCT GTC 
432 
Glu Val Ser Gln Pro Arg Leu Ala Glu Leu As - #n Ser Tyr Val Pro Val 
130 - # 135 - # 140 
- - ACT GCC TAC ACT GGA CCC CTC GTT GAG GAC TT - #C CTT AGT GGT TTC CAG 
480 
Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Ph - #e Leu Ser Gly Phe Gln 
145 1 - #50 1 - #55 1 - 
#60 
- - GTG GTG GTG CTC ACC AAC ACC CCC CTG GAG GA - #C CAG CTG CGA GTG 
GGT 528 
Val Val Val Leu Thr Asn Thr Pro Leu Glu As - #p Gln Leu Arg Val Gly 
165 - # 170 - # 175 
- - GAG TTC TGT CAC AAC CGT GGC ATC AAG CTG GT - #G GTG GCA GAC ACG CGG 
576 
Glu Phe Cys His Asn Arg Gly Ile Lys Leu Va - #l Val Ala Asp Thr Arg 
180 - # 185 - # 190 
- - GGC CTG TTT GGG CAG CTC TTC TGT GAC TTT GG - #A GAG GAA ATG ATC CTC 
624 
Gly Leu Phe Gly Gln Leu Phe Cys Asp Phe Gl - #y Glu Glu Met Ile Leu 
195 - # 200 - # 205 
- - ACA GAT TCC AAT GGG GAG CAG CCA CTC AGT GC - #T ATG GTT TCT ATG GTT 
672 
Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Al - #a Met Val Ser Met Val 
210 - # 215 - # 220 
- - ACC AAG GAC AAC CCC GGT GTG GTT ACC TGC CT - #G GAT GAG GCC CGA CAC 
720 
Thr Lys Asp Asn Pro Gly Val Val Thr Cys Le - #u Asp Glu Ala Arg His 
225 2 - #30 2 - #35 2 - 
#40 
- - GGG TTT GAG AGC GGG GAC TTT GTC TCC TTT TC - #A GAA GTA CAG GGC 
ATG 768 
Gly Phe Glu Ser Gly Asp Phe Val Ser Phe Se - #r Glu Val Gln Gly Met 
245 - # 250 - # 255 
- - GTT GAA CTC AAC GGA AAT CAG CCC ATG GAG AT - #C AAA GTC CTG GGT CCT 
816 
Val Glu Leu Asn Gly Asn Gln Pro Met Glu Il - #e Lys Val Leu Gly Pro 
260 - # 265 - # 270 
- - TAT ACC TTT AGC ATC TGT GAC ACC TCC AAC TT - #C TCC GAC TAC ATC CGT 
864 
Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Ph - #e Ser Asp Tyr Ile Arg 
275 - # 280 - # 285 
- - GGA GGC ATC GTC AGT CAG GTC AAA GTA CCT AA - #G AAG ATT AGC TTT AAA 
912 
Gly Gly Ile Val Ser Gln Val Lys Val Pro Ly - #s Lys Ile Ser Phe Lys 
290 - # 295 - # 300 
- - TCC TTG GTG GCC TCA CTG GCA GAA CCT GAC TT - #T GTG GTG ACG GAC TTC 
960 
Ser Leu Val Ala Ser Leu Ala Glu Pro Asp Ph - #e Val Val Thr Asp Phe 
305 3 - #10 3 - #15 3 - 
#20 
- - GCC AAG TTT TCT CGC CCT GCC CAG CTG CAC AT - #T GGC TTC CAG GCC 
CTG 1008 
Ala Lys Phe Ser Arg Pro Ala Gln Leu His Il - #e Gly Phe Gln Ala Leu 
325 - # 330 - # 335 
- - CAC CAG TTC TGT GCT CAG CAT GGC CGG CCA CC - #T CGG CCC CGC AAT GAG 
1056 
His Gln Phe Cys Ala Gln His Gly Arg Pro Pr - #o Arg Pro Arg Asn Glu 
340 - # 345 - # 350 
- - GAG GAT GCA GCA GAA CTG GTA GCC TTA GCA CA - #G GCT GTG AAT GCT CGA 
1104 
Glu Asp Ala Ala Glu Leu Val Ala Leu Ala Gl - #n Ala Val Asn Ala Arg 
355 - # 360 - # 365 
- - GCC CTG CCA GCA GTG CAG CAA AAT AAC CTG GA - #C GAG GAC CTC ATC CGG 
1152 
Ala Leu Pro Ala Val Gln Gln Asn Asn Leu As - #p Glu Asp Leu Ile Arg 
370 - # 375 - # 380 
- - AAG CTG GCA TAT GTG GCT GCT GGG GAT CTG GC - #A CCC ATA AAC GCC TTC 
1200 
Lys Leu Ala Tyr Val Ala Ala Gly Asp Leu Al - #a Pro Ile Asn Ala Phe 
385 3 - #90 3 - #95 4 - 
#00 
- - ATT GGG GGC CTG GCT GCC CAG GAA GTC ATG AA - #G GCC TGC TCC GGG 
AAG 1248 
Ile Gly Gly Leu Ala Ala Gln Glu Val Met Ly - #s Ala Cys Ser Gly Lys 
405 - # 410 - # 415 
- - TTC ATG CCC ATC ATG CAG TGG CTA TAC TTT GA - #T GCC CTT GAG TGT CTC 
1296 
Phe Met Pro Ile Met Gln Trp Leu Tyr Phe As - #p Ala Leu Glu Cys Leu 
420 - # 425 - # 430 
- - CCT GAG GAC AAA GAG GTC CTC ACA GAG GAC AA - #G TGC CTC CAG CGC CAG 
1344 
Pro Glu Asp Lys Glu Val Leu Thr Glu Asp Ly - #s Cys Leu Gln Arg Gln 
435 - # 440 - # 445 
- - AAC CGT TAT GAC GGG CAA GTG GCT GTG TTT GG - #C TCA GAC CTG CAA GAG 
1392 
Asn Arg Tyr Asp Gly Gln Val Ala Val Phe Gl - #y Ser Asp Leu Gln Glu 
450 - # 455 - # 460 
- - AAG CTG GGC AAG CAG AAG TAT TTC CTG GTG GG - #T GCG GGG GCC ATT GGC 
1440 
Lys Leu Gly Lys Gln Lys Tyr Phe Leu Val Gl - #y Ala Gly Ala Ile Gly 
465 4 - #70 4 - #75 4 - 
#80 
- - TGT GAG CTG CTC AAG AAC TTT GCC ATG ATT GG - #G CTG GGC TGC GGG 
GAG 1488 
Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gl - #y Leu Gly Cys Gly Glu 
485 - # 490 - # 495 
- - GGT GGA GAA ATC ATC GTT ACA GAC ATG GAC AC - #C ATT GAG AAG TCA AAT 
1536 
Gly Gly Glu Ile Ile Val Thr Asp Met Asp Th - #r Ile Glu Lys Ser Asn 
500 - # 505 - # 510 
- - CTG AAT CGA CAG TTT CTT TTC CGG CCC TGG GA - #T GTC ACG AAG TTA AAG 
1584 
Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp As - #p Val Thr Lys Leu Lys 
515 - # 520 - # 525 
- - TCT GAC ACG GCT GCT GCA GCT GTG CGC CAA AT - #G AAT CCA CAT ATC CGG 
1632 
Ser Asp Thr Ala Ala Ala Ala Val Arg Gln Me - #t Asn Pro His Ile Arg 
530 - # 535 - # 540 
- - GTG ACA AGC CAC CAG AAC CGT GTG GGT CCT GA - #C ACG GAG CGC ATC TAT 
1680 
Val Thr Ser His Gln Asn Arg Val Gly Pro As - #p Thr Glu Arg Ile Tyr 
545 5 - #50 5 - #55 5 - 
#60 
- - GAT GAC GAT TTT TTC CAA AAC CTA GAT GGC GT - #G GCC AAT GCC CTG 
GAC 1728 
Asp Asp Asp Phe Phe Gln Asn Leu Asp Gly Va - #l Ala Asn Ala Leu Asp 
565 - # 570 - # 575 
- - AAC GTG GAT GCC CGC ATG TAC ATG GAC CGC CG - #C TGT GTC TAC TAC CGG 
1776 
Asn Val Asp Ala Arg Met Tyr Met Asp Arg Ar - #g Cys Val Tyr Tyr Arg 
580 - # 585 - # 590 
- - AAG CCA CTG CTG GAG TCA GGC ACA CTG GGC AC - #C AAA GGC AAT GTG CAG 
1824 
Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Th - #r Lys Gly Asn Val Gln 
595 - # 600 - # 605 
- - GTG GTG ATC CCC TTC CTG ACA GAG TCG TAC AG - #T TCC AGC CAG GAC CCA 
1872 
Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Se - #r Ser Ser Gln Asp Pro 
610 - # 615 - # 620 
- - CCT GAG AAG TCC ATC CCC ATC TGT ACC CTG AA - #G AAC TTC CCT AAT GCC 
1920 
Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Ly - #s Asn Phe Pro Asn Ala 
625 6 - #30 6 - #35 6 - 
#40 
- - ATC GAG CAC ACC CTG CAG TGG GCT CGG GAT GA - #G TTT GAA GGC CTC 
TTC 1968 
Ile Glu His Thr Leu Gln Trp Ala Arg Asp Gl - #u Phe Glu Gly Leu Phe 
645 - # 650 - # 655 
- - AAG CAG CCA GCA GAA AAT GTC AAC CAG TAC CT - #C ACA GAC CCC AAG TTT 
2016 
Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr Le - #u Thr Asp Pro Lys Phe 
660 - # 665 - # 670 
- - GTG GAG CGA ACA CTG CGG CTG GCA GGC ACT CA - #G CCC TTG GAG GTG CTG 
2064 
Val Glu Arg Thr Leu Arg Leu Ala Gly Thr Gl - #n Pro Leu Glu Val Leu 
675 - # 680 - # 685 
- - GAG GCT GTG CAG CGC AGC CTG GTG CTG CAG CG - #A CCA CAG ACC TGG GCT 
2112 
Glu Ala Val Gln Arg Ser Leu Val Leu Gln Ar - #g Pro Gln Thr Trp Ala 
690 - # 695 - # 700 
- - GAC TGC GTG ACC TGG GCC TGC CAC CAC TGG CA - #C ACC CAG TAC TCG AAC 
2160 
Asp Cys Val Thr Trp Ala Cys His His Trp Hi - #s Thr Gln Tyr Ser Asn 
705 7 - #10 7 - #15 7 - 
#20 
- - AAC ATC CGG CAG CTG CTG CAC AAC TTC CCT CC - #T GAC CAG CTC ACA 
AGC 2208 
Asn Ile Arg Gln Leu Leu His Asn Phe Pro Pr - #o Asp Gln Leu Thr Ser 
725 - # 730 - # 735 
- - TCA GGA GCG CCG TTC TGG TCT GGG CCC AAA CG - #C TGT CCA CAC CCG CTC 
2256 
Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Ar - #g Cys Pro His Pro Leu 
740 - # 745 - # 750 
- - ACC TTT GAT GTC AAC AAT CCC CTG CAT CTG GA - #C TAT GTG ATG GCT GCT 
2304 
Thr Phe Asp Val Asn Asn Pro Leu His Leu As - #p Tyr Val Met Ala Ala 
755 - # 760 - # 765 
- - GCC AAC CTG TTT GCC CAG ACC TAC GGG CTG AC - #A GGC TCT CAG GAC CGA 
2352 
Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Th - #r Gly Ser Gln Asp Arg 
770 - # 775 - # 780 
- - GCT GCT GTG GCC ACA TTC CTG CAG TCT GTG CA - #G GTC CCC GAA TTC ACC 
2400 
Ala Ala Val Ala Thr Phe Leu Gln Ser Val Gl - #n Val Pro Glu Phe Thr 
785 7 - #90 7 - #95 8 - 
#00 
- - CCC AAG TCT GGC GTC AAG ATC CAT GTT TCT GA - #C CAG GAG CTG CAG 
AGC 2448 
Pro Lys Ser Gly Val Lys Ile His Val Ser As - #p Gln Glu Leu Gln Ser 
805 - # 810 - # 815 
- - GCC AAT GCC TCT GTT GAT GAC AGT CGT CTA GA - #G GAG CTC AAA GCC ACT 
2496 
Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Gl - #u Glu Leu Lys Ala Thr 
820 - # 825 - # 830 
- - CTG CCC AGC CCA GAC AAG CTC CCT GGA TTC AA - #G ATG TAC CCC ATT GAC 
2544 
Leu Pro Ser Pro Asp Lys Leu Pro Gly Phe Ly - #s Met Tyr Pro Ile Asp 
835 - # 840 - # 845 
- - TTT GAG AAG GAT GAT GAC AGC AAC TTT CAT AT - #G GAT TTC ATC GTG GCT 
2592 
Phe Glu Lys Asp Asp Asp Ser Asn Phe His Me - #t Asp Phe Ile Val Ala 
850 - # 855 - # 860 
- - GCA TCC AAC CTC CGG GCA GAA AAC TAT GAC AT - #T CCT TCT GCA GAC CGG 
2640 
Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Il - #e Pro Ser Ala Asp Arg 
865 8 - #70 8 - #75 8 - 
#80 
- - CAC AAG AGC AAG CTG ATT GCA GGG AAG ATC AT - #C CCA GCC ATT GCC 
ACG 2688 
His Lys Ser Lys Leu Ile Ala Gly Lys Ile Il - #e Pro Ala Ile Ala Thr 
885 - # 890 - # 895 
- - ACC ACA GCA GCC GTG GTT GGC CTT GTG TGT CT - #G GAA CTG TAC AAG GTT 
2736 
Thr Thr Ala Ala Val Val Gly Leu Val Cys Le - #u Glu Leu Tyr Lys Val 
900 - # 905 - # 910 
- - GTG CAG GGG CAC CGA CAG CTT GAC TCC TAC AA - #G AAT GGT TTC CTC AAC 
2784 
Val Gln Gly His Arg Gln Leu Asp Ser Tyr Ly - #s Asn Gly Phe Leu Asn 
915 - # 920 - # 925 
- - TTG GCC CTG CCT TTC TTT GGT TTC TCT GAA CC - #C CTT GCC GCA CCA CGT 
2832 
Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pr - #o Leu Ala Ala Pro Arg 
930 - # 935 - # 940 
- - CAC CAG TAC TAT AAC CAA GAG TGG ACA TTG TG - #G GAT CGC TTT GAG GTA 
2880 
His Gln Tyr Tyr Asn Gln Glu Trp Thr Leu Tr - #p Asp Arg Phe Glu Val 
945 9 - #50 9 - #55 9 - 
#60 
- - CAA GGG CTG CAG CCT AAT GGT GAG GAG ATG AC - #C CTC AAA CAG TTC 
CTC 2928 
Gln Gly Leu Gln Pro Asn Gly Glu Glu Met Th - #r Leu Lys Gln Phe Leu 
965 - # 970 - # 975 
- - GAC TAT TTT AAG ACA GAG CAC AAA TTA GAG AT - #C ACC ATG CTG TCC CAG 
2976 
Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Il - #e Thr Met Leu Ser Gln 
980 - # 985 - # 990 
- - GGC GTG TCC ATG CTC TAT TCC TTC TTC ATG CC - #A GCT GCC AAG CTC AAG 
3024 
Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pr - #o Ala Ala Lys Leu Lys 
995 - # 1000 - # 1005 
- - GAA CGG TTG GAT CAG CCG ATG ACA GAG ATT GT - #G AGC CGT GTG TCG AAG 
3072 
Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Va - #l Ser Arg Val Ser Lys 
1010 - # 1015 - # 1020 
- - CGA AAG CTG GGC CGC CAC GTG CGG GCG CTG GT - #G CTT GAG CTG TGC TGT 
3120 
Arg Lys Leu Gly Arg His Val Arg Ala Leu Va - #l Leu Glu Leu Cys Cys 
1025 1030 - # 1035 - # 1040 
- - AAC GAC GAG AGC GGC GAG GAT GTC GAG GTT CC - #C TAT GTC CGA TAC ACC 
3168 
Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pr - #o Tyr Val Arg Tyr Thr 
1045 - # 1050 - # 1055 
- - ATC CGC TG - # - # - 
# 3176 
Ile Arg 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1058 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Ar - #g Val Ser Gly Pro Asp 
1 5 - # 10 - # 15 
- - Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala Gl - #n Ser Val Leu Ser Glu 
20 - # 25 - # 30 
- - Val Pro Ser Val Pro Thr Asn Gly Met Ala Ly - #s Asn Gly Ser Glu Ala 
35 - # 40 - # 45 
- - Asp Ile Asp Glu Gly Leu Tyr Ser Arg Gln Le - #u Tyr Val Leu Gly His 
50 - # 55 - # 60 
- - Glu Ala Met Lys Arg Leu Gln Thr Ser Ser Va - #l Leu Val Ser Gly Leu 
65 - # 70 - # 75 - # 80 
- - Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Il - #e Ile Leu Gly Gly Val 
85 - # 90 - # 95 
- - Lys Ala Val Thr Leu His Asp Gln Gly Thr Al - #a Gln Trp Ala Asp Leu 
100 - # 105 - # 110 
- - Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Il - #e Gly Lys Asn Arg Ala 
115 - # 120 - # 125 
- - Glu Val Ser Gln Pro Arg Leu Ala Glu Leu As - #n Ser Tyr Val Pro Val 
130 - # 135 - # 140 
- - Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Ph - #e Leu Ser Gly Phe Gln 
145 1 - #50 1 - #55 1 - 
#60 
- - Val Val Val Leu Thr Asn Thr Pro Leu Glu As - #p Gln Leu Arg Val 
Gly 
165 - # 170 - # 175 
- - Glu Phe Cys His Asn Arg Gly Ile Lys Leu Va - #l Val Ala Asp Thr Arg 
180 - # 185 - # 190 
- - Gly Leu Phe Gly Gln Leu Phe Cys Asp Phe Gl - #y Glu Glu Met Ile Leu 
195 - # 200 - # 205 
- - Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Al - #a Met Val Ser Met Val 
210 - # 215 - # 220 
- - Thr Lys Asp Asn Pro Gly Val Val Thr Cys Le - #u Asp Glu Ala Arg His 
225 2 - #30 2 - #35 2 - 
#40 
- - Gly Phe Glu Ser Gly Asp Phe Val Ser Phe Se - #r Glu Val Gln Gly 
Met 
245 - # 250 - # 255 
- - Val Glu Leu Asn Gly Asn Gln Pro Met Glu Il - #e Lys Val Leu Gly Pro 
260 - # 265 - # 270 
- - Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Ph - #e Ser Asp Tyr Ile Arg 
275 - # 280 - # 285 
- - Gly Gly Ile Val Ser Gln Val Lys Val Pro Ly - #s Lys Ile Ser Phe Lys 
290 - # 295 - # 300 
- - Ser Leu Val Ala Ser Leu Ala Glu Pro Asp Ph - #e Val Val Thr Asp Phe 
305 3 - #10 3 - #15 3 - 
#20 
- - Ala Lys Phe Ser Arg Pro Ala Gln Leu His Il - #e Gly Phe Gln Ala 
Leu 
325 - # 330 - # 335 
- - His Gln Phe Cys Ala Gln His Gly Arg Pro Pr - #o Arg Pro Arg Asn Glu 
340 - # 345 - # 350 
- - Glu Asp Ala Ala Glu Leu Val Ala Leu Ala Gl - #n Ala Val Asn Ala Arg 
355 - # 360 - # 365 
- - Ala Leu Pro Ala Val Gln Gln Asn Asn Leu As - #p Glu Asp Leu Ile Arg 
370 - # 375 - # 380 
- - Lys Leu Ala Tyr Val Ala Ala Gly Asp Leu Al - #a Pro Ile Asn Ala Phe 
385 3 - #90 3 - #95 4 - 
#00 
- - Ile Gly Gly Leu Ala Ala Gln Glu Val Met Ly - #s Ala Cys Ser Gly 
Lys 
405 - # 410 - # 415 
- - Phe Met Pro Ile Met Gln Trp Leu Tyr Phe As - #p Ala Leu Glu Cys Leu 
420 - # 425 - # 430 
- - Pro Glu Asp Lys Glu Val Leu Thr Glu Asp Ly - #s Cys Leu Gln Arg Gln 
435 - # 440 - # 445 
- - Asn Arg Tyr Asp Gly Gln Val Ala Val Phe Gl - #y Ser Asp Leu Gln Glu 
450 - # 455 - # 460 
- - Lys Leu Gly Lys Gln Lys Tyr Phe Leu Val Gl - #y Ala Gly Ala Ile Gly 
465 4 - #70 4 - #75 4 - 
#80 
- - Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gl - #y Leu Gly Cys Gly 
Glu 
485 - # 490 - # 495 
- - Gly Gly Glu Ile Ile Val Thr Asp Met Asp Th - #r Ile Glu Lys Ser Asn 
500 - # 505 - # 510 
- - Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp As - #p Val Thr Lys Leu Lys 
515 - # 520 - # 525 
- - Ser Asp Thr Ala Ala Ala Ala Val Arg Gln Me - #t Asn Pro His Ile Arg 
530 - # 535 - # 540 
- - Val Thr Ser His Gln Asn Arg Val Gly Pro As - #p Thr Glu Arg Ile Tyr 
545 5 - #50 5 - #55 5 - 
#60 
- - Asp Asp Asp Phe Phe Gln Asn Leu Asp Gly Va - #l Ala Asn Ala Leu 
Asp 
565 - # 570 - # 575 
- - Asn Val Asp Ala Arg Met Tyr Met Asp Arg Ar - #g Cys Val Tyr Tyr Arg 
580 - # 585 - # 590 
- - Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Th - #r Lys Gly Asn Val Gln 
595 - # 600 - # 605 
- - Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Se - #r Ser Ser Gln Asp Pro 
610 - # 615 - # 620 
- - Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Ly - #s Asn Phe Pro Asn Ala 
625 6 - #30 6 - #35 6 - 
#40 
- - Ile Glu His Thr Leu Gln Trp Ala Arg Asp Gl - #u Phe Glu Gly Leu 
Phe 
645 - # 650 - # 655 
- - Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr Le - #u Thr Asp Pro Lys Phe 
660 - # 665 - # 670 
- - Val Glu Arg Thr Leu Arg Leu Ala Gly Thr Gl - #n Pro Leu Glu Val Leu 
675 - # 680 - # 685 
- - Glu Ala Val Gln Arg Ser Leu Val Leu Gln Ar - #g Pro Gln Thr Trp Ala 
690 - # 695 - # 700 
- - Asp Cys Val Thr Trp Ala Cys His His Trp Hi - #s Thr Gln Tyr Ser Asn 
705 7 - #10 7 - #15 7 - 
#20 
- - Asn Ile Arg Gln Leu Leu His Asn Phe Pro Pr - #o Asp Gln Leu Thr 
Ser 
725 - # 730 - # 735 
- - Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Ar - #g Cys Pro His Pro Leu 
740 - # 745 - # 750 
- - Thr Phe Asp Val Asn Asn Pro Leu His Leu As - #p Tyr Val Met Ala Ala 
755 - # 760 - # 765 
- - Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Th - #r Gly Ser Gln Asp Arg 
770 - # 775 - # 780 
- - Ala Ala Val Ala Thr Phe Leu Gln Ser Val Gl - #n Val Pro Glu Phe Thr 
785 7 - #90 7 - #95 8 - 
#00 
- - Pro Lys Ser Gly Val Lys Ile His Val Ser As - #p Gln Glu Leu Gln 
Ser 
805 - # 810 - # 815 
- - Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Gl - #u Glu Leu Lys Ala Thr 
820 - # 825 - # 830 
- - Leu Pro Ser Pro Asp Lys Leu Pro Gly Phe Ly - #s Met Tyr Pro Ile Asp 
835 - # 840 - # 845 
- - Phe Glu Lys Asp Asp Asp Ser Asn Phe His Me - #t Asp Phe Ile Val Ala 
850 - # 855 - # 860 
- - Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Il - #e Pro Ser Ala Asp Arg 
865 8 - #70 8 - #75 8 - 
#80 
- - His Lys Ser Lys Leu Ile Ala Gly Lys Ile Il - #e Pro Ala Ile Ala 
Thr 
885 - # 890 - # 895 
- - Thr Thr Ala Ala Val Val Gly Leu Val Cys Le - #u Glu Leu Tyr Lys Val 
900 - # 905 - # 910 
- - Val Gln Gly His Arg Gln Leu Asp Ser Tyr Ly - #s Asn Gly Phe Leu Asn 
915 - # 920 - # 925 
- - Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pr - #o Leu Ala Ala Pro Arg 
930 - # 935 - # 940 
- - His Gln Tyr Tyr Asn Gln Glu Trp Thr Leu Tr - #p Asp Arg Phe Glu Val 
945 9 - #50 9 - #55 9 - 
#60 
- - Gln Gly Leu Gln Pro Asn Gly Glu Glu Met Th - #r Leu Lys Gln Phe 
Leu 
965 - # 970 - # 975 
- - Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Il - #e Thr Met Leu Ser Gln 
980 - # 985 - # 990 
- - Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pr - #o Ala Ala Lys Leu Lys 
995 - # 1000 - # 1005 
- - Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Va - #l Ser Arg Val Ser Lys 
1010 - # 1015 - # 1020 
- - Arg Lys Leu Gly Arg His Val Arg Ala Leu Va - #l Leu Glu Leu Cys Cys 
1025 1030 - # 1035 - # 1040 
- - Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pr - #o Tyr Val Arg Tyr Thr 
1045 - # 1050 - # 1055 
- - Ile Arg 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 458 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..456 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - ATG TCG ACC CCG GCC CGG AGG AGG CTC ATG CG - #G GAT TTC AAG CGG TTA 
48 
Met Ser Thr Pro Ala Arg Arg Arg Leu Met Ar - #g Asp Phe Lys Arg Leu 
1 5 - # 10 - # 15 
- - CAA GAG GAC CCA CCT GTG GGT GTC AGT GGC GC - #A CCA TCT GAA AAC AAC 
96 
Gln Glu Asp Pro Pro Val Gly Val Ser Gly Al - #a Pro Ser Glu Asn Asn 
20 - # 25 - # 30 
- - ATC ATG CAG TGG AAT GCA GTT ATA TTT GGA CC - #A GAA GGG ACA CCT TTT 
144 
Ile Met Gln Trp Asn Ala Val Ile Phe Gly Pr - #o Glu Gly Thr Pro Phe 
35 - # 40 - # 45 
- - GAA GAT GGT ACT TTT AAA CTA GTA ATA GAA TT - #T TCT GAA GAA TAT CCA 
192 
Glu Asp Gly Thr Phe Lys Leu Val Ile Glu Ph - #e Ser Glu Glu Tyr Pro 
50 - # 55 - # 60 
- - AAT AAA CCA CCA ACT GTT AGG TTT TTA TCC AA - #A ATG TTT CAT CCA AAT 
240 
Asn Lys Pro Pro Thr Val Arg Phe Leu Ser Ly - #s Met Phe His Pro Asn 
65 - # 70 - # 75 - # 80 
- - GTG TAT GCT GAT GGT AGC ATA TGT TTA GAT AT - #C CTT CAG AAT CGA TGG 
288 
Val Tyr Ala Asp Gly Ser Ile Cys Leu Asp Il - #e Leu Gln Asn Arg Trp 
85 - # 90 - # 95 
- - AGT CCA ACA TAT GAT GTA TCT TCT ATC TTA AC - #A TCA ATT CAG TCT CTG 
336 
Ser Pro Thr Tyr Asp Val Ser Ser Ile Leu Th - #r Ser Ile Gln Ser Leu 
100 - # 105 - # 110 
- - CTG GAT GAA CCG AAT CCT AAC AGT CCA GCC AA - #T AGC CAG GCA GCA CAG 
384 
Leu Asp Glu Pro Asn Pro Asn Ser Pro Ala As - #n Ser Gln Ala Ala Gln 
115 - # 120 - # 125 
- - CTT TAT CAG GAA AAC AAA CGA GAA TAT GAG AA - #A AGA GTT TCG GCC ATT 
432 
Leu Tyr Gln Glu Asn Lys Arg Glu Tyr Glu Ly - #s Arg Val Ser Ala Ile 
130 - # 135 - # 140 
- - GTT GAA CAA AGC TGG AAT GAT TCA TA - # - # 
458 
Val Glu Gln Ser Trp Asn Asp Ser 
145 1 - #50 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 152 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- - Met Ser Thr Pro Ala Arg Arg Arg Leu Met Ar - #g Asp Phe Lys Arg Leu 
1 5 - # 10 - # 15 
- - Gln Glu Asp Pro Pro Val Gly Val Ser Gly Al - #a Pro Ser Glu Asn Asn 
20 - # 25 - # 30 
- - Ile Met Gln Trp Asn Ala Val Ile Phe Gly Pr - #o Glu Gly Thr Pro Phe 
35 - # 40 - # 45 
- - Glu Asp Gly Thr Phe Lys Leu Val Ile Glu Ph - #e Ser Glu Glu Tyr Pro 
50 - # 55 - # 60 
- - Asn Lys Pro Pro Thr Val Arg Phe Leu Ser Ly - #s Met Phe His Pro Asn 
65 - # 70 - # 75 - # 80 
- - Val Tyr Ala Asp Gly Ser Ile Cys Leu Asp Il - #e Leu Gln Asn Arg Trp 
85 - # 90 - # 95 
- - Ser Pro Thr Tyr Asp Val Ser Ser Ile Leu Th - #r Ser Ile Gln Ser Leu 
100 - # 105 - # 110 
- - Leu Asp Glu Pro Asn Pro Asn Ser Pro Ala As - #n Ser Gln Ala Ala Gln 
115 - # 120 - # 125 
- - Leu Tyr Gln Glu Asn Lys Arg Glu Tyr Glu Ly - #s Arg Val Ser Ala Ile 
130 - # 135 - # 140 
- - Val Glu Gln Ser Trp Asn Asp Ser 
145 1 - #50 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 476 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..474 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- - ATG GCG CGC TTT GAG GAT CCA ACA CGG CGA CC - #C TAC AAG CTA CCT GAT 
48 
Met Ala Arg Phe Glu Asp Pro Thr Arg Arg Pr - #o Tyr Lys Leu Pro Asp 
1 5 - # 10 - # 15 
- - CTG TGC ACG GAA CTG AAC ACT TCA CTG CAA GA - #C ATA GAA ATA ACC TGT 
96 
Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln As - #p Ile Glu Ile Thr Cys 
20 - # 25 - # 30 
- - GTA TAT TGC AAG ACA GTA TTG GAA CTT ACA GA - #G GTA TTT GAA TTT GCA 
144 
Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Gl - #u Val Phe Glu Phe Ala 
35 - # 40 - # 45 
- - TTT AAA GAT TTA TTT GTG GTG TAT AGA GAC AG - #T ATA CCG CAT GCT GCA 
192 
Phe Lys Asp Leu Phe Val Val Tyr Arg Asp Se - #r Ile Pro His Ala Ala 
50 - # 55 - # 60 
- - TGC CAT AAA TGT ATA GAT TTT TAT TCT AGA AT - #T AGA GAA TTA AGA CAT 
240 
Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Il - #e Arg Glu Leu Arg His 
65 - # 70 - # 75 - # 80 
- - TAT TCA GAC TCT GTG TAT GGA GAC ACA TTG GA - #A AAA CTA ACT AAC ACT 
288 
Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Gl - #u Lys Leu Thr Asn Thr 
85 - # 90 - # 95 
- - GGG TTA TAC AAT TTA TTA ATA AGG TGC CTG CG - #G TGC CAG AAA CCG TTG 
336 
Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Ar - #g Cys Gln Lys Pro Leu 
100 - # 105 - # 110 
- - AAT CCA GCA GAA AAA CTT AGA CAC CTT AAT GA - #A AAA CGA CGA TTT CAC 
384 
Asn Pro Ala Glu Lys Leu Arg His Leu Asn Gl - #u Lys Arg Arg Phe His 
115 - # 120 - # 125 
- - AAC ATA GCT GGG CAC TAT AGA GGC CAG TGC CA - #T TCG TGC TGC AAC CGA 
432 
Asn Ile Ala Gly His Tyr Arg Gly Gln Cys Hi - #s Ser Cys Cys Asn Arg 
130 - # 135 - # 140 
- - GCA CGA CAG GAA CGA CTC CAA CGA CGC AGA GA - #A ACA CAA GTA 
- # 474 
Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Gl - #u Thr Gln Val 
145 1 - #50 1 - #55 
- - TA - # - # - # 
476 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 158 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- - Met Ala Arg Phe Glu Asp Pro Thr Arg Arg Pr - #o Tyr Lys Leu Pro Asp 
1 5 - # 10 - # 15 
- - Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln As - #p Ile Glu Ile Thr Cys 
20 - # 25 - # 30 
- - Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Gl - #u Val Phe Glu Phe Ala 
35 - # 40 - # 45 
- - Phe Lys Asp Leu Phe Val Val Tyr Arg Asp Se - #r Ile Pro His Ala Ala 
50 - # 55 - # 60 
- - Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Il - #e Arg Glu Leu Arg His 
65 - # 70 - # 75 - # 80 
- - Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Gl - #u Lys Leu Thr Asn Thr 
85 - # 90 - # 95 
- - Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Ar - #g Cys Gln Lys Pro Leu 
100 - # 105 - # 110 
- - Asn Pro Ala Glu Lys Leu Arg His Leu Asn Gl - #u Lys Arg Arg Phe His 
115 - # 120 - # 125 
- - Asn Ile Ala Gly His Tyr Arg Gly Gln Cys Hi - #s Ser Cys Cys Asn Arg 
130 - # 135 - # 140 
- - Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Gl - #u Thr Gln Val 
145 1 - #50 1 - #55 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2624 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..2622 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- - TCA GGA GAA CCT CAG TCT GAC GAC ATT GAA GC - #T AGC CGA ATG AAG CGA 
48 
Ser Gly Glu Pro Gln Ser Asp Asp Ile Glu Al - #a Ser Arg Met Lys Arg 
1 5 - # 10 - # 15 
- - GCA GCT GCA AAG CAT CTA ATA GAA CGC TAC TA - #C CAC CAG TTA ACT GAG 
96 
Ala Ala Ala Lys His Leu Ile Glu Arg Tyr Ty - #r His Gln Leu Thr Glu 
20 - # 25 - # 30 
- - GGC TGT GGA AAT GAA GCC TGC ACG AAT GAG TT - #T TGT GCT TCC TGT CCA 
144 
Gly Cys Gly Asn Glu Ala Cys Thr Asn Glu Ph - #e Cys Ala Ser Cys Pro 
35 - # 40 - # 45 
- - ACT TTT CTT CGT ATG GAT AAT AAT GCA GCA GC - #T ATT AAA GCC CTC GAG 
192 
Thr Phe Leu Arg Met Asp Asn Asn Ala Ala Al - #a Ile Lys Ala Leu Glu 
50 - # 55 - # 60 
- - CTT TAT AAG ATT AAT GCA AAA CTC TGT GAT CC - #T CAT CCC TCC AAG AAA 
240 
Leu Tyr Lys Ile Asn Ala Lys Leu Cys Asp Pr - #o His Pro Ser Lys Lys 
65 - # 70 - # 75 - # 80 
- - GGA GCA AGC TCA GCT TAC CTT GAG AAC TCG AA - #A GGT GCC CCC AAC AAC 
288 
Gly Ala Ser Ser Ala Tyr Leu Glu Asn Ser Ly - #s Gly Ala Pro Asn Asn 
85 - # 90 - # 95 
- - TCC TGC TCT GAG ATA AAA ATG AAC AAG AAA GG - #C GCT AGA ATT GAT TTT 
336 
Ser Cys Ser Glu Ile Lys Met Asn Lys Lys Gl - #y Ala Arg Ile Asp Phe 
100 - # 105 - # 110 
- - AAA GAT GTG ACT TAC TTA ACA GAA GAG AAG GT - #A TAT GAA ATT CTT GAA 
384 
Lys Asp Val Thr Tyr Leu Thr Glu Glu Lys Va - #l Tyr Glu Ile Leu Glu 
115 - # 120 - # 125 
- - TTA TGT AGA GAA AGA GAG GAT TAT TCC CCT TT - #A ATC CGT GTT ATT GGA 
432 
Leu Cys Arg Glu Arg Glu Asp Tyr Ser Pro Le - #u Ile Arg Val Ile Gly 
130 - # 135 - # 140 
- - AGA GTT TTT TCT AGT GCT GAG GCA TTG GTA CA - #G AGC TTC CGG AAA GTT 
480 
Arg Val Phe Ser Ser Ala Glu Ala Leu Val Gl - #n Ser Phe Arg Lys Val 
145 1 - #50 1 - #55 1 - 
#60 
- - AAA CAA CAC ACC AAG GAA GAA CTG AAA TCT CT - #T CAA GCA AAA GAT 
GAA 528 
Lys Gln His Thr Lys Glu Glu Leu Lys Ser Le - #u Gln Ala Lys Asp Glu 
165 - # 170 - # 175 
- - GAC AAA GAT GAA GAT GAA AAG GAA AAA GCT GC - #A TGT TCT GCT GCT GCT 
576 
Asp Lys Asp Glu Asp Glu Lys Glu Lys Ala Al - #a Cys Ser Ala Ala Ala 
180 - # 185 - # 190 
- - ATG GAA GAA GAC TCA GAA GCA TCT TCC TCA AG - #G ATA GGT GAT AGC TCA 
624 
Met Glu Glu Asp Ser Glu Ala Ser Ser Ser Ar - #g Ile Gly Asp Ser Ser 
195 - # 200 - # 205 
- - CAG GGA GAC AAC AAT TTG CAA AAA TTA GGC CC - #T GAT GAT GTG TCT GTG 
672 
Gln Gly Asp Asn Asn Leu Gln Lys Leu Gly Pr - #o Asp Asp Val Ser Val 
210 - # 215 - # 220 
- - GAT ATT GAT GCC ATT AGA AGG GTC TAC ACC AG - #A TTG CTC TCT AAT GAA 
720 
Asp Ile Asp Ala Ile Arg Arg Val Tyr Thr Ar - #g Leu Leu Ser Asn Glu 
225 2 - #30 2 - #35 2 - 
#40 
- - AAA ATT GAA ACT GCC TTT CTC AAT GCA CTT GT - #A TAT TTG TCA CCT 
AAC 768 
Lys Ile Glu Thr Ala Phe Leu Asn Ala Leu Va - #l Tyr Leu Ser Pro Asn 
245 - # 250 - # 255 
- - GTG GAA TGT GAC TTG ACG TAT CAC AAT GTA TA - #C TCT CGA GAT CCT AAT 
816 
Val Glu Cys Asp Leu Thr Tyr His Asn Val Ty - #r Ser Arg Asp Pro Asn 
260 - # 265 - # 270 
- - TAT CTG AAT TTG TTC ATT ATC GGA ATG GAG AA - #T AGA AAT CTC CAC AGT 
864 
Tyr Leu Asn Leu Phe Ile Ile Gly Met Glu As - #n Arg Asn Leu His Ser 
275 - # 280 - # 285 
- - CCT GAA TAT CTG GAA ATG GCT TTG CCA TTA TT - #T TGC AAA GCG ATG AGC 
912 
Pro Glu Tyr Leu Glu Met Ala Leu Pro Leu Ph - #e Cys Lys Ala Met Ser 
290 - # 295 - # 300 
- - AAG CTA CCC CTT GCA GCC CAA GGA AAA CTG AT - #C AGA CTG TGG TCT AAA 
960 
Lys Leu Pro Leu Ala Ala Gln Gly Lys Leu Il - #e Arg Leu Trp Ser Lys 
305 3 - #10 3 - #15 3 - 
#20 
- - TAC AAT GCA GAC CAG ATT CGG AGA ATG ATG GA - #G ACA TTT CAG CAA 
CTT 1008 
Tyr Asn Ala Asp Gln Ile Arg Arg Met Met Gl - #u Thr Phe Gln Gln Leu 
325 - # 330 - # 335 
- - ATT ACT TAT AAA GTC ATA AGC AAT GAA TTT AA - #C AGT CGA AAT CTA GTG 
1056 
Ile Thr Tyr Lys Val Ile Ser Asn Glu Phe As - #n Ser Arg Asn Leu Val 
340 - # 345 - # 350 
- - AAT GAA TTT AAC AGT CGA AAT CTA GTG AAT GA - #T GAT GAT GCC ATT GTT 
1104 
Asn Glu Phe Asn Ser Arg Asn Leu Val Asn As - #p Asp Asp Ala Ile Val 
355 - # 360 - # 365 
- - GCT GCT TCG AAG TGC TTG AAA ATG GTT TAC TA - #T GCA AAT GTA GTG GGA 
1152 
Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Ty - #r Ala Asn Val Val Gly 
370 - # 375 - # 380 
- - GGG GAA GTG GAC ACA AAT CAC AAT GAA GAA GA - #T GAT GAA GAG CCC ATC 
1200 
Gly Glu Val Asp Thr Asn His Asn Glu Glu As - #p Asp Glu Glu Pro Ile 
385 3 - #90 3 - #95 4 - 
#00 
- - CCT GAG TCC AGC GAG CTG ACA CTT CAG GAA CT - #T TTG GGA GAA GAA 
AGA 1248 
Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Le - #u Leu Gly Glu Glu Arg 
405 - # 410 - # 415 
- - AGA AAC AAG AAA GGT CTT CGA GTG GAC CCC CT - #G GAA ACT GAA CTT GGT 
1296 
Arg Asn Lys Lys Gly Leu Arg Val Asp Pro Le - #u Glu Thr Glu Leu Gly 
420 - # 425 - # 430 
- - GTT AAA ACC CTG GAT TGT CGA AAA CCA CTT AT - #C CCT TTT GAA GAG TTT 
1344 
Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Il - #e Pro Phe Glu Glu Phe 
435 - # 440 - # 445 
- - ATT AAT GAA CCA CTG AAT GAG GTT CTA GAA AT - #G GAT AAA GAT TAT ACT 
1392 
Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Me - #t Asp Lys Asp Tyr Thr 
450 - # 455 - # 460 
- - TTT TTC AAA GTA GAA ACA GAG AAC AAA TTC TC - #T TTT ATG ACA TGT CCC 
1440 
Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Se - #r Phe Met Thr Cys Pro 
465 4 - #70 4 - #75 4 - 
#80 
- - TTT ATA TTG AAT GCT GTC ACA AAG AAT TTG GG - #A TTA TAT TAT GAC 
AAT 1488 
Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gl - #y Leu Tyr Tyr Asp Asn 
485 - # 490 - # 495 
- - AGA ATT CGC ATG TAC AGT GAA CGA AGA ATC AC - #T GTT CTC TAC AGC TTA 
1536 
Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Th - #r Val Leu Tyr Ser Leu 
500 - # 505 - # 510 
- - GTT CAA GGA CAG CAG TTG AAT CCA TAT TTG AG - #A CTC AAA GTT AGA CGT 
1584 
Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Ar - #g Leu Lys Val Arg Arg 
515 - # 520 - # 525 
- - GAC CAT ATC ATA GAT GAT GCA CTT GTC CGG CT - #A GAG ATG ATC GCT ATG 
1632 
Asp His Ile Ile Asp Asp Ala Leu Val Arg Le - #u Glu Met Ile Ala Met 
530 - # 535 - # 540 
- - GAA AAT CCT GCA GAC TTG AAG AAG CAG TTG TA - #T GTG GAA TTT GAA GGA 
1680 
Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Ty - #r Val Glu Phe Glu Gly 
545 5 - #50 5 - #55 5 - 
#60 
- - GAA CAA GGA GTT GAT GAG GGA GGT GTT TCC AA - #A GAA TTT TTT CAG 
CTG 1728 
Glu Gln Gly Val Asp Glu Gly Gly Val Ser Ly - #s Glu Phe Phe Gln Leu 
565 - # 570 - # 575 
- - GTT GTG GAG GAA ATC TTC AAT CCA GAT ATT GG - #T ATG TTC ACA TAC GAT 
1776 
Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gl - #y Met Phe Thr Tyr Asp 
580 - # 585 - # 590 
- - GAA TCT ACA AAA TTG TTT TGG TTT AAT CCA TC - #T TCT TTT GAA ACA GAG 
1824 
Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Se - #r Ser Phe Glu Thr Glu 
595 - # 600 - # 605 
- - GGT CAG TTT ACT CTG ATT GGC ATA GTA CTG GG - #T CTG GCT ATT TAC AAT 
1872 
Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gl - #y Leu Ala Ile Tyr Asn 
610 - # 615 - # 620 
- - AAC TGT ATA CTG GAT GTA CAT TTT CCC ATG GT - #T GTC TAC AGG AAG CTA 
1920 
Asn Cys Ile Leu Asp Val His Phe Pro Met Va - #l Val Tyr Arg Lys Leu 
625 6 - #30 6 - #35 6 - 
#40 
- - ATG GGG AAA AAA GGA CTT TTC GTC GAC TTG GG - #A GAC TCT CAC CCA 
GTT 1968 
Met Gly Lys Lys Gly Leu Phe Val Asp Leu Gl - #y Asp Ser His Pro Val 
645 - # 650 - # 655 
- - CTA TAT CAG AGT TTA AAA GAT TTA TTG GAG TA - #T GTT GGG AAT GTG GAA 
2016 
Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Ty - #r Val Gly Asn Val Glu 
660 - # 665 - # 670 
- - GAT GAC ATG ATG ATC ACT TTC CAG ATA TCA CA - #G ACA AAT CTT TTT GGT 
2064 
Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gl - #n Thr Asn Leu Phe Gly 
675 - # 680 - # 685 
- - AAC CCA ATG ATG TAT GAT CTA AAG GAA AAT GG - #T GAT AAA ATT CCA ATT 
2112 
Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gl - #y Asp Lys Ile Pro Ile 
690 - # 695 - # 700 
- - ACA AAT GAA AAC AGG AAG GAA TTT GTC AAT CT - #T TAT TCT GAC TAC ATT 
2160 
Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Le - #u Tyr Ser Asp Tyr Ile 
705 7 - #10 7 - #15 7 - 
#20 
- - CTC AAT AAA TCA GTA GAA AAA CAG TTC AAG GC - #T TTT CGG AGA GGT 
TTT 2208 
Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Al - #a Phe Arg Arg Gly Phe 
725 - # 730 - # 735 
- - CAT ATG GTG ACC AAT GAA TCT CCC TTA AAG TA - #C TTA TTC AGA CCA GAA 
2256 
His Met Val Thr Asn Glu Ser Pro Leu Lys Ty - #r Leu Phe Arg Pro Glu 
740 - # 745 - # 750 
- - GAA ATT GAA TTG CTT ATA TGT GGA AGC CGC AA - #T CTA GAT TTC CAA GCA 
2304 
Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg As - #n Leu Asp Phe Gln Ala 
755 - # 760 - # 765 
- - CTA GAA GAA ACT ACA GAA TAT GAC GGT GGC TA - #T ACC AGG GAC TCT GTT 
2352 
Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Ty - #r Thr Arg Asp Ser Val 
770 - # 775 - # 780 
- - CTG ATT AGG GAG TTC TGG GAA ATC GTT CAT TC - #A TTT ACA GAT GAA CAG 
2400 
Leu Ile Arg Glu Phe Trp Glu Ile Val His Se - #r Phe Thr Asp Glu Gln 
785 7 - #90 7 - #95 8 - 
#00 
- - AAA AGA CTC TTC TTG CAG TTT ACA ACG GGC AC - #A GAC AGA GCA CCT 
GTG 2448 
Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Th - #r Asp Arg Ala Pro Val 
805 - # 810 - # 815 
- - GGA GGA CTA GGA AAA TTA AAG ATG ATT ATA GC - #C AAA AAT GGC CCA GAC 
2496 
Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Al - #a Lys Asn Gly Pro Asp 
820 - # 825 - # 830 
- - ACA GAA AGG TTA CCT ACA TCT CAT ACT TGC TT - #T AAT GTG CTT TTA CTT 
2544 
Thr Glu Arg Leu Pro Thr Ser His Thr Cys Ph - #e Asn Val Leu Leu Leu 
835 - # 840 - # 845 
- - CCG GAA TAC TCA AGC AAA GAA AAA CTT AAA GA - #G AGA TTG TTG AAG GCC 
2592 
Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Gl - #u Arg Leu Leu Lys Ala 
850 - # 855 - # 860 
- - ATC ACG TAT GCC AAA GGA TTT GGC ATG CTG TA - # - # 
2624 
Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu 
865 8 - #70 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 874 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- - Ser Gly Glu Pro Gln Ser Asp Asp Ile Glu Al - #a Ser Arg Met Lys Arg 
1 5 - # 10 - # 15 
- - Ala Ala Ala Lys His Leu Ile Glu Arg Tyr Ty - #r His Gln Leu Thr Glu 
20 - # 25 - # 30 
- - Gly Cys Gly Asn Glu Ala Cys Thr Asn Glu Ph - #e Cys Ala Ser Cys Pro 
35 - # 40 - # 45 
- - Thr Phe Leu Arg Met Asp Asn Asn Ala Ala Al - #a Ile Lys Ala Leu Glu 
50 - # 55 - # 60 
- - Leu Tyr Lys Ile Asn Ala Lys Leu Cys Asp Pr - #o His Pro Ser Lys Lys 
65 - # 70 - # 75 - # 80 
- - Gly Ala Ser Ser Ala Tyr Leu Glu Asn Ser Ly - #s Gly Ala Pro Asn Asn 
85 - # 90 - # 95 
- - Ser Cys Ser Glu Ile Lys Met Asn Lys Lys Gl - #y Ala Arg Ile Asp Phe 
100 - # 105 - # 110 
- - Lys Asp Val Thr Tyr Leu Thr Glu Glu Lys Va - #l Tyr Glu Ile Leu Glu 
115 - # 120 - # 125 
- - Leu Cys Arg Glu Arg Glu Asp Tyr Ser Pro Le - #u Ile Arg Val Ile Gly 
130 - # 135 - # 140 
- - Arg Val Phe Ser Ser Ala Glu Ala Leu Val Gl - #n Ser Phe Arg Lys Val 
145 1 - #50 1 - #55 1 - 
#60 
- - Lys Gln His Thr Lys Glu Glu Leu Lys Ser Le - #u Gln Ala Lys Asp 
Glu 
165 - # 170 - # 175 
- - Asp Lys Asp Glu Asp Glu Lys Glu Lys Ala Al - #a Cys Ser Ala Ala Ala 
180 - # 185 - # 190 
- - Met Glu Glu Asp Ser Glu Ala Ser Ser Ser Ar - #g Ile Gly Asp Ser Ser 
195 - # 200 - # 205 
- - Gln Gly Asp Asn Asn Leu Gln Lys Leu Gly Pr - #o Asp Asp Val Ser Val 
210 - # 215 - # 220 
- - Asp Ile Asp Ala Ile Arg Arg Val Tyr Thr Ar - #g Leu Leu Ser Asn Glu 
225 2 - #30 2 - #35 2 - 
#40 
- - Lys Ile Glu Thr Ala Phe Leu Asn Ala Leu Va - #l Tyr Leu Ser Pro 
Asn 
245 - # 250 - # 255 
- - Val Glu Cys Asp Leu Thr Tyr His Asn Val Ty - #r Ser Arg Asp Pro Asn 
260 - # 265 - # 270 
- - Tyr Leu Asn Leu Phe Ile Ile Gly Met Glu As - #n Arg Asn Leu His Ser 
275 - # 280 - # 285 
- - Pro Glu Tyr Leu Glu Met Ala Leu Pro Leu Ph - #e Cys Lys Ala Met Ser 
290 - # 295 - # 300 
- - Lys Leu Pro Leu Ala Ala Gln Gly Lys Leu Il - #e Arg Leu Trp Ser Lys 
305 3 - #10 3 - #15 3 - 
#20 
- - Tyr Asn Ala Asp Gln Ile Arg Arg Met Met Gl - #u Thr Phe Gln Gln 
Leu 
325 - # 330 - # 335 
- - Ile Thr Tyr Lys Val Ile Ser Asn Glu Phe As - #n Ser Arg Asn Leu Val 
340 - # 345 - # 350 
- - Asn Glu Phe Asn Ser Arg Asn Leu Val Asn As - #p Asp Asp Ala Ile Val 
355 - # 360 - # 365 
- - Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Ty - #r Ala Asn Val Val Gly 
370 - # 375 - # 380 
- - Gly Glu Val Asp Thr Asn His Asn Glu Glu As - #p Asp Glu Glu Pro Ile 
385 3 - #90 3 - #95 4 - 
#00 
- - Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Le - #u Leu Gly Glu Glu 
Arg 
405 - # 410 - # 415 
- - Arg Asn Lys Lys Gly Leu Arg Val Asp Pro Le - #u Glu Thr Glu Leu Gly 
420 - # 425 - # 430 
- - Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Il - #e Pro Phe Glu Glu Phe 
435 - # 440 - # 445 
- - Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Me - #t Asp Lys Asp Tyr Thr 
450 - # 455 - # 460 
- - Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Se - #r Phe Met Thr Cys Pro 
465 4 - #70 4 - #75 4 - 
#80 
- - Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gl - #y Leu Tyr Tyr Asp 
Asn 
485 - # 490 - # 495 
- - Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Th - #r Val Leu Tyr Ser Leu 
500 - # 505 - # 510 
- - Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Ar - #g Leu Lys Val Arg Arg 
515 - # 520 - # 525 
- - Asp His Ile Ile Asp Asp Ala Leu Val Arg Le - #u Glu Met Ile Ala Met 
530 - # 535 - # 540 
- - Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Ty - #r Val Glu Phe Glu Gly 
545 5 - #50 5 - #55 5 - 
#60 
- - Glu Gln Gly Val Asp Glu Gly Gly Val Ser Ly - #s Glu Phe Phe Gln 
Leu 
565 - # 570 - # 575 
- - Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gl - #y Met Phe Thr Tyr Asp 
580 - # 585 - # 590 
- - Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Se - #r Ser Phe Glu Thr Glu 
595 - # 600 - # 605 
- - Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gl - #y Leu Ala Ile Tyr Asn 
610 - # 615 - # 620 
- - Asn Cys Ile Leu Asp Val His Phe Pro Met Va - #l Val Tyr Arg Lys Leu 
625 6 - #30 6 - #35 6 - 
#40 
- - Met Gly Lys Lys Gly Leu Phe Val Asp Leu Gl - #y Asp Ser His Pro 
Val 
645 - # 650 - # 655 
- - Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Ty - #r Val Gly Asn Val Glu 
660 - # 665 - # 670 
- - Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gl - #n Thr Asn Leu Phe Gly 
675 - # 680 - # 685 
- - Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gl - #y Asp Lys Ile Pro Ile 
690 - # 695 - # 700 
- - Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Le - #u Tyr Ser Asp Tyr Ile 
705 7 - #10 7 - #15 7 - 
#20 
- - Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Al - #a Phe Arg Arg Gly 
Phe 
725 - # 730 - # 735 
- - His Met Val Thr Asn Glu Ser Pro Leu Lys Ty - #r Leu Phe Arg Pro Glu 
740 - # 745 - # 750 
- - Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg As - #n Leu Asp Phe Gln Ala 
755 - # 760 - # 765 
- - Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Ty - #r Thr Arg Asp Ser Val 
770 - # 775 - # 780 
- - Leu Ile Arg Glu Phe Trp Glu Ile Val His Se - #r Phe Thr Asp Glu Gln 
785 7 - #90 7 - #95 8 - 
#00 
- - Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Th - #r Asp Arg Ala Pro 
Val 
805 - # 810 - # 815 
- - Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Al - #a Lys Asn Gly Pro Asp 
820 - # 825 - # 830 
- - Thr Glu Arg Leu Pro Thr Ser His Thr Cys Ph - #e Asn Val Leu Leu Leu 
835 - # 840 - # 845 
- - Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Gl - #u Arg Leu Leu Lys Ala 
850 - # 855 - # 860 
- - Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu 
865 8 - #70 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1181 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1179 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- - ATG GAG GAG CCG CAG TCA GAT CCT AGC GTC GA - #G CCC CCT CTG AGT CAG 
48 
Met Glu Glu Pro Gln Ser Asp Pro Ser Val Gl - #u Pro Pro Leu Ser Gln 
1 5 - # 10 - # 15 
- - GAA ACA TTT TCA GAC CTA TGG AAA CTA CTT CC - #T GAA AAC AAC GTT CTG 
96 
Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pr - #o Glu Asn Asn Val Leu 
20 - # 25 - # 30 
- - TCC CCC TTG CCG TCC CAA GCA ATG GAT GAT TT - #G ATG CTG TCC CCG GAC 
144 
Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Le - #u Met Leu Ser Pro Asp 
35 - # 40 - # 45 
- - GAT ATT GAA CAA TGG TTC ACT GAA GAC CCA GG - #T CCA GAT GAA GCT CCC 
192 
Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gl - #y Pro Asp Glu Ala Pro 
50 - # 55 - # 60 
- - AGA ATG CCA GAG GCT GCT CCC CCC GTG GCC CC - #T GCA CCA GCA GCT CCT 
240 
Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pr - #o Ala Pro Ala Ala Pro 
65 - # 70 - # 75 - # 80 
- - ACA CCG GCG GCC CCT GCA CCA GCC CCC TCC TG - #G CCC CTG TCA TCT TCT 
288 
Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Tr - #p Pro Leu Ser Ser Ser 
85 - # 90 - # 95 
- - GTC CCT TCC CAG AAA ACC TAC CAG GGC AGC TA - #C GGT TTC CGT CTG GGC 
336 
Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Ty - #r Gly Phe Arg Leu Gly 
100 - # 105 - # 110 
- - TTC TTG CAT TCT GGG ACA GCC AAG TCT GTG AC - #T TGC ACG TAC TCC CCT 
384 
Phe Leu His Ser Gly Thr Ala Lys Ser Val Th - #r Cys Thr Tyr Ser Pro 
115 - # 120 - # 125 
- - GCC CTC AAC AAG ATG TTT TGC CAA CTG GCC AA - #G ACC TGC CCT GTG CAG 
432 
Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Ly - #s Thr Cys Pro Val Gln 
130 - # 135 - # 140 
- - CTG TGG GTT GAT TCC ACA CCC CCG CCC GGC AC - #C CGC GTC CGC GCC ATG 
480 
Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Th - #r Arg Val Arg Ala Met 
145 1 - #50 1 - #55 1 - 
#60 
- - GCC ATC TAC AAG CAG TCA CAG CAC ATG ACG GA - #G GTT GTG AGG CGC 
TGC 528 
Ala Ile Tyr Lys Gln Ser Gln His Met Thr Gl - #u Val Val Arg Arg Cys 
165 - # 170 - # 175 
- - CCC CAC CAT GAG CGC TGC TCA GAT AGC GAT GG - #T CTG GCC CCT CCT CAG 
576 
Pro His His Glu Arg Cys Ser Asp Ser Asp Gl - #y Leu Ala Pro Pro Gln 
180 - # 185 - # 190 
- - CAT CTT ATC CGA GTG GAA GGA AAT TTG CGT GT - #G GAG TAT TTG GAT GAC 
624 
His Leu Ile Arg Val Glu Gly Asn Leu Arg Va - #l Glu Tyr Leu Asp Asp 
195 - # 200 - # 205 
- - AGA AAC ACT TTT CGA CAT AGT GTG GTG GTG CC - #C TAT GAG CCG CCT GAG 
672 
Arg Asn Thr Phe Arg His Ser Val Val Val Pr - #o Tyr Glu Pro Pro Glu 
210 - # 215 - # 220 
- - GTT GGC TCT GAC TGT ACC ACC ATC CAC TAC AA - #C TAC ATG TGT AAC AGT 
720 
Val Gly Ser Asp Cys Thr Thr Ile His Tyr As - #n Tyr Met Cys Asn Ser 
225 2 - #30 2 - #35 2 - 
#40 
- - TCC TGC ATG GGC GGC ATG AAC CGG AGG CCC AT - #C CTC ACC ATC ATC 
ACA 768 
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Il - #e Leu Thr Ile Ile Thr 
245 - # 250 - # 255 
- - CTG GAA GAC TCC AGT GGT AAT CTA CTG GGA CG - #G AAC AGC TTT GAG GTG 
816 
Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Ar - #g Asn Ser Phe Glu Val 
260 - # 265 - # 270 
- - CGT GTT TGT GCC TGT CCT GGG AGA GAC CGG CG - #C ACA GAG GAA GAG AAT 
864 
Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Ar - #g Thr Glu Glu Glu Asn 
275 - # 280 - # 285 
- - CTC CGC AAG AAA GGG GAG CCT CAC CAC GAG CT - #G CCC CCA GGG AGC ACT 
912 
Leu Arg Lys Lys Gly Glu Pro His His Glu Le - #u Pro Pro Gly Ser Thr 
290 - # 295 - # 300 
- - AAG CGA GCA CTG CCC AAC AAC ACC AGC TCC TC - #T CCC CAG CCA AAG AAG 
960 
Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Se - #r Pro Gln Pro Lys Lys 
305 3 - #10 3 - #15 3 - 
#20 
- - AAA CCA CTG GAT GGA GAA TAT TTC ACC CTT CA - #G ATC CGT GGG CGT 
GAG 1008 
Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gl - #n Ile Arg Gly Arg Glu 
325 - # 330 - # 335 
- - CGC TTC GAG ATG TTC CGA GAG CTG AAT GAG GC - #C TTG GAA CTC AAG GAT 
1056 
Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Al - #a Leu Glu Leu Lys Asp 
340 - # 345 - # 350 
- - GCC CAG GCT GGG AAG GAG CCA GGG GGG AGC AG - #G GCT CAC TCC AGC CAC 
1104 
Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Ar - #g Ala His Ser Ser His 
355 - # 360 - # 365 
- - CTG AAG TCC AAA AAG GGT CAG TCT ACC TCC CG - #C CAT AAA AAA CTC ATG 
1152 
Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Ar - #g His Lys Lys Leu Met 
370 - # 375 - # 380 
- - TTC AAG ACA GAA GGG CCT GAC TCA GAC TG - # - # 
1181 
Phe Lys Thr Glu Gly Pro Asp Ser Asp 
385 3 - #90 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 393 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- - Met Glu Glu Pro Gln Ser Asp Pro Ser Val Gl - #u Pro Pro Leu Ser Gln 
1 5 - # 10 - # 15 
- - Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pr - #o Glu Asn Asn Val Leu 
20 - # 25 - # 30 
- - Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Le - #u Met Leu Ser Pro Asp 
35 - # 40 - # 45 
- - Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gl - #y Pro Asp Glu Ala Pro 
50 - # 55 - # 60 
- - Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pr - #o Ala Pro Ala Ala Pro 
65 - # 70 - # 75 - # 80 
- - Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Tr - #p Pro Leu Ser Ser Ser 
85 - # 90 - # 95 
- - Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Ty - #r Gly Phe Arg Leu Gly 
100 - # 105 - # 110 
- - Phe Leu His Ser Gly Thr Ala Lys Ser Val Th - #r Cys Thr Tyr Ser Pro 
115 - # 120 - # 125 
- - Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Ly - #s Thr Cys Pro Val Gln 
130 - # 135 - # 140 
- - Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Th - #r Arg Val Arg Ala Met 
145 1 - #50 1 - #55 1 - 
#60 
- - Ala Ile Tyr Lys Gln Ser Gln His Met Thr Gl - #u Val Val Arg Arg 
Cys 
165 - # 170 - # 175 
- - Pro His His Glu Arg Cys Ser Asp Ser Asp Gl - #y Leu Ala Pro Pro Gln 
180 - # 185 - # 190 
- - His Leu Ile Arg Val Glu Gly Asn Leu Arg Va - #l Glu Tyr Leu Asp Asp 
195 - # 200 - # 205 
- - Arg Asn Thr Phe Arg His Ser Val Val Val Pr - #o Tyr Glu Pro Pro Glu 
210 - # 215 - # 220 
- - Val Gly Ser Asp Cys Thr Thr Ile His Tyr As - #n Tyr Met Cys Asn Ser 
225 2 - #30 2 - #35 2 - 
#40 
- - Ser Cys Met Gly Gly Met Asn Arg Arg Pro Il - #e Leu Thr Ile Ile 
Thr 
245 - # 250 - # 255 
- - Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Ar - #g Asn Ser Phe Glu Val 
260 - # 265 - # 270 
- - Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Ar - #g Thr Glu Glu Glu Asn 
275 - # 280 - # 285 
- - Leu Arg Lys Lys Gly Glu Pro His His Glu Le - #u Pro Pro Gly Ser Thr 
290 - # 295 - # 300 
- - Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Se - #r Pro Gln Pro Lys Lys 
305 3 - #10 3 - #15 3 - 
#20 
- - Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gl - #n Ile Arg Gly Arg 
Glu 
325 - # 330 - # 335 
- - Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Al - #a Leu Glu Leu Lys Asp 
340 - # 345 - # 350 
- - Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Ar - #g Ala His Ser Ser His 
355 - # 360 - # 365 
- - Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Ar - #g His Lys Lys Leu Met 
370 - # 375 - # 380 
- - Phe Lys Thr Glu Gly Pro Asp Ser Asp 
385 3 - #90 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 897 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..894 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- - ATT GCG GCG GCG CCA GAG CTG CTG GAG CGC TC - #G GGG TCC CCG GGC GGC 
48 
Ile Ala Ala Ala Pro Glu Leu Leu Glu Arg Se - #r Gly Ser Pro Gly Gly 
1 5 - # 10 - # 15 
- - GGC GGC GGC GCA GAG GAG GAG GCA GGC GGC GG - #C CCC GGT GGC TCC CCC 
96 
Gly Gly Gly Ala Glu Glu Glu Ala Gly Gly Gl - #y Pro Gly Gly Ser Pro 
20 - # 25 - # 30 
- - CCG GAC GGT GCG CGG CCC GGC CCG TCT CGC GA - #A CTC GCG GTG GTC GCG 
144 
Pro Asp Gly Ala Arg Pro Gly Pro Ser Arg Gl - #u Leu Ala Val Val Ala 
35 - # 40 - # 45 
- - CGG CCC CGC GCT GCT CCG ACC CCG GGC CCC TC - #C GCC GCC GCC ATG GCT 
192 
Arg Pro Arg Ala Ala Pro Thr Pro Gly Pro Se - #r Ala Ala Ala Met Ala 
50 - # 55 - # 60 
- - CGG CCG CTA GTG CCC AGC TCG CAG AAG GCG CT - #G CTG CTG GAG CTC AAG 
240 
Arg Pro Leu Val Pro Ser Ser Gln Lys Ala Le - #u Leu Leu Glu Leu Lys 
65 - # 70 - # 75 - # 80 
- - GGG CTG CAG GAA GAG CCG GTC GAG GGA TTC CG - #C GTG ACA CTG GTG GAC 
288 
Gly Leu Gln Glu Glu Pro Val Glu Gly Phe Ar - #g Val Thr Leu Val Asp 
85 - # 90 - # 95 
- - GAG GGC GAT CTA TAC AAC TGG GAG GTG GCC AT - #T TTC GGG CCC CCC AAC 
336 
Glu Gly Asp Leu Tyr Asn Trp Glu Val Ala Il - #e Phe Gly Pro Pro Asn 
100 - # 105 - # 110 
- - ACC TAC TAC GAG GGC GGC TAC TTC AAG GCG CG - #C CTC AAG TTC CCC ATC 
384 
Thr Tyr Tyr Glu Gly Gly Tyr Phe Lys Ala Ar - #g Leu Lys Phe Pro Ile 
115 - # 120 - # 125 
- - GAC TAC CCA TAC TCT CCA CCA GCC TTT CGG TT - #C CTG ACC AAG ATG TGG 
432 
Asp Tyr Pro Tyr Ser Pro Pro Ala Phe Arg Ph - #e Leu Thr Lys Met Trp 
130 - # 135 - # 140 
- - CAC CCT AAC ATC TAC GAG ACG GGG GAC GTG TG - #T ATC TCC ATC CTC CAC 
480 
His Pro Asn Ile Tyr Glu Thr Gly Asp Val Cy - #s Ile Ser Ile Leu His 
145 1 - #50 1 - #55 1 - 
#60 
- - CCG CCG GTG GAC GAC CCC CAG AGC GGG GAG CT - #G CCC TCA GAG AGG 
TGG 528 
Pro Pro Val Asp Asp Pro Gln Ser Gly Glu Le - #u Pro Ser Glu Arg Trp 
165 - # 170 - # 175 
- - AAC CCC ACG CAG AAC GTC AGG ACC ATT CTC CT - #G AGT GTG ATC TCC CTC 
576 
Asn Pro Thr Gln Asn Val Arg Thr Ile Leu Le - #u Ser Val Ile Ser Leu 
180 - # 185 - # 190 
- - CTG AAC GAG CCC AAC ACC TTC TCG CCC GCA AA - #C GTG GAC GCC TCC GTG 
624 
Leu Asn Glu Pro Asn Thr Phe Ser Pro Ala As - #n Val Asp Ala Ser Val 
195 - # 200 - # 205 
- - ATG TAC AGG AAG TGG AAA GAG AGC AAG GGG AA - #G GAT CGG GAG TAC ACA 
672 
Met Tyr Arg Lys Trp Lys Glu Ser Lys Gly Ly - #s Asp Arg Glu Tyr Thr 
210 - # 215 - # 220 
- - GAC ATC ATC CGG AAG CAG GTC CTG GGG ACC AA - #G GTG GAC GCG GAG CGT 
720 
Asp Ile Ile Arg Lys Gln Val Leu Gly Thr Ly - #s Val Asp Ala Glu Arg 
225 2 - #30 2 - #35 2 - 
#40 
- - GAC GGC GTG AAG GTG CCC ACC ACG CTG GCC GA - #G TAC TGC GTG AAG 
ACC 768 
Asp Gly Val Lys Val Pro Thr Thr Leu Ala Gl - #u Tyr Cys Val Lys Thr 
245 - # 250 - # 255 
- - AAG GCG CCG GCG CCC GAC GAG GGC TCA GAC CT - #C TTC TAC GAC GAC TAC 
816 
Lys Ala Pro Ala Pro Asp Glu Gly Ser Asp Le - #u Phe Tyr Asp Asp Tyr 
260 - # 265 - # 270 
- - TAC GAG GAC GGC GAG GTG GAG GAG GAG GCC GA - #C AGC TGC TTC GGG GAC 
864 
Tyr Glu Asp Gly Glu Val Glu Glu Glu Ala As - #p Ser Cys Phe Gly Asp 
275 - # 280 - # 285 
- - GAT GAG GAT GAC TCT GGC ACG GAG GAG TCC TG - #A - 
# 897 
Asp Glu Asp Asp Ser Gly Thr Glu Glu Ser 
290 - # 295 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 298 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- - Ile Ala Ala Ala Pro Glu Leu Leu Glu Arg Se - #r Gly Ser Pro Gly 
Gly 
1 5 - # 10 - # 15 
- - Gly Gly Gly Ala Glu Glu Glu Ala Gly Gly Gl - #y Pro Gly Gly Ser Pro 
20 - # 25 - # 30 
- - Pro Asp Gly Ala Arg Pro Gly Pro Ser Arg Gl - #u Leu Ala Val Val Ala 
35 - # 40 - # 45 
- - Arg Pro Arg Ala Ala Pro Thr Pro Gly Pro Se - #r Ala Ala Ala Met Ala 
50 - # 55 - # 60 
- - Arg Pro Leu Val Pro Ser Ser Gln Lys Ala Le - #u Leu Leu Glu Leu Lys 
65 - # 70 - # 75 - # 80 
- - Gly Leu Gln Glu Glu Pro Val Glu Gly Phe Ar - #g Val Thr Leu Val Asp 
85 - # 90 - # 95 
- - Glu Gly Asp Leu Tyr Asn Trp Glu Val Ala Il - #e Phe Gly Pro Pro Asn 
100 - # 105 - # 110 
- - Thr Tyr Tyr Glu Gly Gly Tyr Phe Lys Ala Ar - #g Leu Lys Phe Pro Ile 
115 - # 120 - # 125 
- - Asp Tyr Pro Tyr Ser Pro Pro Ala Phe Arg Ph - #e Leu Thr Lys Met Trp 
130 - # 135 - # 140 
- - His Pro Asn Ile Tyr Glu Thr Gly Asp Val Cy - #s Ile Ser Ile Leu His 
145 1 - #50 1 - #55 1 - 
#60 
- - Pro Pro Val Asp Asp Pro Gln Ser Gly Glu Le - #u Pro Ser Glu Arg 
Trp 
165 - # 170 - # 175 
- - Asn Pro Thr Gln Asn Val Arg Thr Ile Leu Le - #u Ser Val Ile Ser Leu 
180 - # 185 - # 190 
- - Leu Asn Glu Pro Asn Thr Phe Ser Pro Ala As - #n Val Asp Ala Ser Val 
195 - # 200 - # 205 
- - Met Tyr Arg Lys Trp Lys Glu Ser Lys Gly Ly - #s Asp Arg Glu Tyr Thr 
210 - # 215 - # 220 
- - Asp Ile Ile Arg Lys Gln Val Leu Gly Thr Ly - #s Val Asp Ala Glu Arg 
225 2 - #30 2 - #35 2 - 
#40 
- - Asp Gly Val Lys Val Pro Thr Thr Leu Ala Gl - #u Tyr Cys Val Lys 
Thr 
245 - # 250 - # 255 
- - Lys Ala Pro Ala Pro Asp Glu Gly Ser Asp Le - #u Phe Tyr Asp Asp Tyr 
260 - # 265 - # 270 
- - Tyr Glu Asp Gly Glu Val Glu Glu Glu Ala As - #p Ser Cys Phe Gly Asp 
275 - # 280 - # 285 
- - Asp Glu Asp Asp Ser Gly Thr Glu Glu Ser 
290 - # 295 
- - - - (2) INFORMATION FOR SEQ ID NO:26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- - CTACTAATAG GTAGAAGCGG TGG - # - # 
23 
- - - - (2) INFORMATION FOR SEQ ID NO:27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- - GGTAAACCAA AGCACCGACA GGG - # - # 
23 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- - GCGCGCAAGC TTATGTCCAG CTCGCCGCTG TCCAAG - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- - GCGCGCGGAT CCTCAGCGGA TGGTGTATCG GACATA - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- - GCGCGCAAGC TTATGTCGAC CCCGGCCCGG AGGAGG - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- - GCGCGCGAAT TCTTATGAAT CATTCCAGCT TTGTTC - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- - GCGCGCAAGC TTATGGCGCG CTTTGAGGAT CCAACA - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
- - GCGCGCGAAT TCTTATACTT GTGTTTCTCT GCGTCG - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:34: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
- - GCGCGCAAGC TTTCAGGACC TCAGTCTGAC GAC - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:35: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
- - GCGCGCGGAT CCTTACAGCA TGCCAAATCC TTTGGC - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:36: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
- - GCGCGCGAAT TCGCCATGGA GGAGCCGCAG TCAGATCCT - # 
- # 39 
- - - - (2) INFORMATION FOR SEQ ID NO:37: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
- - GCGCGCAAGC TTTCAGTCTG AGTCAGGCCC TTCTGT - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:38: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
- - Pro Val Gly Asp Asp Leu Phe His Trp Xaa 
1 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:39: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
- - Ile Thr Leu Ala Pro Ser Trp 
1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:40: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
- - TCGACGGACA TGCCCGGGCA TGTCCC - # - # 
26 
- - - - (2) INFORMATION FOR SEQ ID NO:41: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
- - TCGCAGGGAC ATGCCCGGGC ATGTCCG - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:42: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
- - TCGACCACGT GGC - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:43: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
- - TCGAGCCACG TGG - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:44: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
- - TCGACGGGGC GGGC - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:45: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
- - TCGAGCCCGC CCCG - # - # 
- # 14 
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