This invention relates to the identification and molecular characterization of NAD:arginine ADP-ribosyltransferases. Sequences from the rabbit skeletal muscle NAD:arginine ADP-ribosyltransferase and the human NAD:arginine ADP-ribosyltransferase are provided herein. Recombinant protein is expressed from a recombinant gene vector containing at least 15 continuous bases of genes encoding these sequences.

RELATED APPLICATIONS 
The present application is a divisional application of International 
Application No. PCT/US93/11569 which in its United States designation is a 
continuation-in-part of U.S. application Ser. No. 07/985,698, filed Nov. 
30, 1992. The complete disclosures of these related applications are 
hereby incorporated herein by this reference thereto. 
FIELD OF THE INVENTION 
This invention relates to vertebrate ADP-ribosyltransferases and 
specifically to mono-ADP-ribosyltransferases. In particular this invention 
relates to the purification, isolation and identification of 
mono-NAD:arginine ADP-ribosyltransferases. 
BACKGROUND OF THE INVENTION 
Mono-ADP-ribosylation is a post-translational event resulting in the 
covalent modification of proteins. ADP-ribosyltransferases and 
ADP-ribosylarginine hydrolases are responsible for the forward and reverse 
reactions that control the ADP-ribosylation of cellular proteins. In some 
systems, i.e. bacterial toxin ADP-ribosyltransferases, the extent of 
protein ADP-ribosylation is a critical determinant of enzymatic activity. 
Mono-ADP-ribosylation is involved in the action of bacterial toxins and in 
the regulation of cellular processes in prokaryotes and eukaryotes (Moss, 
et al. Adv. Enzymol. 61: 303-379 (1988); Lowery, et al. and Williamson, et 
al. ADP-ribosylating Toxins and G Proteins: Insights into Signal 
Transduction, (1990) eds. Moss, J. et al. pp. 459-477 and pp. 493-510). 
Cholera toxin is a secretory product of the microorganism Vibrio cholera. 
This toxin is responsible for the pathogenesis of cholera. The cholera 
toxin directs the ADP-ribosylation of guanine nucleotide-binding proteins 
which in turn enhances their activity and increases the responsiveness of 
some animal cells to various hormones, neurotransmitters and drugs (Moss, 
et al. supra and Ueda, K., et al. Ann. Rev. Biochem. 54:73-100, 1985). 
Several ADP-ribosylating toxins have been cloned from bacteria (Nicosia, et 
al. Proc. Natl. Acad. Sci. (USA) 83: 4631-4635, 1986 and Nemoto, et al. J. 
Biol. Chem. 266: 19312-19319, 1991) and the crystal structures of some of 
the toxins have been solved (Allured, et al. Proc. Natl. Acad. Sci. (USA) 
83: 1320-1324, 1986 and Sixma, et al. Nature 351: 371-377, 1991). While 
the bacterial toxins have similarities to one another in their amino acid 
sequences, the enzymes differ in the amino acids that they modify. 
Arginine, cysteine, asparagine and diphthamide (modified histidine) serve 
as ADP-ribose acceptors for Cholera toxin, pertussis toxin, botulinum C3 
transferase and diphtheria toxin respectively. 
Within prokaryotic and eukaryotic cells, ADP-ribosylation appears to be a 
reversible modification of proteins. An ADP-ribosylation cycle is involved 
in the regulation of the nitrogenase of the photosynthetic bacterium 
Rhodospirillium rubrum (Lowery, et al. supra). Here, an 
ADP-ribosyltransferase is responsible for the inactivation of the 
nitrogenase, whereas an ADP-ribosylarginine hydrolase releases the 
ADP-ribose moiety and activates the nitrogenase. 
The role of mono-ADP-ribosylation in eukaryotes is less well-characterized; 
however, it is postulated that families of mono-ADP-ribosyltransferases 
will be identified in a given species and that these 
mono-ADP-ribosyltransferases will share homologies within their gene 
sequences. Eukaryotic mono-ADP-ribosyltransferases are believed to be 
involved in a number of physiological processes such as the regulation of 
adenylyl cyclase (Obara, et al. Eur. J. Biochem. 200: 75-80, 1991; Brune, 
et al. Proc. Natl. Acad. Sci. USA 87: 3304-3308, 1990; Fendrick, et al. 
Eur. J. Biochem. 205: 25-31 (1992); and Kharadia, et al. Exp. Cell. Res. 
201: 33-42, 1992). While it is believed that there are families of 
mono-ADP-ribosyltransferases located in different tissues, the gene 
sequences of this invention will be useful for verifying this hypothesis. 
Arginine- and cysteine-specific ADP-ribosyltransferases and 
ADP-ribosylarginine and ADP-ribosylcysteine hydrolases have been 
identified in animal tissues, consistent with the presence of 
ADP-ribosylation cycles responsible for the reversible ADP-ribosylation of 
arginine and cysteine residues in proteins (Moss, et al. Proc. Natl. Acad. 
Sci. USA 82: 5603-5607, 1985; Tanuma, et al. J. Biol. Chem. 263: 
5485-5489, 1988; and Tanuma, et al. FEBS Lett. 261: 381-384, 1990). 
ADP-ribosylarginine hydrolase has been purified from turkey erythrocytes 
and rat brain. Further, the ADP-ribosylarginine hydrolase has been cloned 
from rat brain (Moss, et al. J. Biol. Chem. 267: 10481-10488, 1992). NAD: 
Arginine ADP-ribosyltransferases have been purified from turkey 
erythrocytes (Moss, et al. J. Biol. Chem. 255: 5838-5840, 1980; Yost, et 
al. J. Biol. Chem. 258: 4926-4929, 1983) and rabbit skeletal muscle 
(Taniguchi, et al. Biochem. Biophys. Res. Commun. 164: 128-133, 1989 and 
Peterson, et al. J. Biol. Chem. 265: 17062-17069, 1990). However, the gene 
sequences for these enzymes have remained unidentified until now. In 
turkey erythrocytes there is a family of ADP-ribosyltransferase enzymes 
that differ in their localization within the cell as well as in their 
physical, regulatory and kinetic properties (Williamson, et al., Moss, et 
al. J. Biol. Chem., Yost, et at, all supra, and West, et al. Biochemistry 
25: 8057-8062, 1986). The turkey ADP-ribosyltransferases appear to be 
ubiquitous in their tissue distribution, while the rabbit 
ADP-ribosyltransferase is located primarily within the sarcoplasmic 
reticulum of cardiac and skeletal muscle. Neither the RNA nor the DNA 
sequence of any mono-ADP-ribosyltransferases have been previously 
identified from a eukaryotic system 
There are significant differences between the bacterial 
ADP-ribosyltransferases and eukaryotic ribosyltransferases. For example, 
since the bacterial toxins differ from the animal transferases in 
substrate specificity, therapies directed toward ADP-ribosyltransferases 
cannot rely on cloned bacterial enzymes. Eukaryotic 
ADP-ribosyltransferases are required for this work. In addition, the 
bacterial enzymes differ from their mammalian counterpart in their 
sensitivity to inhibitors. Therefore, it would be more valuable to test 
the effect of different inhibitors on eukaryotic enzymes than on their 
bacterial counterparts. Finally, bacterial transferases are targeted by a 
system different from those used with the animal transferases and 
therefore, the recombinant bacterial enzymes may localize to different 
compartments within animal cells. The bacterial toxins function by binding 
to the outside of a eukaryotic cell and delivering their catalytic subunit 
to the cells. The eukaryotic enzymes are intracellular enzymes that are 
required for effective protein regulation. 
Few ADP-ribosyltransferases have been purified from animal cells. Moss, et 
al. purified an ADP-ribosyltransferase from turkey erythrocytes. In 
another example, Peterson, et al. (supra), purified an enzyme from the 
same organ system and species used in the instant invention. This enzyme 
had an activity in vitro that was predictive of a 
mono-ADP-ribosyltransferase; however, the protein was not purified to a 
level that would permit someone to obtain useful tryptic digest 
information. Prediction of the gene sequence requires tryptic digest 
information. While the protein of Peterson, et al. can be used to study 
the enzymatic properties of a mono-ADP-ribosyltransferase, gene 
therapeutic strategies cannot be pursued nor can studies be conducted to 
assess the effect of the ADP-ribosyltransferase, or a mutated 
ADP-ribosyltransferase, on cell metabolism. Modification of cellular 
metabolism requires an ADP-ribosyltransferase gene, as produced in the 
present invention. Similarly, to develop a therapeutic modality in humans, 
a human enzyme is particularly preferred primarily for immunological 
reasons. 
Once a vertebrate ADP-ribosyltransferase is identified, the gene can be 
used to isolate other ADP-ribosyltransferases, including the human 
counterpart. The human sequence is heretofore undefined. Therefore, it is 
an object of the present invention to identify the gene sequence for 
vertebrate mono-ADP-ribosyltransferases in general and for human 
mono-ADP-ribosyltransferase in particular. 
SUMMARY OF THE INVENTION 
This invention provides the amino acid and nucleotide sequence of a rabbit 
and human mono-ADP-ribosyltransferase. Oligonucleotide fragments from 
these sequences are useful for the further identification and isolation of 
homologous mono-ADP-ribosyltransferases isolated from other vertebrates. 
Nucleotide and peptide fragments derived from these sequences are useful 
for the development of assays to detect the presence of the enzyme in a 
tissue or fluid sample from a vertebrate. 
In one embodiment of the present invention, Applicants disclose an assay 
method for identifying a mono-ADP-ribosyltransferase gene sequence from a 
vertebrate comprising (a) harvesting tissue containing 
ADP-ribosyltransferase activity from a vertebrate, (b) purifying the 
mono-ADP-ribosyltransferase from the tissue, (c) obtaining fragments of 
the mono-ADP-ribosyltransferase, (d) sequencing peptides obtained from the 
fragments, (e) preparing degenerate oligonucleotides corresponding to the 
amino acid sequence of the peptides, (f) using the oligonucleotides in at 
least one polymerase chain reaction to generate nucleic acid sequences, 
wherein the resulting fragments correspond to at least a portion of the 
mono-ADP-ribosyltransferase sequence, (g) generating the nucleic acid 
sequence of the polymerase chain reaction fragments, (h) identifying 
different oligonucleotides corresponding to the 
mono-ADP-ribosyltransferase sequence, and (i) repeating steps (f) through 
(h) until the complete nucleic acid sequence is identified. 
In another embodiment of the present invention an assay method is disclosed 
for identifying a mono-ADP-ribosyltransferase gene sequence in a 
vertebrate comprising (a) identifying tissue from the vertebrate that 
contains ADP-ribosyltransferase activity, (b) isolating mRNA from the 
tissue, (c) preparing cDNA from the mRNA, (d) preparing an oligonucleotide 
pair suitable for use in a polymerase chain reaction, one oligonucleotide 
of the pair having a sequence substantially the same as a first portion of 
SEQ ID NO:1, and the other oligonucleotide of the pair having a sequence 
substantially complementary to a second portion of SEQ ID NO: 1, (e) 
performing the polymerase chain reaction on the cDNA using the 
oligonucleotide pair of step (d) to generate PCR-amplified fragments, (f) 
sequencing the fragments generated from the polymerase chain reaction, and 
(g) repeating steps (e) through (g) until the cDNA is completely 
sequenced. 
In another preferred embodiment of the present invention, an isolated or 
purified nucleic acid fragment encoding rabbit skeletal muscle 
ADP-ribosyltransferase is provided that corresponds to SEQ ID NO:1. 
Alternatively, the gene sequence is provided wherein the sequence is 
mutated in vitro to contain at least one nucleotide change in the 
sequence. Purified or isolated oligonucleotide is also provided that 
comprises at least 15 continuous bases of SEQ ID NO:1. In addition 
recombinant gene vectors are provided that contain at least a 15 base 
portion of SEQ ID NO:1 as well as recombinant protein expressed from the 
recombinant gene vector. Preferably, this protein is essentially pure and 
the protein exhibits ADP-ribosyltransferase activity. The recombinant 
protein is preferably expressed in eukaryotes or prokaryotes. In another 
preferred embodiment, gene sequences are disclosed that have at least an 
85% homology to SEQ ID NO:1. 
In another preferred embodiment a gene sequence is provided that encodes 
human ADP-ribosyltransferase, consisting essentially of the sequence 
corresponding to SEQ ID NO:37. In addition, recombinant gene vectors are 
provided that contain at least 15 continuous bases of SEQ ID NO:37. 
Recombinant protein expressed from the recombinant gene vector are 
disclosed. Preferably, the recombinant protein is essentially pure. More 
preferably, the recombinant protein exhibits ADP-ribosyltransferase 
activity. The recombinant protein is expressed in eukaryotes or 
prokaryotes. 
In yet another preferred embodiment purified antibody is provided that is 
capable of specifically binding to the recombinant protein encoded by SEQ 
ID NO:1. In another embodiment, purified antibody is provided that is 
capable of specifically binding to the recombinant protein encoded by SEQ 
ID NO:37. 
In another aspect of the present invention, there is provided an assay 
method for detecting an ADP-ribosyltransferase gene sequence homologous to 
SEQ ID NO:37 in a vertebrate comprising (a) obtaining at least one 
oligonucleotide pair from SEQ ID NO:37 suitable for a polymerase chain 
reaction and a third oligonucleotide pair selected from SEQ ID NO:3 
positioned between the oligonucleotide pair, (b) isolating a tissue sample 
from a vertebrate, (c) processing the tissue to obtain nucleic acid 
suitable as a template for use in a polymerase chain reaction, (d) 
performing a polymerase chain reaction using the oligonucleotide pair to 
generate at least one DNA fragment, (e) hybridizing the third 
oligonucleotide with the DNA fragment and (f) detecting hybridization 
between the third oligonucleotide and the DNA fragment.

DETAILED DESCRIPTION OF THE INVENTION 
The gene sequence for rabbit and human muscle NAD: Arginine 
ADP-ribosyltransferase is disclosed. In addition, methods are disclosed 
for isolating and identifying sequences corresponding to NAD: Arginine 
ADP-ribosyltransferase from other vertebrates. 
Knowledge of the gene sequence is required in order to study the effects of 
the enzyme on cells both in vivo and in vitro. Recombinant NAD: Arginine 
ADP-ribosyltransferase nucleic acid can be introduced into cells to alter 
the level of protein ADP-ribosylation and to modify intracellular protein 
activity in general. The ADP-ribosyltransferase gene, when overexpressed, 
can also be used to study the effect of pharmacological agents on 
endogenous ADP-ribosylation. Further, the identification of the gene 
sequence and the expression of this sequence in appropriate eukaryotic or 
prokaryotic cells permits the isolation of this protein in amounts 
suitable for purification for antibody production, the development of 
diagnostic reagents, and sensitive tests to detect the activity of this 
enzyme in cell lysates. Nucleic acid fragments of this sequence are useful 
as genetic probes for assessing differences in ADP-ribosyltransferase 
expression within a population and for the identification of 
ADP-ribosyltransferase mutants. The isolation of purified recombinant 
protein facilitates production of tests to identify inhibitors and 
activators of the ADP-ribosyltransferase. These agents would likely have 
therapeutic value in the medical community. 
To identify the gene sequence, the enzyme is first purified from mammalian 
muscle. In Example 1, the source of enzyme was rabbit skeletal muscle. It 
is contemplated that the procedures disclosed herein are suitable for a 
variety of muscle tissue from a variety of vertebrates. While Example 1 
provides a specific exemplary method, there are also a number of methods 
recognized in the art to purify active enzyme from tissue homogenates. The 
purification scheme selected should yield suitable quantities of enzyme 
(at least 100 picomoles) at a suitable level of purity (at least 80% 
pure). 
The initial purification steps used in this invention (through concanavalin 
A agarose) were those described by Peterson, et al. (supra), with several 
important modifications (Table 1 and Example 1). The specific activity of 
the transferase identified by Peterson, et al., used two chromatographic 
steps (DE52 cellulose and concanavalin A agarose) to generate enzymatic 
activity ranging from 0.13 to 5.1 .mu.mol-min.sup.-1 -mg.sup.-1 measured 
with 2 mM NAD and 10 mM L-arginine methyl ester. The assay to measure the 
specific activity of the enzyme is described by Larew, et al. (J. Biol. 
Chem. 266: 52-57 1991). Analysis of the enzyme fraction purified on DE52 
cellulose (Whatman, Maidstone, England and concanavalin A agarose, 
revealed a significant level of impurity as determined by sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This level of 
impurity was too high to permit effective amino acid sequencing as 
evidenced by contaminating protein of equal molecular weight. Therefore, 
two additional purification steps were introduced; high resolution DEAE 
chromatography and gel filtration-high pressure liquid chromatography 
(HPLC). This combination dramatically improved the purity of the 
transferase, as described in Example 1. The final purity was assessed by 
Gel filtration-HPLC (not shown). Tryptic digestion and amino acid 
sequencing of tryptic peptides were performed following nondenaturing gel 
filtration HPLC. 
TABLE 1 
__________________________________________________________________________ 
PURIFICATION SUMMARY OF ADP-RIBOSYLTRANSFERASE 
FROM RABBIT SKELETAL MUSCLE 
Specific 
Protein 
Units activity Purification 
Yield 
Purification step 
(mg) 
(mmol/min) 
(mmol.min.sup.1.mg.sup.-1) 
(-fold) 
(%) 
__________________________________________________________________________ 
15,000 g supernatant 
26,000 
1.70 0.000065 1 100 
KCl-washed pellet 
1,400 
1.20 0.00086 13 71 
DE52 120 0.82 0.0068 105 48 
Concanavalin A agarose 
4 0.57 0.14 2,150 34 
DEAE Memsep 
0.067 
0.29 4.3 66,150 
17 
Gel filtration HPLC 
0.030 
0.27 9.0 138,500 
16 
Gel filtration HPLC 
0.005 
0.07 14 215,400 
4 
(+1% SDS) 
__________________________________________________________________________ 
While there are a variety of purification schemes that can be used to 
obtain the purified enzyme corresponding to the amino acid sequence of 
this invention, those with skill in the art will recognize that the 
purification scheme should maximize protein yield and maintain protein 
integrity thereby maximizing enzymatic activity. Following the methods 
disclosed in Example 1, the ADP-ribosyltransferase was purified about 
215,000-fold with respect to the 15,000 g supernatant and at least 
16,000-fold with respect to the membrane fraction. The overall yield, 
which will vary according to the methods selected, was about 4% of the 
starting material using the purification strategy of Example 1 (see Table 
1). The specific activity of the enzyme preparation was 14 
.mu.mol-min.sup.-1 -mg.sup.-1 when assayed with 0.1 mM NAD and 20 mM 
agmatine, and 68 .mu.mol-min.sup.-1 -mg.sup.-1 with 2 mM NAD. The 
transferase apparently represented approximately 90% of the purified 
protein. 
There was a significant discrepancy between the molecular size of the 
transferase, estimated from the mobility of the enzyme on gel 
filtration-HPLC (61 kDa), and the estimated molecular size predicted from 
SDS-PAGE (38 kDa). This discrepancy is consistent with the interaction of 
the protein with CHAPS, a zwitterionic detergent (Calbiochem, La Jolla, 
Calif.) or alternatively with protein dimerization. 
The purified enzyme preparation was subjected to tryptic digestion as 
described in Example 2. After running SDS-PAGE, proteins were 
electroblotted onto a nitrocellulose membrane and a band, corresponding to 
the ADP-ribosyltransferases, was excised and sent to Dr. William Lane 
(Harvard Microchem, Boston, Mass.). In situ tryptic digestions were 
performed as described (Aebersold, et al. Proc. Natl. Acad. Sci. USA 84: 
6970-6974 (1987)). Trypsin was incubated with the piece of nitrocellulose 
(enzyme to substrate ratio of about 1:20). Cleaved peptides, released from 
the membrane, were separated by reverse-phase HPLC. Peptide-containing 
fractions were collected. Seven peptides, which had the highest absorption 
at 215 nm, were derivatized with phenylthiohydantoin and amino acid 
sequence analysis was performed in a gas-phase sequenator. The amino acid 
sequences of several tryptic peptides was determined and these are 
provided in Table 3 as underlined sequences. The amino acid sequence of 
one of the tryptic peptides of the purified ADP-ribosyltransferase (amino 
acids 74-87) was used to synthesize two sets of degenerate 
oligonucleotides, which were used as nested primers in PCR amplifications 
from a rabbit skeletal muscle cDNA library. 
Cloning of an ADP-ribosyltransferase cDNA 
As noted above, the sequence of a tryptic peptide corresponding to amino 
acids 74-87 in Table 3, was used to synthesize degenerate antisense 
oligonucleotides. Other primers, corresponding to the other tryptic 
peptides, could similarly be used in nested PCR reactions to identify the 
sequence of interest. The oligonucleotides B2, B3 and B4 (SEQ ID 
NOS:14-16, see also Table 2) were used in two sequential polymerase chain 
reactions (PCR) with oligonucleotides derived from the pBluescript plasmid 
sequence (BSC1, SEQ ID NO:17 and BSC2, SEQ ID NO:18, see Table 2) to 
identify candidate sequences from a Lambda ZAPII rabbit skeletal muscle 
library (see Example 2). Although both orientations of the primers were 
used, significant amounts of PCR product were obtained with the antisense 
primers (B2, B3 and B4, SEQ ID NOS: 14-16), based on amino acids 74-82, 
and sense plasmid primers BSC1 and BSC2, SEQ ID NOS:17 and 18, 
respectively. PCR fragments corresponded to the 5'-coding and untranslated 
region of the clone (positions -91 to 239 in Table 3). This PCR fragment 
was cloned into a suitable cloning vector (see Example 3) using methods 
well known in the art and subjected to dideoxynucleotide sequencing. Those 
with skill in the art will recognize that any number of commercially 
available cloning vectors could similarly be used to facilitate DNA 
sequencing. The deduced amino acid sequence of the cloned DNA fragment 
included a sequence that corresponded to one of the tryptic peptides 
(amino acids 31-58, see Table 3 and SEQ ID NO:2), thus confirming the 
identity of the clone. 
TABLE 2 
______________________________________ 
Amplification Primers 
Sep. 
Name ID No. Description 
______________________________________ 
B2 14 Inverse complement of nucleotides encoding 
amino acids 74-80 
B3 15 Inverse complement of nucleotides encoding 
amino acids 76-82 
B4 16 Inverse complement of nucleotides encoding 
amino acids 76-82 
BSC1 17 Specific to pBluescript sequence 
BSC2 18 Specific to pBluescript sequence, 3' to BSC1 
TG 19 Inverse complement of nucleotides encoding 
amino acids 52-58 
CAU-AC 20 Inverse complement of nucleotides encoding 
amino acids 45-51 (underlined) and a subcloning 
sequence at 5'-end 
R.sub.0 R.sub.1 T 
21 (dT) 17 adaptor primer for 5'-RACE 
R.sub.0 22 Outer adaptor primer for 5'-RACE 
CUA-R.sub.1 
23 Inner adaptor primer for 5'-RACE (underlined) 
and subcloning sequence at 5'-end 
5Ndel 24 Corresponding to amino acids 24-30 (underlined), 
a Ndel site (italics) plus subcloning sequence at 
5'-end 
3BamHI 25 Inverse complement of nucleotides encoding 
amino acids 297-303 (underlined), a stop codon 
(double underlined), a BamHI site (italics), and a 
subcloning sequence at 5'-end 
5PRM 26 Inverse complement of nucleotides (-90)-(-43) 
48SP 27 Inverse complement of nucleotides encoding 
amino acids 31-46 
3PRM 28 Inverse complement of nucleotides 960-1007 
HSM-5 29 Inverse complement of nucleotides 283-306 
HSM- 30 Inverse complement of nucleotides 250-268 and 
CAUN contains a subcloning sequence at 5'-end 
HSM-30 31 Inverse complement of nucleotides 212-242 
HSM-1F 32 Corresponding to nucleotides 857-876 
CAUHSM- 33 Corresponding to nucleotides 881-901 and 
2F contains a subcloning sequence at 5'-end 
P-RT 34 Inverse complement of nucleotides 1099-1122 
HSM-1 N 35 Corresponding to nucleotides (-79)-(-49) 
containing a subcloning sequence at 5'-end 
HSM-RN 36 Inverse complement of nucleotides 1057-1074 
containing a subcloning sequence at 5'-end 
______________________________________ 
Table 2 provides the sequences for the oligonucleotides that were used to 
identify the full length rabbit nucleic acid sequence of this invention. 
The letter "N" denotes any nucleotide A, C, G or T. Oligonucleotides are 
listed from their 5' to 3' end. 
Based on the partial cDNA sequence, an oligonucleotide (48SP, SEQ ID 
:NO:27, see Table 2) was synthesized for use as a probe to screen a 
skeletal muscle cDNA library. An exemplary screening strategy is provided 
in Example 4. Several clones were obtained, one of which contained a 
sequence that overlapped with the PCR product described above. This 
sequence extended from position -14 to 1020 (Table 3 and SEQ ID NO:1) and 
contained a 981-bp open reading frame, encoding a 36, 134-kDa protein. The 
deduced amino acid sequence (SEQ ID NO:2, GenBank accession no. M98764) of 
this protein includes all seven amino acid sequences identified by tryptic 
digest from the purified transferase. Numbering is relative to the 
initiating methionine codon. Sequences identified by tryptic digest are 
underlined. Stop codons are double underlined. Asterisks identify 
potential N-glycosylation sites. 
TABLE 3 
__________________________________________________________________________ 
Rabbit Skeletal Muscle NAD; 
arginine ADP - ribosyltransferan 
__________________________________________________________________________ 
-105GACCATCACATGAAGCCAACACCAGCTCCCCTGCCCCGGACAAGG 
-60CCTAGATGAGGAAAGTAAGAGTCAAAAGGAGAGAGAAACTGGCCTGGGGTGGCCCCAACC 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
##STR15## 
##STR16## 
##STR17## 
__________________________________________________________________________ 
The sequence was also obtained from poly (A).sup.+ RNA isolated from rabbit 
skeletal muscle. A preferred exemplary procedure for obtaining the 
sequence from poly (A).sup.+ RNA is provided in Example 5. The sequence of 
the ribosyltransferase was obtained by hybridizing primer TG, SEQ ID NO: 
19 (Table 2) to the RNA to generate cDNA using an avian myeloblastosis 
virus (AMV) reverse transcriptase (Invitrogen, San Diego, Calif.) under 
conditions described by Frohman, et al. (Proc Natl. Acad. Sci. USA 85: 
8998-9002, 1988. A 3' tail was added to the product using terminal 
deoxynucleotidyl transferase and the second strand was prepared by 
annealing primer R.sub.O R.sub.I T, SEQ ID NO:21, to the RNA and extending 
the primer with Taq DNA polymerase. Further amplification of the 
fragments, by PCR, was performed using primers TG, SEQ ID NO:19, and 
CAU-AC, SEQ ID NO:20, and primer pair R.sub.O, SEQ ID NO:22, and 
CUA-R.sub.I, SEQ ID NO:23. The final product was cloned into a suitable 
cloning vector such as pAMP1 (CloneAmp subcloning system, GIBCO-BRL, 
Gaithersburg, Md.) and sequenced. The sequence corresponded to positions 
-105 to 152 of Table 3 and was generated from 5'RACE techniques as 
outlined in Example 5. 
Since the amino acid sequence of the N terminus of the 
ADP-ribosyltransferase was not identified using the techniques described 
above, supplemental techniques can be used to identify the position 1 
methionine (Table 3 and SEQ ID NO:1). Northern analysis is used in Example 
6 as one example of a method to determine the initiating methionine. The 
sequence of the PCR product obtained with degenerate primers B2, B3 and 
B4, SEQ ID NOS:14-16 (position -91 to 239) and the sequence of the 5'-RACE 
product (position -105 to 152) contained two in-frame stop codons upstream 
from the methionine codon at positions -54 to -52 and -45 to -43. Northern 
blot analysis indicated that oligonucleotide probes 5PRM, SEQ ID NO:26 
(specific to the 5'-untranslated region, containing the two putative stop 
codons), 48SP, SEQ ID NO:27 (specific to the coding region) and 3PRM, SEQ 
ID NO:28 (specific to the 3'-end of the coding region) hybridized to RNA 
of the same size (about 4 kb), consistent with the conclusion that the 
5'-untranslated region is present in transferase mRNA. 
Once the enzyme has been cloned and sequenced it is possible to use 
specific probes identified from the cloned sequence, or degenerate probes 
with substantial homology to the cloned sequence, to assess the tissue 
distribution of the ADP-ribosyltransferase in other tissues (see Example 
6). An ADP-ribosyltransferase specific probe was hybridized to RNA 
isolated from a variety of rabbit tissues. The probe recognized a 4-kb 
mRNA expressed primarily in skeletal and cardiac muscle tissues. The 
northern blots assessed the tissue distribution of the rabbit 
ribosyltransferase. 
It is contemplated that a similar analysis could be performed on tissues 
derived from other vertebrates using probes derived from the rabbit 
ADP-ribosyltransferase sequence. Similarly, degenerate probes 
corresponding to the rabbit ADP-ribosyltransferase sequence, hybridization 
at lower temperature, washes at reduced stringencies, or the like can be 
used to identify ribosyltransferases from tissues of other vertebrates. 
In mammals, cell lysates and partially purified protein preparations from 
cells indicate that arginine-specific ADP-ribosyltransferase enzymatic 
activity is predominantly found in skeletal muscle and cardiac tissues 
(Soman, et al. Biochem. Biophys. Res. Commun. 120: 973-980, 1984). 
Recently, activity was also found in murine T-cell hybridoma, thymoma and 
lymphoma cells (Soman, et al. Biochem. Biophys. Res. Commun. 176: 301-308, 
1991). 
The sequence and amino acid data from the ADP-ribosyltransferase 
facilitates an analysis of the hydrophilicity and hydrophobicity of the 
enzyme. This analysis helps to identify functional regions of the protein 
and is necessary for structurally analyzing the catalytic core of the 
enzyme. The hydrophilicity plot of the ribosyltransferase (FIG. 1) 
indicates that the enzyme has strongly hydrophobic amino and carboxyl 
termini and a hydrophilic center. These characteristics permit one with 
skill in the art to compare the functional regions of the protein with 
other enzymes known in the art. Here, the hydrophobic and hydrophilic 
pattern is common to glycophosphatidylinositol (GPI)-anchored membrane 
proteins (Ferguson, M.A.J. Biochem. Soc. Trans. 20: 243-256, 1992 and 
Udenfriend, et al. Cell. Mol. Biol. 38: 11-16, 1992). Hydrophilicity 
values were obtained with the MacVector program (IBI, a division of Kodak, 
New Haven, Connecticut) using the Kyte-Doolittle algorithm (provided in 
the MacVector program) using a window setting of 16 amino acids. 
The hydrophobic N-terminal portion serves as a leader sequence, directing 
the enzyme into the endoplasmic reticulum. The hydrophobic sequence at the 
C terminus is recognized inside the ER as a signal for 
glycophosphatidylinositol modification. 
Two potential sites for N-linked glycosylation were found in the deduced 
amino acid sequence of the transferase. These are Asp.sup.65 and 
Asp.sup.253. Since the protein binds to a lectin column (concanavalin A 
agarose) and because phosphatidylinositol-linked proteins are often 
heavily glycosylated, it is likely that the ADP-ribosyltransferase is 
subject to these posttranslational modifications. 
To conclusively show that the cloned enzyme is an arginine-specific 
mono-ADP-ribosyltransferase, the sequence was cloned into a suitable 
expression vector and expressed in either bacteria or eukaryotes. Examples 
7 and 8 outline strategies for the expression of the 
ADP-ribosyltransferase in E. coli and eukaryotic cells, respectively. 
Since eukaryotic cells carry endogenous levels of ADP-ribosyltransferase, 
the levels of enzymatic activity identified in transfected eukaryotic 
cells should be compared with non-transfected or mock-transfected cells. 
Expression of the full length ADP-ribosyltransferase in E. coli was 
attempted using constructs of the ADP-ribosyltransferase either as a 
fusion protein of glutathione S-transferase or as a non-fusion protein. 
The protein was inactive using both constructs. ADP-ribosyltransferase 
activity was obtained in transformed E. coli using a construct that 
included amino acids 24-303 of the ADP-ribosyltransferase ligated as a 
non-fusion protein in pET3a (Novagen, Madison, Wis.). The truncated form 
of the protein lacked both the hydrophobic amino and carboxyl termini. In 
assays to assess the enzymatic activity of the protein, a product was 
formed that comigrated on an anion exchange HPLC column with the product 
(ADP-ribosylagmatine) formed by native rabbit skeletal muscle 
ADP-ribosyltransferase in the presence of NAD and agmatine. 
In transformed rat mammary adenocarcinoma (NMU) cells transformed with the 
rabbit skeletal muscle ADP-ribosyltransferase cDNA seqence of SEQ ID NO: 
1, significant ADP-ribosyltransferase activity was observed, with 62% 
occurring in the membrane fraction. ADP-ribosyltransferase activity was 
negligible in control NMU cells and cells transformed with either the 
vector alone or with the vector containing an antisense insert (Example 
8). 
Comparison of the Deduced Amino Acid Sequence of ADP-ribosyltransferase 
with Other Protein Sequences 
A homology search of the deduced amino acid sequence of the transferase was 
done at the National Center for Biotechnology Information Bethesda, 
Maryland using the BLAST network service. The highest homology score was 
obtained for rat and mouse RT6.2 protein. This protein is expressed 
exclusively on postthymic T cells (Koch, et al. Proc. Natl. Acad. Sci. USA 
87: 964-967, 1990). The regions of greatest similarity were amino acids 
39-88 (42% identity), 214-254 (46% identity), 107-124 (72% identity), 
148-166 (52% identity) and 194-206 (61% identity). RT6.2 is a 26-kDa 
phosphatidylinositol-linked protein, with hydrophobic amino and carboxyl 
termini. The predicted amino acid sequence of the RT6.2 protein begins 
with a leader of 20 hydrophobic amino acids and ends with a hydrophobic 
stretch of 29 residues. 
No significant homology was found between rabbit skeletal muscle 
transferase and various bacterial ADP-ribosylating toxins, the 
ADP-ribosyltransferase from Rhodospirillium rubrum or poly (ADP-ribose) 
polymerase. Thus, the skeletal muscle transferase is an unique enzyme, 
distinct from the bacterial transferases in structure and perhaps in 
substrate specificity. 
Use of the Mammalian Sequence to Obtain Human mono-ADP ribosyltransferase 
Based on the rabbit sequence provided in Table 3 and SEQ ID NO:1, two sets 
of nested degenerate primers were designed for use in two consecutive PCR 
amplifications to obtain the human ADP-ribosyltransferase sequence from 
isolated human skeletal muscle poly (A).sup.+ RNA. Although a preferred 
method for isolating SEQ ID NO: 3 is provided in Example 10, other primer 
pairs for both 5'-RACE and 3'-RACE are contemplated including 5' 
GCTGTCTGCATACACCTGGTTGGC 3' (SEQ ID NO: 10; inverse complement of bases 
80-103 in the human fragment) and 5' GTGGTTGAGATCCGGGAGAGC 3' (SEQ ID NO: 
11; inverse complement of bases 47-67 in the human fragment) for 5'-RACE 
and 5' CCCGCATCTACCTCCGAGCC 3' (SEQ ID NO: 12; bases 54-73 in the human 
fragment) and 5' CAAGCACAGCACCTATAATT 3' (SEQ ID NO: 13; bases 679-698 in 
the human fragment). A partial cDNA sequence, encoding a 224 amino acid 
fragment of human skeletal muscle mono-ADP-ribosyltransferase, was 
obtained by PCR using primers based on the rabbit 
mono-ADP-ribosyltransferase sequence. 
__________________________________________________________________________ 
Primers for the first PCR reaction: 
1A: (ACGT)TT(AG)GA(TC)ATGGC(ACGT)CC(ACGT)GC 
SEQ ID NO:5 
1B: (ACGT)CT(ACGT)GA(TC)ATGGC(ACGT)CC(ACGT)GC 
SEQ ID NO:6 
2: (TC)TT(AG)CA(TC)TGCAT(TC)TC(TC)TT 
SEQ ID NO:7 
Primers for the second PCR reaction: 
3: (AGCT)TT(TC)GA(TC)GA(TC)CA(AG)TA(TC)GT 
SEQ ID NO:8 
4: (AGT)AT(AG)TA(TC)TC(AG)CA(AG)TT(AG)TA 
SEQ ID NO:9 
__________________________________________________________________________ 
The bases in parentheses represent degenerate positions. Primers 1A and 1B 
correspond to amino acids 38-44 in the rabbit ADP-ribosyltransferase 
sequence and primer 2 is an inverse complement of nucleotides encoding 
amino acids 281-286. Primer 3 is internal to 1A and 1B and its sequence 
corresponds to amino acids 45-51. Primer 4 is internal to primer 2 and is 
an inverse complement of nucleotides encoding amino acids 275-280. 
The resulting PCR product (about 670 base pairs) was subcloned into a 
vector (pAmp1, GIBCO-BRL) and sequenced by the dideoxy sequencing methods 
previously described. Rapid amplification of cDNA ends (RACE) was used to 
determine sequentially the 5' and 3' ends of the human transferase mRNA as 
described in Example 11. The full length human sequence (SEQ ID NO: 37) is 
shown in Table 4. The deduced amino acid sequence of the 224 amino acid 
fragment of SEQ ID NO:37, as determined from the nucleotide sequence, was 
87% identical to that of the rabbit mono-ADP-ribosyltransferase (see Table 
5, SEQ ID NO:4). 
Completion of the Human ADP-ribosyltransferase sequence 
The human ADP-ribosyltransferase sequence provided in Table 4, SEQ ID NO:3 
and SEQ ID NO:4 is a partial sequence. Both the amino and carboxyl ends 
remain unidentified. The remaining sequence of the gene was obtained using 
5'-RACE and 3'-RACE methods. These techniques are disclosed in the art and 
permit the rapid amplification of the 5' end and the 3' end of the cDNA. 
For a detailed protocol see Frohman, et al. Technique- A Journal of 
Methods in Cell and Molecular Biology 1: 165-170 1989). The 5'-RACE 
methodology is described in Example 5 and both the 5'-RACE and 3'-RACE 
methods are detailed in Example 11. 
TABLE 4 
__________________________________________________________________________ 
NUCLEOTIDE AND DEDUCED AMINO ACID SEQUENCE OF HUMAN ADP-RIBOSYLTRANSFERSAS 
__________________________________________________________________________ 
TTCCACCAGGACAGGCCTAGATGAGGAAACTGAGACCCAAAAAGAGACAGCAACTGGCCC60 
##STR18## 
##STR19## 
##STR20## 
##STR21## 
##STR22## 
##STR23## 
##STR24## 
##STR25## 
##STR26## 
##STR27## 
##STR28## 
##STR29## 
##STR30## 
##STR31## 
##STR32## 
##STR33## 
##STR34## 
##STR35## 
##STR36## 
##STR37## 
##STR38## 
CGGGACAGCCTCGCCTGCTGCCTCTGCCCATCCTGAGGATGTTGGCCATGTGTGCTTCAG1127 
TGTAACCAAGATTCCTGTCAATCCCATCTGCAGGGAACTCTGGGACCTTCTCTGGTAGCT1187 
GCCAGACCGGCTGGTGGAGAAACAGGAGACAATCTGGGGACTGAACCTTACCCAGGGCTG1247 
TAGGAGTGAGACTCTGAATAAAGGGTTGGGCCGGCAAAAAAAAAAAAAAAAAAAAAAA1305 
__________________________________________________________________________ 
TABLE 5 
__________________________________________________________________________ 
COMISON OF AMINO ACID SEQUENCES OF MONO-ADP- 
RIBOSYLTRANSFERASES FROM RABBIT AND HUMAN 
__________________________________________________________________________ 
A: mono-ADP-ribosyltransferase from rabbit 
B: residues 51-274 of mono-ADP-ribosyltransferase from human 
The character to show that two aligned residues are identical is 
`.vertline.` 
A- MWVPAVANLLLLSLGLLEAIQAQSHLVTRRDLFSQETPLDMAPASFDDQY-50 
##STR39## 
##STR40## 
##STR41## 
##STR42## 
##STR43## 
A- RLSTAWSLLLLLAFLAVGPFPGSPGLF-327 
__________________________________________________________________________ 
Identity: 195 (87.1%) 
Number of gaps inserted in A: 0 
Number of gaps inserted in B: 0 
Diagnostic Tests to Assess the Presence or Absence of 
ADP-ribosyltransferase Transcripts in Cell Preparations. 
Northern Blots are used to detect the presence of ADP-ribosyltransferase 
specific transcripts in cell samples from a patient. Tissue biopsies are 
obtained from a patient, washed briefly in sterile saline and lysed in 
guanidine isothiocyanate. RNA is isolated from the lysate using 
commercially available kits such as the RNA isolation kits available from 
Invitrogen. Purified total RNA or oligo (dT) column purified mRNA is 
blotted onto nylon membranes in a range of from 0.05 .mu.g to 5 .mu.g per 
blot. Probes complementary to the human ADP-ribosyltransferase gene 
sequence such as primers 5-1 and 5-2 (see Example 11) are end labelled 
with .sup.32 P using polynucleotide kinase (Pharmacia) or commercially 
available kits. The probes are hybridized to the blotted RNA using 
conditions provided in Example 6 and developed at -80.degree. C. using 
Kodak X-Omat film. Developed spots indicate the presence of human 
ADP-ribosyltransferase transcripts. 
Gene Therapy using the mono-ADP-ribosyltransferase 
It is contemplated that the human sequence encoding 
mono-ADP-ribosyltransferase can be used in a number of gene therapeutic 
strategies recognized in the art. For example, the full length sequence or 
a portion of the sequence encoding an enzymatically active fragment is 
incorporated into a suitable gene delivery vehicle. There are a number of 
gene delivery vehicles recognized in the art that are useful for 
delivering a gene sequence to a cell. RNA and DNA gene sequences can be 
incorporated into viral vectors such as retroviral vectors, influenza 
vectors and adenovirus vectors. Similarly, RNA and DNA gene sequences can 
be introduced to cells in vivo as naked gene sequences or associated with 
membrane fusion promoting agents such as Lipofectin.RTM., or the like. 
Introduction of the gene into patients in need of increased levels of 
mono-ADP-ribosyltransferase can be accommodated by in vitro gene therapy. 
Samples of patient cells are removed and digested into single cell 
suspensions. The single cell suspension is then transfected with the 
mono-ADP-ribosyltransferase gene that is incorporated into a suitable 
mammalian expression vector such as those available from Stratagene, La 
Jolla, Calif.; New England Biolabs, Beverly, Mass; or Promega, Madison, 
Wis. The expression vectors preferably contain suitable promoters such as 
an SV40 promoter, the Cytomegalovirus immediate early promoter, or the 
like, as well as a selection mechanism such as thymidine kinase or 
neomycin. Selection of transformants in vitro is followed by the 
re-introduction of the cells into, preferably, the same patient in need of 
increased levels of mono-ADP-ribosyltransferase. 
It is additionally contemplated that antisense molecules may be prepared 
from the gene sequence and introduced into cells in need of 
ADP-ribosyltransferase down-regulation. Antisense technology is known in 
the art, for detailed applications of antisense technologies see U.S. Pat. 
No. 4,948,882 to Ruth and European Patent Publication no. EP-387775 to 
Beug, et al. 
Mutagenesis of NAD: Arginine ADP-ribosyl transferase 
The identification of the gene sequence in mammals and humans facilitates 
further structure/function studies to assess the interaction of the enzyme 
with proteins within the cell. Since the ADP-ribosyltransferases are 
localized within different cellular compartments, it will be possible to 
modify the cellular targeting of the transferase gene, through in vitro 
mutagenesis, and thereby alter the localization of the expressed protein 
and its contact with cell substrates. Transfection of mammalian cells is 
currently being performed in the laboratory both with intact sequence and 
sequence subjected to mutagenesis. 
There are a variety of commercial kits available for generating 
site-directed mutants or random mutants of the ADP-ribosyltransferase 
(Bio-Rad, Richmond, Calif., Stratagene and Invitrogen, San Diego, Calif.). 
Once the nucleic acid sequence is incorporated into a suitable vector, the 
sequence is modified by oligonucleotides containing the random or 
site-directed mutation. Incorporation of the oligonucleotide into the 
unmodified sequence may occur by PCR, ligase chain reaction, single-strand 
mutagenesis or the like. Mutagenesis techniques are well known in the art 
and commercially available as kits from Bio Rad, Invitrogen, and 
Stratagene. These kits include extensive directions and protocols 
therefore no further detail is necessary to enable one with skill in the 
art of molecular biology to use the sequences provided herein to generate 
mutation in the ADP-ribosyltransferase gene. 
Generation of Antibodies 
ADP-ribosyltransferase gene sequence incorporated into a eukaryotic or 
prokaryotic expression vector is useful for generating large quantities of 
the enzyme that cannot otherwise be harvested easily from vertebrate 
tissue. Large quantities of the enzyme are useful for crystallography, for 
in vitro enzyme studies and for antibody preparation. 
Example 11 provides methods for generating microgram/ml quantities of the 
enzyme that are suitable for immunization. Mice, rats or rabbits are 
immunized and boosted with the enzyme preparation in the presence of a 
suitable adjuvant such as complete or incomplete Freund's adjuvant. 
Polyclonal antibodies prepared by the mtthod of Example 12 and monoclonal 
antibodies prepared from the methods of Example 13 are used for diagnostic 
assays to assess the presence of the enzyme within a cell sample. 
Antibodies reactive with the enzyme permit the generation of enzyme linked 
immunosorbent assays (ELISA), western blots, and radioimmunoassays or the 
like. Example 13 details the production of an ELISA assay to detect the 
presence of ADP-ribosyltransferase in a cell sample. 
Identification of other NAD: Arginine ADP-ribosyltransferase from other 
vertebrates: 
It is contemplated that the methods disclosed herein are suitable for the 
isolation and sequence identification of mono-ADP-ribosyltransferase from 
any vertebrate. Tissue homogenates can be used to isolate intact enzyme 
that is purified and subjected to tryptic digestion to identify the amino 
acid sequence. Alternatively, RNA isolated from tissue homogenates is 
useful for direct identification of the ADP-ribosyltransferase sequence 
using degenerate primers in PCR reactions as disclosed for the human 
ADP-ribosyltransferase sequence. 
Particular embodiments of the invention will be discussed in detail and 
reference will be made to possible variations within the scope of the 
invention. There are a variety of alternative techniques and procedures 
available to those of skill in the art which would similarly permit one to 
successfully perform the intended invention. 
EXAMPLE 1 
Purification of ADP-ribosyltransferase 
Frozen rabbit skeletal muscle (1 kg, Pel-Freeze, Rodgers, Arkansas was 
thawed, ground and homogenized in a Waring blender for 1 minute at 
4.degree. C. in 3 liters of buffer A (10% sucrose/10 mM histidine, pH 
7.0/1 mM EDTA/1 mM benzamidine/1 mM iodoacetamide/0.25 mM PMSF/leupeptin, 
pepstatin and aprotinin, each 0.5 .mu.g/ml, Sigma, St. Louis, Mo.). The 
homogenate was centrifuged at 15,000 g for 30 minutes and the resulting 
supernatant was centrifuged at 100,000 g for 2 hours. The pellet 
containing 1.5 g of protein as determined by BCA protein quantitation 
assay (Pierce Biochemicals, Rockford, Ill.) was washed once with 400 ml of 
buffer B (0.6M KCl/10 mM histidine, pH 7.0/1 mM EDTA/1 mM benzamidine/1 mM 
iodoacetamide/0.25 mM PMSF/leupeptin, pepstatin and aprotinin, each 0.5 
.mu.g/ml) and centrifuged at 100,000 g for 1 hour. The pellet (1.4 g of 
protein) was suspended in 200 ml of buffer A supplemented with 0.3% sodium 
deoxycholate (Sigma), stirred for 30 minutes at 4.degree. C. and 
centrifuged at 100,000 g for 2 hours. The supernatant, containing 0.6 g of 
protein was applied to a column (5.times.55 cm) of DE52, equilibrated with 
buffer C (10 mM potassium phosphate, pH 7.5/10% glycerol/0.05% sodium 
deoxycholate/1 mM EDTA/1 mM benzamidine). 
The column was washed and eluted with a linear gradient of 0-1M NaCl in 
buffer C (total volume 4 liters; flow rate 6 ml/min; 20-ml fractions). 
Transferase activity was eluted as a single peak with maximal activity at 
0.4M NaCl. Active fractions were pooled and applied to a column 
(1.4.times.4 cm) of concanavalin A agarose (Sigma) equilibrated with 
buffer D (50 mM Tris-Cl, pH 7.5/0.2M NaCl/1% CHAPS/0.01% NaN.sub.3), 
followed by washing with buffer D and eluted with 25 ml of buffer D plus 
0.3M methylmannopyranoside. The eluate was dialyzed at 4.degree. C. 
against buffer E (10 mM Tris-Cl, pH 7.5, 1%CHAPS/0.01% NaN.sub.3) and 
applied (4 ml/min) to a high resolution DEAE column (MemSep.RTM. 
cartridge, 1.4 ml bed volume, Millipore, Medford, Mass.), previously 
equilibrated with buffer E. After washing with buffer E, the column was 
eluted with a linear gradient of 0-0.3M NaCl in 60 ml of buffer E (flow 
rate 2 ml/min). Four 20 ml fractions that eluted at 0.025 to 0.075M NaCl 
and contained transferase activity were pooled and concentrated to 0.8 ml 
(Centricon 30 microconcentrators, Amicon, Beverly, Mass.). The resulting 
solution was loaded successively in 200-.mu.l samples onto a TSK 3000 HPLC 
gel filtration column (TosoHaas, Philadelphia, Pa.). The column was eluted 
with buffer F (50 mM Tris-Cl, pH 7.0/0.2M NaCl/1% CHAPS/0.01% NaN.sub.3) 
at a flow rate of 0.9 ml/min and 0.45-ml fractions were collected. 
15-.mu.l samples of fractions 31-41 were analyzed by SDS-PAGE in 10% 
acrylamide gel. Samples obtained from the HPLC gel filtration column were 
passed through a gel filtration column a second time in the presence of 1% 
SDS. 20-.mu.l aliquots of fractions 27-35 that had passed through two gel 
filtration columns were analyzed by SDS-PAGE in a 12% acrylamide gel. The 
electrophoretic profile of the gel filtration HPLC-purified 
ADP-ribosyltransferase samples indicated that the second HPLC purification 
was useful in obtaining essentially pure ADP-ribosyltransferase. Fractions 
37 and 38, containing the peak of transferase activity, were subjected to 
SDS-PAGE (without reducing agent). The lane corresponding to fraction 32 
from the second polyacrylamide gel contained 0.1 .mu.g of protein. Both 
gels were silver stained according to the methods of Rabilloud, et al. 
Electrophoresis 9: 288-291, 1988. The gel was sliced into 2-mm fragments 
and proteins were eluted by shaking the slices overnight at room 
temperature in 50 mM Tris-Cl, pH 7.5 with 1% CHAPS. Transferase activity 
was found in slices corresponding to the 38-kDa protein band as identified 
by the kDa markers used in the polyacrylamide gels. Most of the high 
molecular weight contaminating protein was removed by reloading the 
factions containing transferase activity on the same HPLC column and 
eluting with buffer F plus 1% SDS. Before assaying the fractions, SDS was 
removed by precipitation with 0.2M potassium phosphate followed by 
repeated concentration and dilution with buffer lacking SDS using a 
Centricon 30 microconcentrator. 
EXAMPLE 2 
Amino Acid Sequence Analysis of the ADP-ribosyltransferase 
Proteins present in fractions 37 and 38 from the HPLC gel filtration 
protocol, in the absence of SDS, were separated by SDS-PAGE in a 10% gel 
and transferred to PVDF membrane. The band corresponding to the 
transferase (38 kDa, 10 .mu.g of protein) was excised and subjected to in 
situ tryptic digestion. Peptides were HPLC-purified and seven were 
sequenced (Harvard Microchemistry Facility, Dr. William Lane). The 
procedure followed the protocol of Aebersold, et al. Proc. Natl. Acad. 
Sci. USA 84: 6970-6974, 1987. The yield of the amino acids detected in 
each cycle ranged from 60 pmol in early cycles to 1 pmol in later cycles. 
The sequences of the peptides generated from these experiments are 
underlined in Table 3. 
EXAMPLE 3 
Generation of Partial ADP-ribosyltransferase Sequences by PCR 
Sequence of a tryptic peptide (amino acids 74-87) was used to synthesize 
degenerate antisense oligonucleotides B2, B3, and B4 (SEQ ID NOS:14-16, 
see Table 2). A partial cDNA sequence was generated using a nucleotide 
sequencing kit employing Sequenase T7 DNA polymerase (United States 
Biochemical, Cleveland, Ohio) in two successive polymerase chain 
reactions. In the first amplification, a 5-.mu.l sample of a Lambda ZAPII 
(Stratagene, La Jolla, Calif.) rabbit skeletal muscle cDNA library 
(8.5.times.10.sup.7 pfu) was used as a template. The reaction was 
performed with mixed B3 and B4 primers, SEQ ID NOS:15 and 16, respectively 
(50 pmol of each) and BSC1 primer (SEQ ID NO:17, 10 pmol of primer, 
complementary to pBluescript sequence present in the Lambda ZAP vector 
near the cloning site). Amplification was performed in 100.mu.l volume for 
35 cycles at 94.degree. C. for 1 minute, 57.degree. C. for 1 minute and 
72.degree. C. for 1 minute using the PCR reagent kit with AmpliTaq DNA 
polymerase from Perkin-Elmer (Norwalk, Conn.). The final cycle was 
followed by an extension at 72.degree.C. for 7 minutes. 
The product from the first amplification (1 .mu.l) was used as a template 
in a second round of PCR, together with B2 primer (SEQ ID NO:14) 50 pmol, 
5' to B3 and B4 (SEQ ID NOS:15 and 16, respectively) and BSC2 primer (SEQ 
ID NO:18, 10 pmol, pBluescript specific, 3' to BSC1, SEQ ID NO:17). 
Amplification conditions were the same except for the annealing 
temperature, which was raised to 63.degree. C. The major product (330-bp) 
was subcloned into a TA cloning plasmid vector (Invitrogen, San Diego, 
Calif.) and sequenced by the Sanger dideoxy chain termination method using 
the Sequenase sequencing kit using deoxyadenosine 5'.alpha.-.sup.35 
s!thiotriphosphate (.sup.1233 Ci/mmol, NEN-DuPont). 
EXAMPLE 4 
Screening of the cDNA Library 
A Lambda ZAPII rabbit skeletal muscle cDNA library (Stratagene, 
1.7.times.10.sup.10 pfu/ml) was screened in E. coli XL-1 Blue host cells 
(Stratagene) by plaque hybridization (about 5.times.10.sup.5 plaques) with 
the 48SP oligonucleotide probe, SEQ ID NO:27, labeled with 
.alpha.-.sup.32 -P! dATP (New England Nuclear, Beverly, Mass.) and 
terminal deoxynucleotidyl transferase (GIBCO-BRL, Gaithersburg, Md.) to a 
specific activity of 5.times.10.sup.7 cpm/pmol. Duplicate lifts of 2 
minutes and 4 minutes were performed using nylon colony/plaque 
hybridization filters. Filters were prehybridized for 4 hours at 
42.degree. C. in 5.times.SSC (1.times.SSC=0.15M NaCl/0.015M sodium 
citrate, pH 7.0), 5.times. Denhardt's solution (1.times.=0.02% Ficol, 
0.02% polyvinylpyrrolidone,0.02% bovine serum albumin), 10 mM Tris-Cl (pH 
7.4), 10% dextran sulfate, 0.5% SDS and salmon sperm DNA (100 .mu.g/ml, 
Lofstrand Laboratories, Gaithersburg, Md.). Hybridization was performed in 
the same solution, supplemented with radiolabeled probe (2.times.10.sup.6 
cpm/filter). Filters were washed twice in 2.times.SSC/0.5% SDS at room 
temperature and twice in 0.5.times.SSC/0.5% SDS at 42.degree. C. and 
exposed to Kodak X-OMAT film for 24 hours at -80.degree. C. with 
intensifying screens. After three rounds of screening, several positive 
clones were identified. pBluescript plasmids carrying the cloned cDNA 
insert were excised in vivo, purified, and sequenced. 
EXAMPLE 5 
Rapid Amplification of 5'-end of cDNA (5'-RACE) 
Amplification was performed as described (Frohman, et al. Proc. Natl. Acad. 
Sci. USA 85: 8998-9002 1988 with some modifications. Poly (A).sup.+ RNA 
from rabbit skeletal muscle was denatured with methyl mercury hydroxide 
and the first cDNA strand was synthesized by extension of primer TG, SEQ 
ID NO:19 (Table 2) with AMV reverse transcriptase (Invitrogen). After 
3'-end tailing of the product with dATP and terminal deoxynucleotidyl 
transferase, the second cDNA strand was synthesized by annealing and 
extending primer R.sub.O R.sub.I T, SEQ ID NO:21 with Taq DNA polymerase. 
Two rounds of PCR amplifications were then performed using primers TG, SEQ 
ID NO:19 and CAU-AC, SEQ ID NO:20, on one side of the cDNA fragment and 
R.sub.O, SEQ ID NO:22 and CUA-R.sub.I, SEQ ID NO:23 on the other side. The 
final product was subcloned into the pAMP1 vector using the CloneAmp 
system (GIBCO-BRL) and sequenced. 
EXAMPLE 6 
Northern Blot Analysis 
Total RNA was isolated from rabbit tissues as described by Chomczynski and 
Sacchi (Anal. Biochem. 162: 156-159, 1987). Poly (A).sup.+ RNA was 
purified from total RNA using oligo(dT) columns (Clontech, Palo Alto, 
Calif.). For Northern blot analysis, 20-30 .mu.g of total RNA or 5 .mu.g 
of poly(A).sup.+ RNA was subjected to electrophoresis in a denaturing 1.2% 
agarose gel containing formaldehyde and ethidium bromide and then 
transferred to Nytran membrane. After prehybridization for 12 hours at 
42.degree. C. in 5.times.SSC/10.times.Denhardt's reagent/40% 
formamide/0.1% SDS/10% dextran sulfate/and 100 .mu.g/ml of salmon sperm 
DNA, hybridization was performed for 16 hours at 42.degree. C. in 
5.times.SSC/2.times.Denhardt's/40% formamide/3% SDS/10% dextran 
sulfate/100 .mu.g/ml of salmon sperm DNA and an oligonucleotide probe 
(2.times.10.sup.6 cpm/ml), radiolabeled as described in Example 4. Blots 
were washed twice in 2.times.SSC/0.1% SDS and once in 0.5.times.SSC/0.1% 
SDS at room temperature and once in 0.1 .times.SSC/0.1% SDS at 60.degree. 
C. and exposed to Kodak X-OMAT film at -80.degree. C. for 24 hours with 
intensifying screens. Transferase specific probe 5PRM, SEQ ID NO:26 (see 
Table 2 for probe sequence) was specific to the 5'-untranslated region of 
the cDNA; Probe 48SP, SEQ ID NO:27, was specific to the coding region; and 
probe 3PRM, SEQ ID NO:28, was specific to the 3'-end of the coding region. 
This procedure was also used to assess the distribution of 
ADP-ribosyltransferase specific RNA in different tissues. These tissues 
included skeletal muscle, smooth muscle, heart, brain, lung, kidney, 
spleen and liver. 20-30 .mu.g of total RNA from the indicated tissues were 
hybridized with the transferase-specific probe, 48SP, SEQ ID NO:27. Total 
RNA was visualized on the gel following ethidium bromide staining using UV 
transillumination. 
EXAMPLE 7 
Expression of ADP-ribosyltransferase in E. coli 
ADP-ribosyltransferase cDNA was amplified by PCR using the primers 5Ndel, 
SEQ ID NO:24 and 3BamHI, SEQ ID NO:25. The PCR product was gel-purified, 
digested with Ndel and BamHI restriction enzymes (Promega) and the 
resulting 875-bp fragment was ligated to Ndel- and BamHI-digested pET3a 
(Novagen) with T4 DNA ligase (Promega) at 16.degree. C. for 16 hours. BL21 
(DE3) cells (Novagen) were transformed with the ligation product and 
applied to LB/ampicillin plates. After incubation overnight at 37.degree. 
C., colonies were screened by hybridization with the 48SP oligonucleotide 
probe, SEQ ID NO:27. One positive colony was grown at 37.degree. C. for 4 
hours in LB/ampicillin medium. The culture was then diluted 1:10 in 5 ml 
of the same medium, grown for 1 hour and induced with 0.4 mM IPTG 
(isopropyl-.beta.-D-thiogalactopyranoside) for 1.5 hours. After 
centrifugation at 10,000 g for 2 minutes the pellet was dispersed in 10 mM 
Tris-Cl, pH 8.0/1 mM EDTA/0.5 mM PMSF/leupeptin, aprotinin and pepstatin, 
each 0.5 .mu.g/ml. Following a 30s sonication on ice, samples were used 
for SDS-PAGE or transferase assay. 
Protein concentration was determined either by BCA assay or ISS protein 
gold (Integrated Protein Systems, Natick, Mass.) with bovine serum albumin 
as the standard. SDS-polyacrylamide gels were stained with Coomassie Blue 
or with silver stain (Rabilloud, et al., supra). 
EXAMPLE 8 
Expression of ADP-ribosyltransferase in mammalian cells 
Rat mammary adenocarcinoma (NMU) cells were grown in Eagle's Modified 
Essential Medium (EMEM) containing 10% fetal calf serum. Subconfluent NMU 
cells on 100.times.20 mm dishes were transformed with 15 .mu.g of purified 
pMAMneo (Higuchi, (1989) in PCR Technology: Principles and Applications 
for DNA Amplification, Ehrlich, H. A., ed., pp. 61-70, Stockton press, New 
York), pM-T, pM-AT or pM-3'T constructs by the calcium phosphate 
precipitation method (Ausubel et al., (1990) Current Protocols in 
Molecular Biology, Vol. I, p. 9.1.1., John Wiley & Sons, New York). 
To generate the pM-T construct, Nhel and Xhol restriction sites were added 
to the 5' and 3' ends, respectively, of the rabbit skeletal muscle 
ADP-ribosyltransferase cDNA during PCR amplification for ligation into 
pMAMneo. The PCR product and pMAMneo vector were digested with Nhel and 
Xhol and ligated using T4 DNA ligase. In the pM-AT construct, the 
ADP-ribosyltransferase cDNA was ligated into the pMAMneo vector in the 
reverse orientation. To generate the pM-3'T construct, the truncated form 
of the ADP-ribosyltransferase, from which 75 bases were removed at the 
3'-end of the cDNA coding region, was cloned into pMAMneo. All cloning 
steps were methods well known in the art. 
Cells were allowed to double before plating in selective medium (EMEM 
containing 10% FCS and 500 .mu.g/ml G418). Expression of stably 
incorporated ADP-ribosyltransferase was induced by incubation of cells 
with 1 .mu.M dexamethasone sodium phosphate for 48 hours (Sardet et al., 
(1989) Cell, 56: 271-280). 
EXAMPLE 9 
Assay to Detect ADP-ribosyltransferase Activity 
ADP-ribosyltransferase activity was assayed in 300 .mu.l of 50 mM potassium 
phosphate, pH 7.5, with 20 mM agmatine (Sigma, St. Louis, Mo.) and 0.1 mM 
adenine-U-.sup.14 C!NAD (1.7 mCi/mmol) (Amersham, Arlington Heights, 
Ill.) and cold NAD (Sigma). After incubation at 30.degree. C., a 100-.mu.l 
sample was applied to a 1-ml column of Dowex AG 1-X2 (Bio-Rad, Richmond, 
Calif.). .sup.14 C!ADP-ribosylagmatine was eluted with 5 ml of H.sub.2 O 
for radioassay. The elution profiles of .sup.14 C!ADP-ribosylagmatine 
with 0.1M sodium phosphate, pH 4.5 (flow rate 1 ml/minute) after 
incubation with native transferase, recombinant enzyme or control E. coli 
cells, transformed with expression vector lacking insert, with 0.1 mM 
adenine-U-.sup.14 C!NAD or without or with 20 mM agmatine. The elution 
times for adenosine, nicotinamide (Nic) and NAD did not vary between the 
native and recombinant enzyme. 
EXAMPLE 10 
Identification of the Human mono-ADP-ribosyltransferase sequence 
Human skeletal muscle mRNA (0.5 .mu.g, Clontech) was reverse transcribed 
(Invitrogen) using a mixed oligo(dT) primer (0.2 .mu.g) and random hexamer 
primers (1 .mu.g) (total volume 20 .mu.l) (Invitrogen). Techniques for 
isolating mRNA are disclosed in Example 6. The first strand of cDNA was 
used as a template in a PCR reaction employing mixed primers 1A, 1B and 2, 
SEQ ID NOS:5-7, (50 pmol of each). Amplification products (1% of the 
reaction volume) were reamplified in a second PCR reaction, using primers 
3 and 4, SEQ ID NOS 8 and 9, respectively (50 pmol of each). Both PCR 
amplifications were performed under the same conditions (35 cycles of 
94.degree. C. for one minute, 72.degree. for two minutes; followed by 
extension at 72.degree. for 7 minutes). 
EXAMPLE 11 
Completion of the Human ADP-ribosyltransferase Sequence 
The fragment of human ADP-ribosyltransferase as provided in Table 5 and SEQ 
ID NO:3 consisted of 224 amino acids. The full length nascent protein is 
likely to be about 330 amino acids long. It was estimated that about 70% 
of the human sequence was known and about 30% of the sequence still 
remained to be identified. The 5' and 3' remaining portions of the 
sequence were identified using 5'-RACE and 3'-RACE methods (rapid 
amplifications of 5'-end and 3'-end of cDNA, respectively) see Frohman, et 
al. Technique- A Journal of Methods in Cell and Molecular Biology 1: 
165-170 (1989). These procedures are easily performed by those skilled in 
the art and are used routinely in our laboratory. One example of the 
5'-RACE methodology is provided in Example 5. 
Human skeletal muscle poly(A).sup.+ RNA (1 .mu.g) was denatured with 
methylmercury hydroxide and reverse transcribed with MoMLV reverse 
transcriptase and 100 ng of transferase-specific primers; HSM-5 for 5'RACE 
(SEQ ID NO: 29), and R.sub.O primer (SEQ ID NO: 22) for 3'-RACE. 
The first cDNA strand product from the 5' end was incubated with dATP and 
terminal deoxynucleotidyl transferase to add a 3' deoxyadenosine tail as 
described (Frohman and Martin, 1989). The second DNA strand was 
synthesized using 100 ng of primers R.sub.O (SEQ ID NO: 22) and R.sub.O 
R.sub.I T (SEQ ID NO: 21) with Taq DNA polymerase according to the GeneAmp 
PCR Kit protocol (Perkin-Elmer, Norwalk, Conn.). Amplification was 
performed for 30 cycles at 94.degree. C. for 1 minute; 72.degree. C. for 2 
minutes followed by a 7 minute extension at 72.degree. C. The 50 .mu.l 
reaction mix was diluted to 1 ml with TE buffer (10 mM Tris-HCl, pH 7.5, 1 
mM EDTA) and the PCR product was separated from the primers using a 
Centricon 100 microconcentrator (Amicon, Beverly, Mass.). A second 
amplification was performed with 1 .mu.l of the first amplification 
product as a template, and 100 ng of nested primers HSM-CAUN (SEQ ID NO: 
30) and HSM-30 (SEQ ID NO: 31). Reaction conditions were the same as 
above. The PCR product was ethanol precipitated and 5'-phosphorylated 
using T4 polynucleotide kinase (1 .mu.l; Promega, Madison, Wis.) according 
to the manufacturer's protocol. The phosphorylated product was analyzed by 
electrophoresis on a low melting point 1% agarose gel, excised from the 
gel and subcloned into the pGEM-72(+) cloning vector (Promega, Madison, 
Wis.). Plasmid DNA was purified and sequenced as described previously. 
The first cDNA strand from the 3' cDNA end was amplified by PCR using 
primers R.sub.O (SEQ ID NO: 22) and HSM-1F (SEQ ID NO: 32). After 
separating the PCR product from the primers, a second round of 
amplification was performed using primers CAUHSM-2F (SEQ ID NO: 33) and 
CUA-RI (SEQ ID NO: 23). Amplification conditions were identical to those 
for the 5'-RACE procedure, except that the reaction was continued for 35 
cycles instead of 30. The amplified product was cloned into the pAMP1 
vector using the CloneAmp system and sequenced. 
To confirm the sequence of the entire human skeletal muscle transferase 
cDNA, poly(A)+RNA (1 .mu.g) was reverse transcribed as described above 
using primer P-RT (SEQ ID NO: 34) followed by two rounds of PCR 
amplification using primers HSM-1 and HSM-3 and subsequently nested 
primers HSM-1N (SEQ ID NO: 35) and HSM-RN (SEQ ID NO: 36). The final PCR 
product was subcloned and sequenced. 
EXAMPLE 12 
Preparation of anti-ADP-ribosyltransferase antibodies 
A truncated form of the rabbit muscle transferase lacking the hydrophobic 
amino- and carboxy-termini was expressed as a non-fusion protein in E. 
coli as described (Zolkiewska et al., (1992) Proc. Natl. Acad. Sci. 
U.S.A., 89: 11352-11356). Expression of the transferase was induced with 
isopropylthiogalactoside (IPTG) in a 20 ml suspension of E. coli. The 
suspension was sonified followed by the addition of 1% CHAPS, then 
centrifuged at 14,000.times.g for 5 minutes. The pellet, containing 500 
.mu.g protein, was emulsified in 1 ml of PBS and 1 ml of Freund's complete 
adjuvant and injected subcutaneously into rabbits having a body weight of 
1-1.5 kg. Rabbits were injected every two weeks with transferase emusified 
in Freund's incomplete adjuvant. The rabbits were bled after the fourth 
injection and antibody titer was assessed against ADP-ribosyltransferase 
on Western blots. 
EXAMPLE 13 
ELISA assay to Detect the Presence of ADP-ribosyltransferase in a Cell 
Sample 
Techniques for generating monoclonal antibodies are well known in the art. 
For a review of monoclonal antibody production, selection and screening 
see Davis, et al. Basic Methods in Molecular Biology. 1986. Elsevier 
Press, N.Y. pp. 348-354. Briefly, the purified protein preparation (50 
.mu.g per injection) of Example 1 or Example 7 is combined with an equal 
volume of complete freund's adjuvant. The remaining injections use between 
20-50 .mu.g purified protein per injection with an equal volume of 
incomplete freund's adjuvant. Injections are given to the mice at weekly 
intervals for approximately 6 weeks. 
The spleens are removed, teased and the splenocytes are isolated. 
Erythrocytes are lysed and the mouse splenocytes are mixes at a cell ratio 
of 4 spleen cells to 1 myeloma cell (cell line SP2/0, or the like, 
American type Culture Collection, Rockville, Md.). 50% polyethyleneglycol 
is added to the cell pellet containing the myeloma and splenocytes slowly 
over 1 minute. This is followed with 1 ml of cell culture medium. Cells 
are selected in hypoxanthine, aminopterin and thymidine as described by 
Davis, et al. (supra). Positive colonies are screened by ELISA. Antibody 
produced by these methods is purified using column chromatography, 
ammonium sulphate cuts or other methods known in the art of immunology. 
ELISA strategies are well known in the art. As one preferred example of an 
ELISA assay, 200 .mu.g of purified protein at 1 .mu.g/ml. in phosphate 
buffered saline (PBS) is incubated in each well of a 96 well ELISA plate 
overnight at 4.degree. C. The wells are washed with PBS containing 0.05% 
Tween 20. Media from the cell fusions, mouse or patient sera is serially 
diluted 1:5 in PBS containing 0.05% Tween 20 and Bovine Serum Albumin (0.1 
mg/ml) in serial dilutions. 200 .mu.l of each dilution are added in 
duplicate to the 96 well plate. Controls are added as well. Plates are 
incubated for 1 hr at room temperature and the wells are washed in PBS 
containing Tween, as described above. Aliquots of goat anti-mouse or human 
IgG conjugated to alkaline phosphatase diluted 1:400 in PBS containing 
Tween is added to each well. Plates are incubated for 1 hr at room 
temperature. Following a wash step, 200 .mu.l of a suitable chromogenic 
substrate are added with hydrogen peroxide according to directs contained 
in the substrate. Color development indicative of the presence of antibody 
to the purified protein in monitored on an ELISA reader. 
While particular embodiments of the invention have been described in 
detail, it will be apparent to those skilled in the art that these 
embodiments are exemplary rather than limiting, and the true scope of the 
invention is that defined in the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 38 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1140 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 106..1086 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GACCATCACATGAAGCCAACACCAGCTCCCTTGCCCCGGACAAGGCCTAGATGAGGAAAG60 
TAAGAGTCAAAAGGAGAGAGAAACTGGCCTGGGGTGGCCCCAACCATGTGGGTT114 
MetTrpVal 
CCTGCCGTGGCGAATCTGCTCCTTCTGTCCCTGGGCCTTCTGGAAGCA162 
ProAlaValAlaAsnLeuLeuLeuLeuSerLeuGlyLeuLeuGluAla 
51015 
ATTCAGGCCCAGAGCCACCTGGTCACACGTCGAGACCTCTTCTCTCAA210 
IleGlnAlaGlnSerHisLeuValThrArgArgAspLeuPheSerGln 
20253035 
GAGACACCGCTGGACATGGCCCCGGCCTCCTTTGATGACCAGTACGTC258 
GluThrProLeuAspMetAlaProAlaSerPheAspAspGlnTyrVal 
404550 
GGCTGTGCAGCAGCCATGACAGCTGCCCTCCCGCATCTCAACCTCACG306 
GlyCysAlaAlaAlaMetThrAlaAlaLeuProHisLeuAsnLeuThr 
556065 
GAGTTCCAGGTCAACAAAGTGTATGCGGACGGCTGGGCACTGGCAAGC354 
GluPheGlnValAsnLysValTyrAlaAspGlyTrpAlaLeuAlaSer 
707580 
AGCCAGTGGCGGGAGCGCTCGGCCTGGGGGCCCGAGTGGGGCCTCAGC402 
SerGlnTrpArgGluArgSerAlaTrpGlyProGluTrpGlyLeuSer 
859095 
ACAACCCGGCTCCCCCCGCCGCCTGCGGGATTTCGGGATGAACACGGG450 
ThrThrArgLeuProProProProAlaGlyPheArgAspGluHisGly 
100105110115 
GTGGCCCTGCTGGCCTACACGGCCAACAGCCCCCTACACAAGGAGTTC498 
ValAlaLeuLeuAlaTyrThrAlaAsnSerProLeuHisLysGluPhe 
120125130 
AATGCCGCGGTACGCCAGGCGGGCCGCTCCCGAGCCCACTACCTCCAG546 
AsnAlaAlaValArgGlnAlaGlyArgSerArgAlaHisTyrLeuGln 
135140145 
CACTTCTCCTTCAAGACCCTGCACTTCCTGCTGACCGAGGCCCTGCAG594 
HisPheSerPheLysThrLeuHisPheLeuLeuThrGluAlaLeuGln 
150155160 
CTGCTGGGCAGGGATCAGCGAATGCCCAGATGCCGTCAGGTGTTCCGG642 
LeuLeuGlyArgAspGlnArgMetProArgCysArgGlnValPheArg 
165170175 
GGGGTGCATGGACTGCGCTTCCGGCCAGCAGGGCCCGGGACCACTGTC690 
GlyValHisGlyLeuArgPheArgProAlaGlyProGlyThrThrVal 
180185190195 
AGGCTGGGGGGCTTTGCCTCTGCGTCACTGAAAAATGTAGCAGCCCAG738 
ArgLeuGlyGlyPheAlaSerAlaSerLeuLysAsnValAlaAlaGln 
200205210 
CAGTTTGGCGAGGACACGTTCTTTGGCATCTGGACCTGCCTTGGGGTC786 
GlnPheGlyGluAspThrPhePheGlyIleTrpThrCysLeuGlyVal 
215220225 
CCTATCCAGGGCTACTCCTTTTTCCCTGGGGAGGAGGAGGTTCTGATC834 
ProIleGlnGlyTyrSerPhePheProGlyGluGluGluValLeuIle 
230235240 
CCCCCCTTTGAGACCTTCCAGGTCATCAACGCCAGCAGACCTGCCCAG882 
ProProPheGluThrPheGlnValIleAsnAlaSerArgProAlaGln 
245250255 
GGCCCTGCCCGCATCTACCTGAAGGCGCTGGGCAAGCGCAGCTCATAC930 
GlyProAlaArgIleTyrLeuLysAlaLeuGlyLysArgSerSerTyr 
260265270275 
AACTGCGAGTACATCAAAGAAATGCAGTGCAAGTCTAGGCCCTGCCAC978 
AsnCysGluTyrIleLysGluMetGlnCysLysSerArgProCysHis 
280285290 
CTGGACAATTCAGCCTCGGCTCAGGAGCGCCTCTCCACAGCCTGGTCC1026 
LeuAspAsnSerAlaSerAlaGlnGluArgLeuSerThrAlaTrpSer 
295300305 
CTCCTGCTGCTGCTCGCGTTCCTTGCGGTGGGGCCCTTCCCAGGAAGC1074 
LeuLeuLeuLeuLeuAlaPheLeuAlaValGlyProPheProGlySer 
310315320 
CCAGGCCTCTTCTGACCCCCCAGACTCTGGACATTCCTGCCTGCTGCCTCTG1126 
ProGlyLeuPhe 
325 
CCCACTCTGTGGAT1140 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 327 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetTrpValProAlaValAlaAsnLeuLeuLeuLeuSerLeuGlyLeu 
151015 
LeuGluAlaIleGlnAlaGlnSerHisLeuValThrArgArgAspLeu 
202530 
PheSerGlnGluThrProLeuAspMetAlaProAlaSerPheAspAsp 
354045 
GlnTyrValGlyCysAlaAlaAlaMetThrAlaAlaLeuProHisLeu 
505560 
AsnLeuThrGluPheGlnValAsnLysValTyrAlaAspGlyTrpAla 
65707580 
LeuAlaSerSerGlnTrpArgGluArgSerAlaTrpGlyProGluTrp 
859095 
GlyLeuSerThrThrArgLeuProProProProAlaGlyPheArgAsp 
100105110 
GluHisGlyValAlaLeuLeuAlaTyrThrAlaAsnSerProLeuHis 
115120125 
LysGluPheAsnAlaAlaValArgGlnAlaGlyArgSerArgAlaHis 
130135140 
TyrLeuGlnHisPheSerPheLysThrLeuHisPheLeuLeuThrGlu 
145150155160 
AlaLeuGlnLeuLeuGlyArgAspGlnArgMetProArgCysArgGln 
165170175 
ValPheArgGlyValHisGlyLeuArgPheArgProAlaGlyProGly 
180185190 
ThrThrValArgLeuGlyGlyPheAlaSerAlaSerLeuLysAsnVal 
195200205 
AlaAlaGlnGlnPheGlyGluAspThrPhePheGlyIleTrpThrCys 
210215220 
LeuGlyValProIleGlnGlyTyrSerPhePheProGlyGluGluGlu 
225230235240 
ValLeuIleProProPheGluThrPheGlnValIleAsnAlaSerArg 
245250255 
ProAlaGlnGlyProAlaArgIleTyrLeuLysAlaLeuGlyLysArg 
260265270 
SerSerTyrAsnCysGluTyrIleLysGluMetGlnCysLysSerArg 
275280285 
ProCysHisLeuAspAsnSerAlaSerAlaGlnGluArgLeuSerThr 
290295300 
AlaTrpSerLeuLeuLeuLeuLeuAlaPheLeuAlaValGlyProPhe 
305310315320 
ProGlySerProGlyLeuPhe 
325 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 669 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..669 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGCTGTGCTGCTGCCATGACAGCTGCTCTCCCGGATCTCAACCACACG48 
GlyCysAlaAlaAlaMetThrAlaAlaLeuProAspLeuAsnHisThr 
151015 
GAGTTCCAGGCCAACCAGGTGTATGCAGACAGCTGGACACTGGCAAGC96 
GluPheGlnAlaAsnGlnValTyrAlaAspSerTrpThrLeuAlaSer 
202530 
AGCCAATGGCAGGAGCGTCAGGCCAGGTGGCCAGAGTGGAGTCTCAGC144 
SerGlnTrpGlnGluArgGlnAlaArgTrpProGluTrpSerLeuSer 
354045 
CCCACCCGTCCATCCCCGCCACCCCTGGGCTTCCGCGATGAGCATGGG192 
ProThrArgProSerProProProLeuGlyPheArgAspGluHisGly 
505560 
GTGGCCCTCCTGGCCTACACAGCCAACAGCCCCCTGCACAAGGAGTTC240 
ValAlaLeuLeuAlaTyrThrAlaAsnSerProLeuHisLysGluPhe 
65707580 
AATGCAGCCGTGCGTGAGGCGGGCCGCTCCCGGGCCCACTACCTCCAC288 
AsnAlaAlaValArgGluAlaGlyArgSerArgAlaHisTyrLeuHis 
859095 
CACTTCTCCTTCAAGACACTCCATTTCCTGCTGACTGAGGCCCTGCAG336 
HisPheSerPheLysThrLeuHisPheLeuLeuThrGluAlaLeuGln 
100105110 
CTCCTGGGCAGCGGCCAGCGTCCACCCCGGTGCCACCAGGTGTTCCGA384 
LeuLeuGlySerGlyGlnArgProProArgCysHisGlnValPheArg 
115120125 
GGTGTGCACGGCCTGCGCTTCCGGCCAGCGGGGCCCCGGGCCACCGTG432 
GlyValHisGlyLeuArgPheArgProAlaGlyProArgAlaThrVal 
130135140 
AGGTTGGGGGGCTTTGCTTCTGCCTCCCTGAAGCATGTTGCAGCCCAG480 
ArgLeuGlyGlyPheAlaSerAlaSerLeuLysHisValAlaAlaGln 
145150155160 
CAGTTTGGTGAGGACACCTTCTTCGGCATCTGGACCTGCCTTGGGGCC528 
GlnPheGlyGluAspThrPhePheGlyIleTrpThrCysLeuGlyAla 
165170175 
CCTATCAAGGGCTACTCCTTCTTCCCTGGAGAGGAAGAGGTGCTGATC576 
ProIleLysGlyTyrSerPhePheProGlyGluGluGluValLeuIle 
180185190 
CCCCCCTTTGAGACCTTCCAAGTGATCAATGCCAGCAGACCGGCCCAG624 
ProProPheGluThrPheGlnValIleAsnAlaSerArgProAlaGln 
195200205 
GGCCCCGCCCGCATCTACCTCCGAGCCCTGGGCAAGCACAGCACC669 
GlyProAlaArgIleTyrLeuArgAlaLeuGlyLysHisSerThr 
210215220 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 223 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GlyCysAlaAlaAlaMetThrAlaAlaLeuProAspLeuAsnHisThr 
151015 
GluPheGlnAlaAsnGlnValTyrAlaAspSerTrpThrLeuAlaSer 
202530 
SerGlnTrpGlnGluArgGlnAlaArgTrpProGluTrpSerLeuSer 
354045 
ProThrArgProSerProProProLeuGlyPheArgAspGluHisGly 
505560 
ValAlaLeuLeuAlaTyrThrAlaAsnSerProLeuHisLysGluPhe 
65707580 
AsnAlaAlaValArgGluAlaGlyArgSerArgAlaHisTyrLeuHis 
859095 
HisPheSerPheLysThrLeuHisPheLeuLeuThrGluAlaLeuGln 
100105110 
LeuLeuGlySerGlyGlnArgProProArgCysHisGlnValPheArg 
115120125 
GlyValHisGlyLeuArgPheArgProAlaGlyProArgAlaThrVal 
130135140 
ArgLeuGlyGlyPheAlaSerAlaSerLeuLysHisValAlaAlaGln 
145150155160 
GlnPheGlyGluAspThrPhePheGlyIleTrpThrCysLeuGlyAla 
165170175 
ProIleLysGlyTyrSerPhePheProGlyGluGluGluValLeuIle 
180185190 
ProProPheGluThrPheGlnValIleAsnAlaSerArgProAlaGln 
195200205 
GlyProAlaArgIleTyrLeuArgAlaLeuGlyLysHisSerThr 
210215220 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
NTTRGAYATGGCNCCNGC18 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
NCTNGAYATGGCNCCNGC18 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
YTTRCAYTGCATYTCYTT18 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
NTTYGAYGAYCARTAYGT18 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
DATRTAYTCRCARTTRTA18 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GCTGTCTGCATACACCTGGTTTGGC25 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GTCGTTGAGATCCGGGAGAGC21 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CCCGCATCTACCTCCGAGCC20 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CAAGCACAGCACCTATAATT20 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GCCCANCCATCNGCATANAC20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GCTAANGCCCANCCATCNGC20 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GCNAGNGCCCANCCATCNGC20 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CAAAAGCTGGAGCTCCACCGCGGTG25 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GCTCTAGAACTAGTGGATCCC21 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
TGTCATGGCTGCTGCACAGC20 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
CAUCAUCAUCAUACGTACTGGTCATCAAAGGA32 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGATTTTTTTTTTTTTTTTT57 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
AAGGATCCGTCGACATC17 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CUACUACUACUAGACATCGATAATACGACTCACTATA37 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CTGGTTCCGGCGACATATGAGCCACCTGGTCACACGTCG39 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CTCGCTCCGGCGAGGATCCTCAGGAGAGGCGCTCCTGAGCCG42 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
TTACTTTCCTCATCTAGGCCTTGTCCGGGGCAGGGGAGCTGGTGTTGG48 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
AAAGGAGGCCGGGGCCATGTCCAGCGGTGTCTCTTGAGAGAAGAGGTC48 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
AGGAATGTCCAGAGTCTGGGGGGTCAGAAGAGGCCTGGGCTTCCTGGG48 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
CGGTTGGTCCACATACGTCTGTCG24 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CAUCAUCAUCAUGTGGTTGAGATCCGGGAGAGC33 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
ACTAGTTATGCAACCGACACGACGACGGTA30 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CCCGCATCTACCTCCGAGCC20 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
CAUCAUCAUCAUCAAGCACAGCACCTATAATT32 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
GGACTCCTACAACGGGTACACACG24 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
CUACUACUACUAAGCAACTGGCCCAGGGTCACCAGC36 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
ACGTACTCTGTGCCCTGTCAUCAUCAUCAU30 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1305 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 73..1047 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
TTCCACCAGGACAGGCCTAGATGAGGAAACTGAGACCCAAAAAGAGACAGCAACTGGCCC60 
AGGGTCACCAGCATGCAGATGCCTGCTATGATGTCTCTGCTTCTTGTG108 
MetGlnMetProAlaMetMetSerLeuLeuLeuVal 
1510 
TCTGTGGGCCTCATGGAAGCACTTCAGGCCCAGAGCCACCCCATCACA156 
SerValGlyLeuMetGluAlaLeuGlnAlaGlnSerHisProIleThr 
152025 
CGACGAGACCTCTTCTCTCAAGAGATTCAGCTGGACATGGCCCTGGCC204 
ArgArgAspLeuPheSerGlnGluIleGlnLeuAspMetAlaLeuAla 
303540 
TCCTTTGATGACCAGTACGCTGGCTGTGCTGCTGCCATGACAGCTGCT252 
SerPheAspAspGlnTyrAlaGlyCysAlaAlaAlaMetThrAlaAla 
45505560 
CTCCCGGATCTCAACCACACGGAGTTCCAGGCCAACCAGGTGTATGCA300 
LeuProAspLeuAsnHisThrGluPheGlnAlaAsnGlnValTyrAla 
657075 
GACAGCTGGACACTGGCAAGCAGCCAATGGCAGGAGCGTCAGGCCAGG348 
AspSerTrpThrLeuAlaSerSerGlnTrpGlnGluArgGlnAlaArg 
808590 
TGGCCAGAGTGGAGTCTCAGCCCCACCCGTCCATCCCCGCCACCCCTG396 
TrpProGluTrpSerLeuSerProThrArgProSerProProProLeu 
95100105 
GGCTTCCGCGATGAGCATGGGGTGGCCCTCCTGGCCTACACAGCCAAC444 
GlyPheArgAspGluHisGlyValAlaLeuLeuAlaTyrThrAlaAsn 
110115120 
AGCCCCCTGCACAAGGAGTTCAATGCAGCCGTGCGTGAGGCGGGCCGC492 
SerProLeuHisLysGluPheAsnAlaAlaValArgGluAlaGlyArg 
125130135140 
TCCCGGGCCCACTACCTCCACCACTTCTCCTTCAAGACACTCCATTTC540 
SerArgAlaHisTyrLeuHisHisPheSerPheLysThrLeuHisPhe 
145150155 
CTGCTGACTGAGGCCCTGCAGCTCCTGGGCAGCGGCCAGCGTCCACCC588 
LeuLeuThrGluAlaLeuGlnLeuLeuGlySerGlyGlnArgProPro 
160165170 
CGGTGCCACCAGGTGTTCCGAGGTGTGCACGGCCTGCGCTTCCGGCCA636 
ArgCysHisGlnValPheArgGlyValHisGlyLeuArgPheArgPro 
175180185 
GCAGGGCCCCGGGCCACCGTGAGGCTGGGGGGCTTTGCTTCTGCCTCC684 
AlaGlyProArgAlaThrValArgLeuGlyGlyPheAlaSerAlaSer 
190195200 
CTGAAGCATGTTGCAGCCCAGCAGTTTGGTGAGGACACCTTCTTCGGC732 
LeuLysHisValAlaAlaGlnGlnPheGlyGluAspThrPhePheGly 
205210215220 
ATCTGGACCTGCCTTGGGGCCCCTATCAAGGGCTACTCCTTCTTCCCT780 
IleTrpThrCysLeuGlyAlaProIleLysGlyTyrSerPhePhePro 
225230235 
GGAGAGGAAGAGGTGCTGATCCCCCCCTTTGAGACCTTCCAAGTGATC828 
GlyGluGluGluValLeuIleProProPheGluThrPheGlnValIle 
240245250 
AATGCCAGCAGACCGGCCCAGGGCCCCGCCCGCATCTACCTCCGAGCC876 
AsnAlaSerArgProAlaGlnGlyProAlaArgIleTyrLeuArgAla 
255260265 
CTGGGCAAGCACAGCACCTACAACTGCGAGTACATCAAAGACAAGAAG924 
LeuGlyLysHisSerThrTyrAsnCysGluTyrIleLysAspLysLys 
270275280 
TGCAAGTCTGGGCCTTGCCATCTGGATAATTCAGCCATGGGTCAGAGC972 
CysLysSerGlyProCysHisLeuAspAsnSerAlaMetGlyGlnSer 
285290295300 
CCCCTCTCTGCAGTCTGGTCTTTGCTGCTGCTGCTCTGGTTCCTCGTG1020 
ProLeuSerAlaValTrpSerLeuLeuLeuLeuLeuTrpPheLeuVal 
305310315 
GTGAGGGCCTTTCCAGATGGTCCAGGCCTCCTTTGATGCATGAGACA1067 
ValArgAlaPheProAspGlyProGlyLeuLeu 
320325 
CGGGACAGCCTCGCCTGCTGCCTCTGCCCATCCTGAGGATGTTGGCCATGTGTGCTTCAG1127 
TGTAACCAAGATTCCTGTCAATCCCATCTGCAGGGAACTCTGGGACCTTCTCTGGTAGCT1187 
GCCAGACCGGCTGGTGGAGAAACAGGAGACAATCTGGGGACTGAACCTTACCCAGGGCTG1247 
TAGGAGTGAGACTCTGAATAAAGGGTTGGGCCGGCAAAAAAAAAAAAAAAAAAAAAAA1305 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 327 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
MetGlnMetProAlaMetMetSerLeuLeuLeuValSerValGlyLeu 
151015 
MetGluAlaLeuGlnAlaGlnSerHisProIleThrArgArgAspLeu 
202530 
PheSerGlnGluIleGlnLeuAspMetAlaLeuAlaSerPheAspAsp 
354045 
GlnTyrAlaGlyCysAlaAlaAlaMetThrAlaAlaLeuProAspLeu 
505560 
AsnHisThrGluPheGlnAlaAsnGlnValTyrAlaAspSerTrpThr 
65707580 
LeuAlaSerSerGlnTrpGlnGluArgGlnAlaArgTrpProGluTrp 
859095 
SerLeuSerProThrArgProSerProProProLeuGlyPheArgAsp 
100105110 
GluHisGlyValAlaLeuLeuAlaTyrThrAlaAsnSerProLeuHis 
115120125 
LysGluPheAsnAlaAlaValArgGluAlaGlyArgSerArgAlaHis 
130135140 
TyrLeuHisHisPheSerPheLysThrLeuHisPheLeuLeuThrGlu 
145150155160 
AlaLeuGlnLeuLeuGlySerGlyGlnArgProProArgCysHisGln 
165170175 
ValPheArgGlyValHisGlyLeuArgPheArgProAlaGlyProArg 
180185190 
AlaThrValArgLeuGlyGlyPheAlaSerAlaSerLeuLysHisVal 
195200205 
AlaAlaGlnGlnPheGlyGluAspThrPhePheGlyIleTrpThrCys 
210215220 
LeuGlyAlaProIleLysGlyTyrSerPhePheProGlyGluGluGlu 
225230235240 
ValLeuIleProProPheGluThrPheGlnValIleAsnAlaSerArg 
245250255 
ProAlaGlnGlyProAlaArgIleTyrLeuArgAlaLeuGlyLysHis 
260265270 
SerThrTyrAsnCysGluTyrIleLysAspLysLysCysLysSerGly 
275280285 
ProCysHisLeuAspAsnSerAlaMetGlyGlnSerProLeuSerAla 
290295300 
ValTrpSerLeuLeuLeuLeuLeuTrpPheLeuValValArgAlaPhe 
305310315320 
ProAspGlyProGlyLeuLeu 
325 
__________________________________________________________________________