Nucleoside cotransporter protein cDNA

The complete cDNA sequence encoding the amino acid sequence corresponding to mammalian Na.sup.+ /nucleoside cotransporter protein (SNST) is disclosed. Methods for obtaining the gene encoding SNST and for obtaining recombinantly produced SNST are described. Antibodies, an inhibitor of nucleoside transport by SNST, and methods for detecting other inhibitors are also described. Methods for inhibiting uptake of nucleosides by SNST using the compositions of the invention are also included.

FIELD OF THE INVENTION 
The present invention relates to the identification and production of 
cotransporter membrane protein, and more particularly to the mammalian 
sodium dependent nucleoside cotransporter protein, to methods of using the 
cotransporter protein and to inhibitors of sodium dependent nucleoside 
transport into mammalian tissues. 
BACKGROUND OF THE INVENTION 
Cotransporter proteins are membrane-bound proteins that actively transport 
substances into cells. For example, organic substrates such as sugars, 
amino acids, carboxylic acids and neurotransmitters, are transported into 
eucaryotic cells by sodium (Na.sup.+) cotransporter proteins. Some 
transport proteins have been identified, for example, Na.sup.+ /glucose 
and Na.sup.+ /proline transporters (Peerce and Wright, Proc. Natl. Acad. 
Sci. USA 8:2223-2226 (1984); Wright and Peerce, J. Biol. Chem. 
259:14993-14996 (1984)), and the brain Na.sup.+ Cl/GABA transporter 
(Radian et al., J. Biol. Chem. 261:15437-15441 (1986)), and progress has 
been made in locating their active sites and probing their conformational 
states (Peerce and Wright, supra; Wright and Peerce, supra; Peerce and 
Wright, J. Biol. Chem. 259:14105-14112 (1984); J. Biol. Chem. 
260:6026-6031 (1985); Proc. Natl. Acad. Sci. USA 83:8092-8096 (1986); 
Biochem. 26:4272-4276 (1987)). 
There appears to be a gene family of sodium (Na+) dependent transporters 
related to the mammalian intestinal Na+/glucose cotransporter, SGLT1. 
Na+/glucose cotransporters have been cloned and sequenced from rabbit 
intestine (Hediger, et al, Nature 330:379-381 (1987)) and kidney (Coady, 
et al, Am. J. Physiol. 259:C605-C610 (1990)), human intestine (Hediger, et 
al, Proc. Natl. Acad. Sci. USA 86:5748-5752, and LLC-PK.sup.1 cells (Ohta, 
et al, Mol. Cell. Biol. 10:6491-6499 (1990)). All of these transporters 
share greater than 80% identity of amino acid sequence. The only other 
proteins with significant homology to the mammalian SGLT1 are bacterial 
Na+-dependent transporters for proline (Nakao, et al, Mol. Gen. Genet. 
208:70-75 (1987)) and pantothenate (Jackowski & Alix, Bacteriol. 
172:3842-3848 (1990)). This suggests that the relationship between members 
of this multigene family is at the level of the transport mechanism, the 
Na+ coupling, rather than the substrate being transported. 
Defects in Na.sup.+ -driven transporters may be associated with diseases. 
For example, a defect in the intestinal brush border Na.sup.+ /glucose 
cotransporter (SGLT1) is the origin of the congenital glucose-galactose 
malabsorption syndrome (Turk et al. Nature 350:354-356 (1991)). 
It has been difficult to clone mammalian cotransport proteins in part 
because of the difficulties in purification of low abundance, hydrophobic 
membrane proteins that constitute less than 0.2% of the membrane protein. 
Hediger et al., Nature 330:379-381 (1987) described a new strategy for 
cloning rabbit intestinal Na.sup.+ /glucose cotransporter for expression 
without the use of antibodies or synthetic oligonucleotide probes. 
Nucleosides direct a number of important physiological activities, 
particularly in the mammalian cardiovascular and central nervous systems. 
For example, adenosine is a potent vasoactive molecule in the coronary and 
cerebral vessels and a modulator of potassium and calcium channels in 
neurons and cardiac muscle (Belardinelli et al., Prog. Cardiovasc. 
Diseases 32:73-97 (1989)). Furthermore, nucleosides and their analogs are 
known to be potent cytotoxic and anti-retroviral agents. Nucleosides and 
their analogs, and nucleoside transport inhibitors are being used or 
proposed for use as broad spectrum anti-retroviral drugs, anti-cancer 
drugs, for treating ischemia and reperfusion-induced cell injury in the 
heart, and anti-arrhythmic drugs. For example, nucleoside transporter 
proteins may be useful to transport drugs to treat diseases such as AIDS 
(see, Yarchoan and Broder, New Engl. J. Med. 316:557-564 (1987)). In 
addition, membrane transport proteins are involved in the regulation of 
the uptake of nucleosides into cells. Nucleosides are required for normal 
growth, metabolism and function of cells. Some cells, such as bone marrow, 
leukocytes and brain cells, are deficient in purine biosynthesis and 
dependent on uptake of preformed purines. 
Phloridzin, 
(1-[2-.beta.-D-Glucopyranosyloxy)-4,6-dihydroxyphenyl]-3]-4-hydroxyphenyl) 
-1-propanone, No. 7300, p. 1163, Merck Index, 11th Edition, (1989)) a 
phloretin-2'-.beta.-glucoside, is a natural substance occurring in all 
parts of the apple tree, and is known to be a very specific inhibitor of 
sugar transport in the mammalian intestine and kidney (see Newey et al., 
J. Physiol. 169:229-236 (1963) and Diedrich, Methods in Enzymology 
191:755-780 (1990)). These effects are known to be due to competitive 
inhibition of active, i.e. sodium dependent, sugar transport. Fifty 
percent inhibition is achieved by phloridzin concentrations as low as 
5.times.10.sup.-6 molar. Phloridzin is very specific for the Na.sup.+ 
/glucose cotransporter protein because the glucose moiety is recognized by 
the active site on the membrane transport protein. There are no other 
known effects of phloridzin on biological. systems. 
No other specific inhibitors of sodium dependent cotransporter proteins are 
known. 
Nucleoside cotransporter proteins have been identified (Plageman et al., 
Biochim. Biophys. Acta. 947:405-554 (1988)), but have not been isolated, 
cloned and expressed. Therefore, these proteins are available in small 
quantities only from mammalian cell membranes. Thus, it would be desirable 
to have available a method for producing practical quantities of 
nucleoside cotransporter protein for use alone or with drugs that can be 
delivered into cells by the protein and to identify inhibitors that will 
specifically block the activity of the cotransporter protein. 
SUMMARY OF THE INVENTION 
The invention provides a means for obtaining mammalian nucleoside 
cotransporter protein in quantity. 
Thus, in one aspect, the invention relates to recombinantly produced 
mammalian nucleoside cotransporter protein (SNST). This protein has an 
amino acid sequence substantially similar to that shown in FIG. 2 (SEQ ID 
NO:1). The invention further relates to a cDNA sequence and a genomic DNA 
sequence encoding mammalian SNST, to expression vectors suitable for 
production of this protein, to recombinant host cells transformed with 
these vectors, and to methods for producing recombinant SNST. In other 
aspects, the invention relates to the identification and use of inhibitors 
of the activity of SNST, and to compositions containing mammalian SNST or 
inhibitors of SNST, and to methods of using these compositions.

DETAILED DESCRIPTION OF THE INVENTION 
In order that the invention herein described may be more fully understood, 
the following description is set forth. 
Definitions 
As used herein, "Na.sup.+ /nucleoside cotransporter protein (SNST or 
SNST1)" refers to the mammalian sodium nucleoside cotransporter membrane 
protein expressed from a clone obtained as described below. SNST1 has the 
amino acid sequence shown in FIG. 2. This protein has substantial homology 
with the amino acid sequence of mammalian intestinal Na.sup.+ /glucose 
cotransporter (SGLT1). The mammalian SNST1 recombinant protein of this 
invention has an amino acid sequence substantially similar to that shown 
in FIG. 2, but minor modifications of this sequence which do not destroy 
activity also fall within the definition and within the protein of the 
invention. Also included within the definition are fragments of the entire 
sequence encoding SNST1 which retain activity. 
As is the case for all proteins, SNST1 can occur in neutral form or in the 
form of basic or acid addition salts, depending on its mode of 
preparation, or, if in solution, upon its environment. In addition, the 
protein may be modified by combination with other biological materials 
such as lipids and saccharides, or by side chain modification, such as 
acetylation of amino groups, phosphorylation of hydroxyl side chains, or 
oxidation of sulfhydryl groups, or other modification of the encoded 
primary sequence. In its native form, SNST1 is probably a glycosylated 
protein and is associated with phospholipids. Included within the 
definition of SNST1 herein are glycosylated and unglycosylated forms, 
hydroxylated and nonhydroxylated forms, and any composition of an amino 
acid sequence substantially similar to that shown in FIG. 2 which retains 
the ability of the protein to transport nucleosides and nucleoside analogs 
across cell membranes. 
It is further understood that minor modifications of primary amino acid 
sequence may result in proteins that have substantially equivalent or 
enhanced activity as compared to the sequence set forth in FIG. 2. These 
modifications may be deliberate, as by site-directed mutagenesis, or may 
be accidental, for example by mutation of hosts that are SNST1 producing 
organisms. All of these modification are included in the definition 
provided that activity of SNST1 is retained. 
"SNST or SNST1 activity" is defined as sodium stimulated nucleoside uptake 
into cells. For example RNA encoding SNST1 is injected into Xenopus 
oocytes, and uptakes of labeled nucleosides as substrate, are measured 
after several days as a function of Na.sup.+ concentration. Transport is 
stopped by washing with choline solution containing excess unlabeled 
substrate. The oocytes are individually dissolved in SDS and assayed for 
radioactivity (see Coady et al., Arch. Biochem. Biophys. 283 (1):130-134 
(1990)). 
"Control sequence" refers to a DNA sequence or sequences which are capable, 
when properly ligated to a desired coding sequence, of effecting its 
expression in hosts compatible with such sequences. Such control sequences 
include promoters in both procaryotic and eucaryotic hosts, and in 
procaryotic organisms also include ribosome binding site sequences, and, 
in eucaryotes, termination signals. Additional factors necessary or 
helpful in effecting expression may subsequently be identified. As used 
herein, "control sequences" simply refers to whatever DNA sequence may be 
required to effect expression in the particular host employed. 
"Operably linked" refers to a positional arrangement wherein the components 
are configured so as to perform their usual function. Thus, control 
sequences operably linked to coding sequences are capable of effecting the 
expression of the coding sequence. 
"Cells" or "recombinant host cells" or "host cells" are often used 
interchangeably herein as will be clear from the context. These terms 
include the immediate subject cell, and the progeny thereof. It is 
understood that not all progeny are exactly identical to the parental 
cell, due to chance mutations or differences in environment. However, such 
altered progeny are included when the above terms are used. 
General Description 
The methods illustrated below to obtain a cDNA sequence encoding mammalian 
SNST1, the gene for SNST and the SNST1 protein, are merely for purposes of 
illustration and are typical of those that might be used. However, other 
procedures may also be employed, as is understood in the art. 
Cloning of Coding Sequences for Mammalian SNST1 
The entire cDNA sequence encoding mammalian SNST1 protein has been cloned 
and expressed in Xenopus oocytes as set forth in Example 1, infra. 
Complementary DNAs encoding seven different proteins related to the rabbit 
intestinal Na.sup.+ /glucose cotransporter protein, SGLT1, designated 
RK-A-R-KI, were isolated from a rabbit renal cDNA library by high 
stringency hybridization with a fragment of the rabbit renal SGLT1 cDNA. 
One of the most abundant renal cDNAs, RK-C, was selected for more detailed 
characterization, and was found to encode most of SNST1 (nucleotides 
66-2150). The composite sequence of SNST1 shown in FIG. 2 was obtained 
from two cDNAs, RK-C (nucleotides 66-2150) and RK-44 (nucleotides 20-2238, 
obtained by rescreening the library with RK-C) and by direct sequencing of 
rabbit renal mRNA. The full sequence of SNST1 (FIG. 2) encodes a protein 
of 672 amino acids. The sequence of SNST1 has significant homology with 
the amino acid sequence of rabbit intestinal SGLT1 (FIG. 3). 
Expression of Mammalian SNST1 
With the complete nucleotide sequence encoding mammalian SNST1 provided 
herein, the sequence may be expressed in a variety of systems. In Example 
1, infra, the SNST1 RNA is used directly in a Xenopus oocyte expression 
system. To effect functional expression, a chimeric plasmid, SNST1c, was 
constructed because the SNST1 cDNAs lacked a start codon and poly(A.sup.+) 
tail. SNST1c RNA was then expressed in Xenopus ooyctes. Expression of 
SNST1c in Xenopus oocytes resulted in nucleoside-stimulated .sup.22 Na 
uptake and sodium-dependent .sup.3 H-uridine uptake. The uptake of 
H-uridine was inhibited by a range of nucleosides, including the anti-HIV 
drug, dideoxycytidine. 
Standard Methods 
The techniques for sequencing, cloning and expressing DNA sequences 
encoding the amino acid sequences corresponding to the Na.sup.+ 
/nucleoside cotransporter protein, e.g PCR, synthesis of oligonucleotides, 
probing a cDNA library, transforming cells, constructing vectors, 
extracting messenger RNA, preparing cDNA libraries, and the like are 
well-established in the art, and most practitioners are familiar with the 
standard resource materials for specific conditions and procedures. 
However, the following paragraphs are provided for convenience and 
notation of modifications where necessary, and may serve as a guideline. 
Sequencing: 
Isolated cDNA and RNA encoding the SNST1 protein is analyzed by using a T7 
sequencing kit (Pharmacia, Piscataway, N.J.) and synthetic 
oligonucleotides (Genosys Inc., San Diego, Calif.) as sequencing primers. 
Alternatively, cDNA is analyzed by restriction and/or sequenced by the 
dideoxy method of Sanger et al., Proc. Nat. Acad. Sci. USA 74:5463 (1977) 
as further described by Messing et al., Nucleic Acids Res. 9:309 (1981) or 
by the method of Maxam et al., Methods in Enzymol. 65:499 (1980). 
Hosts and Control Sequences: 
Both procaryotic and eucaryotic systems may be used to express the SNST1 
protein; procaryotic hosts are the most convenient for cloning procedures. 
If procaryotic systems are used, an intronless coding sequence should be 
used, along with suitable control sequences. The cDNA of mammalian SNST1 
can be excised using suitable restriction enzymes and ligated into 
procaryotic vectors along with suitable control sequences for such 
expression. 
Procaryotes most frequently are represented by various strains of E. coli; 
however, other microbial strains may also be used. Commonly used 
procaryotic control sequences which are defined herein to include 
promoters for transcription initiation, optionally with an operator, along 
with ribosome binding site sequences, include such commonly used promoters 
as the beta-lactamase (penicillinase) and lactose (lac) promoter systems 
(Chang et al., Nature 198:1056 (1977)) and the tryptophan (trp) promoter 
system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda 
derived P.sub.L promoter and N-gene ribosome binding site (Shimatake et 
al., Nature 292:128 (1981)). 
In addition to bacteria, eucaryotic microbes, such as yeast, may also be 
used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's 
yeast, are most used although a number of other strains are commonly 
available. Vectors employing, for example, the 2 .mu. origin of 
replication of Broach, Meth. Enz. 101:307 (1983), or other yeast 
compatible origins of replications (see, for example, Stinchcomb et al., 
Nature 282:39 (1979)); Tschempe et al., Gene 10:157 (1980); and Clarke et 
al., Meth. Enz. 101:300 (1983)) may be used. Control sequences for yeast 
vectors include promoters for the synthesis of glycolytic enzymes (Hess et 
al., J. Adv. Enzyme Reg. 7:149 (1968); Holland et al., Biochemistry 
17:4900 (1978)). Additional promoters known in the art include the 
promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 
255:2073 (1980)), and those for other glycolytic enzymes. Other promoters, 
which. have the additional advantage of transcription controlled by growth 
conditions are the promoter regions for alcohol dehydrogenase 2, 
isocytochrome C, acid phosphatase, degradative enzymes associated with 
nitrogen metabolism, and enzymes responsible for maltose and galactose 
utilization. It is also believed terminator sequences are desirable at the 
3' end of the coding sequences. Such terminators are found in the 3' 
untranslated region following the coding sequences in yeast-derived genes. 
It is also, of course, possible to express genes encoding polypeptides in 
eucaryotic host cell cultures derived from multicellular organisms. (See, 
for example, Tissue Cultures, Academic Press, Cruz and Patterson, Eds. 
(1973)). These systems have the additional advantage of the ability to 
splice out introns and thus can be used directly to express genomic 
fragments. Useful host cell lines include amphibian oocytes such as 
Xenopus oocytes, COS cells, VERO and HeLa cells, Chinese hamster ovary 
(CHO) cells and insect cells such as SF9 cells. Expression vectors for 
such cells ordinarily include promoters and control sequences compatible 
with mammalian cells such as, for example, the commonly used early and 
late promoters from baculovirus, vaccinia virus, Simian Virus 40 (SV 40) 
(Fiers, et al., Nature 273:113 (1973)), or other viral promoters such as 
those derived from polyoma, Adenovirus 2, bovine papilloma virus, or avian 
sarcoma viruses. The controllable promoter, hMTII (Karin, et al., Nature 
299:797-802 (1982)) may also be used. General aspects of mammalian cell 
host system transformations have been described by Axel (U.S. Pat. No. 
4,399,216 issued Aug. 16, 1983). It now appears, that "enhancer" regions 
are important in optimizing expression; these are, generally, sequences 
found upstream or downstream of the promoter region in noncoding DNA 
regions. Origins of replication may be obtained, if needed, from viral 
sources. However, integration into the chromosome is a common mechanism 
for DNA replication in eucaryotes. 
Transformations: 
Depending on the host cell used, transformation is done using standard 
techniques appropriate to such cells. The treatment employing calcium 
chloride, as described by Cohen, Proc. Natl. Acad. Sci. USA (1972) 69:2110 
(1972) or the CaCl.sub.2 method described in Maniatis, et al., Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbor Press, Sambrook et al., 
2nd edition, (1989)) may be used for procaryotes or other cells which 
contain substantial cell wall barriers. For mammalian cells without such 
cell walls, the calcium phosphate precipitation method of Graham and van 
der Eb, Virology 52:546 (1978), optionally as modified by Wigler et al., 
Cell 16:777-785 (1979), may be used. Transformations into yeast may be 
carried out according to the method of Van Solingen et al., J. Bact. 
130:946 (1977), or of Hsiao et al., Proc. Natl. Acad. Sci. USA 76:3829 
(1979). 
Other representative transfection methods include viral transfection, 
DEAE-dextran mediated transfection techniques, lysozyme fusion or 
erythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock, 
direct microinjection, indirect microinjection such as via 
erythrocyte-mediated techniques, and/or by subjecting host cells to 
electric currents. The above list of transfection techniques is not 
considered to be exhaustive, as other procedures for introducing genetic 
information into cells will no doubt be developed. 
Cloning: 
The cDNA sequences encoding SNST1 were obtained from screening of a rabbit 
kidney cDNA library using high stringency hybridization with rabbit renal 
SGLT1 cDNA. 
Alternatively, the cDNA sequences encoding SNST1 are obtained from a cDNA 
library prepared from mRNA isolated from cells expressing SNST1 according 
to procedures described in Molecular Cloning: A Laboratory Manual, Cold 
Spring Harbor, second edition, Sambrook et al. (1989), with particular 
reference to Young et al., Nature, 316:450-452 (1988). The cDNA insert 
from the successful clone, excised with a restriction enzyme such as 
EcoRI, is then used as a probe of the original cDNA library to obtain the 
additional clones containing inserts encoding other regions of SNST1, 
that, together with this probe, span the nucleotides containing the 
complete coding sequence of SNST1. 
Probing cDNA: 
A cDNA library is screened using high stringency conditions as described by 
Ausubel et al., in Current Protocols in Molecular Biology, Greene 
Publishing and Wiley-Interscience, New York. (1990) or using methods 
described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 
Sambrook et al., eds., second edition (1989), with particular reference to 
Young et al., Nature, 316:450-452 (1988), or using the colony 
hybridization procedure with a fragment of the rabbit renal SGLT1. 
cDNA Library Production: 
Double-stranded cDNA is synthesized and prepared for insertion into a 
plasmid vector such as Bluescript.RTM. or Lambda ZAP.RTM. (Stratagene, San 
Diego, Calif.) using standard procedures (see Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor, Sambrook et al., eds. second 
edition (1989). 
Vector Construction: 
Construction of a suitable vector containing the desired coding and control 
sequences employs standard ligation and restriction techniques which are 
well understood in the art. (Young et al., Nature 316:450-452 (1988)). 
Site specific DNA cleavage is performed by treating with the suitable 
restriction enzyme, such as EcoRI, (or enzymes) under conditions which are 
generally understood in the art, and the particulars of which are 
specified by the manufacturer of these commercially available restriction 
enzymes. See, e.g., New England Biolabs, Product Catalog. In general, 
about 1 .mu.g of phage DNA sequence is cleaved by one unit of enzyme in 
about 20 .mu.l of buffer solution; in the examples herein, typically, an 
excess of restriction enzyme is used to ensure complete digestion of the 
DNA substrate. Incubation times of about one hour to two hours at about 
37.degree. C. are workable, although variations can be tolerated. After 
each incubation, protein is removed by extraction with phenol/chloroform, 
and may be followed by ether extraction and the nucleic acid recovered 
from aqueous fractions by precipitation with ethanol. 
In vector construction employing "vector fragments", the vector fragment is 
commonly treated with bacterial alkaline phosphatase (BAP) or calf 
intestinal alkaline phosphatase (CIP) in order to remove the 5' phosphate 
and prevent religation of the vector. Digestions are conducted at pH 8 in 
approximately 150 mM Tris, in the presence of Na+ and Mg+.sup.2 using 
about 1 unit of BAP or CIP per .mu.g of vector at 60.degree. C. or 
37.degree. C., respectively, for about one hour. In order to recover the 
nucleic acid fragments, the preparation is extracted with 
phenol/chloroform and ethanol precipitated. Alternatively, religation can 
be prevented in vectors which have been double digested by additional 
restriction enzyme digestion of the unwanted fragments. 
Ligations are performed in 15-50 .mu.l volumes under the following standard 
conditions and temperatures: 20 mM Tris-Cl, pH 7.5, 10 mM MgCl.sub.2, 10 
mM DTT, 33 .mu.g/ml BSA, 10 mM-50 mM NaCl, and either 40 .mu.M ATP, 
0.01-0.02 (Weiss) units T4 DNA ligase at 0.degree. C. (for "sticky end" 
ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. 
C. (for "blunt end" ligation). Intermolecular "sticky end" ligations are 
usually performed at 33-100 .mu.g/ml total DNA concentrations (5-100 nM 
total end concentration). Intermolecular blunt end ligations (usually 
employing a 10-30 fold molar excess of linkers) are performed at 1 .mu.M 
total ends concentration. 
Verification of Construction: 
Correct ligations for vector construction are confirmed according to the 
procedures of Young et al., Nature, 316:450-452 (1988). 
Isolation of Gene Encoding SNST1: 
The cDNA of SNST1 obtained as described above is then used as a probe of a 
genomic mammalian cDNA library to obtain clones containing the complete 
gene coding sequence of mammalian SNST1. Alternatively, sets of synthetic 
oligonucleotides encoding SNST1 are used to probe a genomic cDNA library. 
Successful hybridizing clones are sequenced, and those containing the 
correct N-terminal sequence for SNST1 are obtained. 
Expression: 
The SNST1 protein may be expressed in a variety of systems as set forth 
below. The cDNA may be excised by suitable restriction enzymes and ligated 
into procaryotic or eucaryotic expression vectors for such expression. 
Protein Recovery: 
SNST1 protein may be produced either as a mature protein or as a fusion 
protein, or may be produced along with a signal sequence in cells capable 
of processing this sequence for secretion. It may be advantageous to 
obtain secretion of the protein as this minimizes the difficulties in 
purification. Cultured mammalian cells are able to cleave and process 
heterologous mammalian proteins containing signal sequences and to secrete 
them into the medium (McCormick et al., Mol. Cell. Biol. 4:166 (1984)). 
The protein is recovered using standard protein purification techniques 
including immunoaffinity purification. If secreted, the purification 
process is simplified, because relatively few proteins are secreted into 
the medium, and the majority of the secreted protein will, therefore 
already be SNST1. However, it is also known in the art to purify the 
protein from membranes of cells in which it is produced in mature or fully 
processed form. 
USE 
SNST1 protein, when expressed in functional form in a host cells such as a 
Xenopus oocyte, can be used to screen compounds, e.g. nucleoside analogs 
and other drugs to identify those capable of more effective uptake into 
appropriate cells. 
SNST1 may also be used to screen compounds for inhibition of sodium 
dependent nucleoside transport. Such inhibitors include phloridzin, 
described above, and phloridzin analogs. Phloridzin analogs may be 
isolated from natural sources (Newey et al., supra) or are synthesized by 
standard chemical procedures (Diedrich, supra). For example, different 
phloretin sugar glycosides are made by mixing phloretin and 
acetobromo-D-sugars (e.g. ribose) in cold 0.25N KOH, incubating in the 
deoxygenated solution in the dark for 24 hours and then adding acetic 
acid. The sticky solid product is collected, washed, dried and extracted 
with CHCl.sub.3. The tetraacetylglucosides are then separated using 
conventional chromatography, and saponified in cold methanolic sodium 
methoxide. Different phloretin analogs are prepared as described by 
Diedrich, (1990) supra; Diedrich, Biochim. Biophys. Acta. 71:688-700 
(1968); Diedrich, Arch. Biochem. Biophys. 117:248-256 (1966) and Lin et 
al., Biochim. Biophys. Acta 693:379-388 (1982)). 
In addition, inhibitors of nucleoside transport, consisting of phloridzin, 
phloridzin analogs, nucleoside analogs and other reagents, may be designed 
using the nucleoside transport assay described herein and the Na.sup.+ 
/nucleoside cotransporter protein of the invention to identify effective 
inhibitor compounds. 
In addition, SNST1 protein may be used to prepare antibodies, including 
polyclonal and monoclonal antibodies that bind to the SNST1 protein. These 
antibodies may be used to purify SNST1 protein. They may also be screened 
using the nucleoside transport assay described herein to identify those 
capable of inhibiting nucleoside transport by SNST1. 
Monoclonal antibodies reactive with SNST1, may be produced by hybridomas 
prepared using known procedures, such as those introduced by Kohler and 
Milstein (see Kohler and Milstein, Nature, 256:495-97 (1975)), and 
modifications thereof, to regulate cellular interactions. 
These techniques involve the use of an animal which is primed to produce a 
particular antibody. The animal can be primed by injection of an immunogen 
(e.g. the SNST1 protein or fusion proteins) to elicit the desired immune 
response, i.e. production of antibodies from the primed animal. A primed 
animal is also one which is expressing a disease. Lymphocytes derived from 
the lymph nodes, spleens or peripheral blood of primed, diseased animals 
can be used to search for a particular antibody. The lymphocyte 
chromosomes encoding desired immunoglobulins are immortalized by fusing 
the lymphocytes with myeloma cells, generally in the presence of a fusing 
agent such as polyethylene glycol (PEG). Any of a number of myeloma cell 
lines may be used as a fusion. partner according to standard techniques; 
for example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653, Sp2/0-Ag14, or HL1-653 
myeloma lines. These myeloma lines are available from the ATCC, Rockville, 
Md. 
The resulting cells, which include the desired hybridomas, are then grown 
in a selective medium such as HAT medium, in which unfused parental 
myeloma or lymphocyte cells eventually die. Only the hybridoma cells 
survive and can be grown under limiting dilution ccnditions to obtain 
isolated clones. The supernatants of the hybridomas are screened for the 
presence of the desired specificity, e.g. by immunoassay techniques using 
the SNST1 protein that has been used for immunization. Positive clones can 
then be subcloned under limiting dilution conditions, and the monoclonal 
antibody produced can be isolated. 
Various conventional methods can be used for isolation and purification of 
the monoclonal antibodies so as to obtain them free from other proteins 
and contaminants. Commonly used methods for purifying monoclonal 
antibodies include ammonium sulfate precipitation, ion exchange 
chromatography, and affinity chromatography (see Zola et al., in 
Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurell (ed.) 
pp. 51-52 (CRC Press, 1982)). Hybridomas produced according to these 
methods can be propagated in vitro or in vivo (in ascites fluid) using 
techniques known in the art (see generally Fink et al., Prog, Clin. 
Pathol., 9:121-33 (1984), FIG. 6-1 at p. 123). 
Generally, the individual cell line may be propagated in vitro, for 
example, in laboratory culture vessels, and the culture medium containing 
high concentrations of a single specific monoclonal antibody can be 
harvested by decantation, filtration, or centrifugation. 
In addition, fragments of these antibodies. containing the active binding 
region reactive with the SNST1 protein, such as Fab, F(ab').sub.2 and Fv 
fragments may be produced. Such fragments can be produced using techniques 
well established in the art (see e.g. Rousseaux et al., in Methods 
Enzymol., 121:663-69, Academic Press (1986)). 
Polyclonal antibodies may be produced, for example polyclonal peptide 
antibodies as described by Hirayama et al., in Am. J. Physiol. 
261:C296-C304 (1991). Briefly, peptides are synthesized, e.g. as described 
by Kent and Clark-Lewis, in Synthetic Peptides in Biology and Medicine, 
Amsterdam, Elsevier, p. 29-57 (1985), and are purified using reverse-phase 
high-performance liquid chromatography on a preparative C.sub.8 column in 
a gradient of 17.5-32.5% acetonitrile with 0.1% trifluoroacetic acid 
(TFA). The purity of the product is verified by isocratic elution on a 
C.sub.18 column in 25.5% acetonitrile and 0.1% TFA and by mass 
spectroscopy before lyophilization. Polyclonal antibodies are then raised 
in rabbits following standard procedures using the peptides as immunogen. 
These procedures permit the production of antibodies that bind to defined 
regions of the SNST1 amino acid sequence, using peptides or portions of 
peptides of the SNST1 protein as immunogen. 
The following examples are presented to illustrate the present invention 
and to assist one of ordinary skill in the art in making and using the 
invention. The examples are not intended in any way to otherwise limit the 
scope of the invention. 
EXAMPLE 1 
Cloning and Expression of Mammalian Na.sup.+ /Nucleoside Cotransporter 
Protein 
This example describes the cloning and expression of a mammalian Na.sup.+ 
/nucleoside cotransporter protein. 
A rabbit renal cDNA library was screened for clones related to the Na.sup.+ 
/glucose cotransporter protein, SGLT1, because the kidney contains a 
number of Na.sup.+ -dependent cotransporters. 
cDNA Library screening. A rabbit kidney cDNA library in Lambda ZAP.RTM., 
provided by Drs. Philpot and Ryan (National Institute of Environmental 
Health Sciences (NIEHS, Triangle Park, N.C.) was screened under high 
stringency conditions (hybridization at 42.degree. C. in 50% formamide and 
washing at 50.degree. C. in 0.1XSSC, as described by Ausubel et al. 
(eds.), in Current Protocols in Molecular Biology, Greene Publishing and 
Wiley-Interscience, New York (1990), incorporated by reference herein, 
with a .sup.32 p-labeled 1.6 kb HindIII/XhoI fragment of the rabbit renal 
Na+/glucose cotransporter (Coady et al., Am. J. Physiol. 259:C605-C610 
(1990)). pBluescript SK.RTM. (Stratagene), containing cDNA for SNST1, was 
excised from positive phage following the manufacturer's directions. All 
subsequent experiments were conducted using the plasmids. The rabbit 
kidney library was also screened with RK-C, one of the cDNAs isolated 
during the first round. 
RNA Preparation and Northern Blots. Poly(A+) RNA preparation and Northern 
transfers were as follows. The samples used for preparation of RNA were 
immediately frozen in liquid nitrogen and then stored until use, up to one 
week at -80.degree. C. The RNA was prepared by CsCl centrifugation 
(Ausubel et al., supra, incorporated by reference herein) using a modified 
homogenization buffer (Chomczynski and Sachhi, Anal. Biochem. 162:156-159 
(1987), incorporated by reference herein). Poly(A.sup.+)RNA was selected 
by oligo(dT) chromatography (Jacobson, Meths. Enzymol. 152:254-261 (1987), 
incorporated by reference herein) but LiCl replaced NaCl in all solutions. 
Before the final precipitation, the mRNA was centrifuged through a Costar 
0.45 .mu.m filter to remove any particulates from the oligo(dT) column 
that would otherwise clog an oocyte injection needle. RNA samples were 
stored at -80.degree. C. 
Two Northern blots containing mRNA from a range of rabbit tissues and 
rabbit kidney were probed in turn with cDNAs for SGLT1 and SGLT1-related 
clones. Whole organs were used except for the intestine and kidney 
samples. The intestinal RNA was prepared from jejunal mucosal scrapings. 
The outer cortex sample represents the outer 1 mm of renal cortex, and 
inner renal medulla represents papilla. The outer/mid-cortex sample 
contained approximately the outer 2 mm of kidney, and the outer medulla 
sample was prepared from outer strips of medulla. The RNA from each sample 
was separated in a 1% agarose gel containing 0.66M formaldehyde (Davis et 
al., in Basic Methods in Molecular Biology,. New York, Elsevier Science 
(1986), incorporated by reference herein), transferred to reinforced 
nitrocellulose filters (Duralose.RTM., Stratagene) and fixed to the 
filters by ultraviolet crosslinking (Stratalinker.RTM., Stratagene). 
Filters were prehybridized at least six hours at 42.degree. C. in 50% 
formamide, 5XSSC (Davis et al, supra), 3X Denhart's (Davis et al., supra), 
25 mM sodium phosphate buffer (pH 6.5), 0.2% sodium dodecyl sulfate (SDS), 
10% Dextran sulfate, and 250 .mu.g/ml Prehybe-HS (Lofstrand Labs, 
Gaithersburg, Md.). Gel-purified cDNA excised with EcoRI consisting of the 
coding region of the rabbit intestinal Na.sup.+ /glucose cotransporter 
(Coady et al., Am. J. Physiol. 259:C605-C610 (1990)) was labelled with 
.sup.32 P-dCTP using an Oligolabelling kit (Pharmacia) and used as the 
probe. The blots were hybridized at 42.degree. C. overnight. Washes were 
as follows: 15 min at room temperature in 5XSSC; 0.1% SDS; 0.05% sarkosyl; 
15 min at 60.degree. C. in 5XSSC, 0.1% SDS, 0.05% sarkosyl; three 15 min 
washes at 60.degree. C. in 0.1XSSC, 0.1% SDS (Coady et al., supra). Each 
lane contained 5 .mu.g of mRNA. Size standards are indicated in FIG. 1. 
Autoradiography was carried out at -80.degree. C. and autoradiograms were 
scanned with a Hoefer GS300 (San Francisco, Calif.) densitometer. 
cDNA and RNA sequencing. Double-stranded sequencing of both strands of cDNA 
was carried out using a T7 sequencing kit (Pharmacia) and synthetic 
oligonucleotides (Genosys Inc., San Diego, Calif.) as sequencing primers. 
Deaza nucleotide mixes were used when necessary to resolve compressions. 
Nucleotides 1-80 of SNST1 were directly sequenced from renal RNA according 
to Geliebter, Focus 9:5-8 (1989), incorporated by reference herein, with a 
reaction temperature of 50.degree. C., avian myeloblastosis virus (AMV) 
reverse transcriptase (Life Sciences, Bethesda, Md.) and gel-purified 
primers and end-labelled with .sup.32 p. 
Construction of chimera pSNST1c. In order to determine the function of 
SNST1, a cDNA chimera, SNST1c, was constructed for functional expression 
of SNST1. SNST1c provided the missing start codon and poly(A+) tail of the 
SNST1 cDNAs (RK-C and RK44). SNST1c consisted of nucleotides 243-2150 of 
SNST1 (amino acids 80-672) together with the 5' end (5' untranslated 
region and amino acids 1-79) and the 3' untranslated region of the rabbit 
SGLT1. The 5' end of pRK-C (to nucleotide 178, MscI site) was replaced by 
nucleotides 1-268 (also at an MscI site) of the rabbit intestinal SGLT1, 
pMC424 (Hediger et al., Nature 330:379-381 (1987)). The 3' end of pMC424 
was cut at the XhoI site and nucleotides 2014-2225, containing the entire 
3' untranslated region and poly(A+) tail, were attached to the end of the 
3'UTR of pRKC. This construct was difficult to grow in pBluescript.TM. and 
was subcloned into the HindIII/KpnI sites of pT7/T3-18 (BRL Laboratories, 
Bethesda, Md.), which lacked the lacZ gene. 
Expression of pSNST1c. The chimeric plasmid pSNST1c was linearized using 
KpnI and RNA was synthesized in vitro with an RNA transcription kit 
(Stratagene). The function of SNST1c was determined by expression of RNA 
in Xenopus oocytes as described below. 
Oocyte injections. Xenopus oocytes were dissected and injected, and 
transport was measured as described below. Stage V and VI oocytes (Dumont, 
J. Morphol. 136:153-180 (1972)) from Xenopus laevis (Xenopus One, Ann 
Arbor, Mich.) were dissected and defolliculated as described by Hediger et 
al., Proc. Natl. Acad. Sci. USA 84:2634-2637 (1987) and Coady et al., 
Arch. Biochem. Biophys. 265:73-81 (1990), both of which are incorporated 
by reference herein. Briefly, oocytes were hand-dissected from ovarian 
tissue of adult female Xenopus laevis. Individual oocytes were obtained by 
gentle agitation in Barth's solution (Silbernagl, Physiol. Rev. 
68:912-1007 (1988)) 1.0% collagenase (to remove follicular cells) and 0.1% 
trypsin inhibitor for 60 min followed by a 30 to 60 min incubation in 100 
mM K.sub.2 HP0.sub.4, pH 6.5, 0.1% BSA (modified from Dumont, J. Morphol. 
136:153-179 (1972)). Oocytes were maintained in Barth's solution at 
18.degree. C. After 16 to 24 hr, the healthy oocytes were injected with 50 
nl of water or mRNA (0.2 to 1 mg/ml) and incubated in Barth's solution 
with gentamicin at 18.degree. C. for another 3 to 8 days. 
Assay of Transporter Activity 
Na.sup.+ /nucleoside transporter activity was assayed by measuring the 
Na.sup.+ -dependent uptake by the chimeric construct, SNST1c. Initial 
screening of SNST1c function utilized radiolabelled substrates, including 
sugars (.alpha.-methyl-D-glucopyranose [.alpha.MDG],D-glucose, 
3-O-methylglucose), L-amino acids (alanine, glutamate, lysine, 
methyl-aminoisobutyric acid, phenylalanine, proline, taurine), carboxylic 
acids (succinate, lactate) and vitamins (biotin, pantothenate), but none 
of these was transported. Because of the sequence similarity to SGLT1, it 
was hypothesized that the transport would be Na.sup.+ -dependent. 
Therefore, the uptake of .sup.22 Na.sup.+ in the presence of potential 
substrates was monitored. 
Measurements were made of [.sup.3 H] uridine uptake in oocytes expressing 
SNST1c (FIG. 4A and 4B). Uptakes were measured over 5 min in a buffer 
containing either 100 mM NaCl, choline chloride or LiCl as described by 
Coady et al., Arch. Biochem. Biophys. 283:130-134 (1990), incorporated by 
reference herein. [All of the solutions contained 2 mM KCL, 1 mM 
MgCl.sub.2, 1 mM CaCl.sub.2, 10 mM HEPES/Tris, pH 7.5 and either 100 mM 
NaCl (Na.sup.+ transport buffer) or 100 mM choline-Cl (choline transport 
buffer). The uptake solutions contained radiolabeled substrate while the 
wash solutions contained nonradioactive substrate. Oocytes were injected 
with water as a control, or SNST1c cRNA (50 ng). Oocytes were preincubated 
for 30 min in substrate-free choline transport buffer when uptakes in 
Na.sup.+ and choline were to be compared. Transport of substrate was 
assayed by placing 4-6 oocytes in 0.5 ml of radioactive uptake solution, 
preceded by a 30 min preincubation in a Na.sup.+ -free (100 mM choline-Cl) 
solution when Na.sup.+ -dependent transport was to be measured. Transport 
was stopped by washing the oocytes five times with 4 ml of ice-cold 
choline solution containing excess unlabeled substrate. The oocytes were 
individually dissolved in 1.0 ml of 10% SDS and assayed for radioactivity. 
All data are expressed as mean .+-.SD. FIG. 4A indicates cation dependence 
of 1 .mu.M [.sup.3 H]uridine uptake in oocytes expressing SNST1c 4 days 
post-injection. In addition, inhibition of 0.6 .mu.M [.sup.3 H] uridine 
uptake in oocytes by 1 mM nucleosides was determined (FIG. 4B). Uptakes 
were measured in Na.sup.+ containing buffer (Coady et al., supra) Oocytes 
were injected with water as a control, or SNST1c cRNA (50 ng) . 
Results 
Seven distinct cDNAs, RK-A through RK-I, were isolated from the rabbit 
kidney library by high stringency hybridization with the rabbit renal 
SGLT1 cDNA as described above. The renal cDNAs were different from one 
another as shown by partial restriction mapping and DNA slot blot 
hybridization. The tissue distribution of mRNA encoding SGLT1 and the 
SGLT1-related clones is shown in FIG. 1. Rabbit tissues are shown on the 
left, and rabbit kidney is shown on the right. Panels from top to bottom 
were probed with SGLT1, RK-A, RK-C (nucleotides 66-2150 of SNST1) and 
RK-1. 
As has been observed previously, the rabbit SGLT1 mRNA was predominantly a 
single species of 2.3 kb found in intestine and kidney (Hediger et al., 
Nature 330:379-381 (1987); and Coady et al., Am. J. Physiol. 259:C605-C610 
(1990)). The signal was stronger in outer renal medulla than cortex. No 
other tissue tested gave a signal with SGLT1. The message size of RK-A was 
approximately 4 kb and was distributed predominantly in brain, lung, 
liver, intestine and also kidney. There was a single mRNA species for RK-C 
at approximately 2.3 kb, which was more abundant in heart than in kidney. 
RK-C appeared to be absent from the outer cortex but present in other 
parts of the kidney. Four other renal cDNAs (RK-B, RK-D, RK-E, RK-F) had 
similar tissue distributions as RK-C mRNA, although the message size for 
RK-D was smaller, about 2.2 kb. Finally, RK-I mRNA was approximately 3 kb 
and was found in all tissues tested: brain, lung, heart, pancreas, liver, 
intestine, kidney. 
One of the renal cDNAs, RK-C, was selected for more detailed 
characterization and, based on results of expression studies, this clone 
encoded most of the SNST1 protein. The composite sequence of SNST1 is 
shown in FIG. 2. The sequence was obtained from two cDNAs: RK-C 
(nucleotides 66-2150) and RK-44 (nucleotides 20-2238, obtained by 
rescreening the library with RK-C), and by direct sequencing of rabbit 
renal mRNA (nucleotides 1-80). All overlapping sequences were identical. 
The two cDNAs contain an ATG (nucleotides 136-138) which was initially 
considered as a putative start codon. In vitro translation experiments 
showed that RK-C and the rabbit intestinal SGLT1 both made proteins of 47 
kDa (in 8% acrylamide), which increased by 6 kDa to 53 kDa in the presence 
of pancreatic microsomes. The putative start codon was rejected for three 
reasons: (1) there was high identity between the sequence upstream of this 
ATG and the coding region of SGLT1 (FIG. 3); 2) the equivalent ATG was not 
used for initiation of translation in SGLT1, as there was no functional 
expression of a 5'-truncated SGLT1, although protein was detected in in 
vitro translation experiments; and (3) the sequences flanking the ATG at 
nucleotides 136-138 do not form a consensus initiation sequence (Kozak, 
Microbiol. Revs., 47:1-45 (1983)). 
The full sequence of SNST1 (FIG. 2) contains a single open reading frame 
which encodes a protein of 672 amino acids. The 5' untranslated region is 
approximately 30 nucleotides long and there is a consensus initiation. 
sequence, YNNAUGG (Kozak, M., Microbiological reviews, 47:1-45 (1983)). 
The 3' untranslated region contains a consensus polyadenylation sequence, 
AAUAAA, (underlined in FIG. 2) but there is no poly(A.sup.+) tail. 
The sequence similarity between SNST1 and the rabbit SGLT1 is shown in 
FIGS. 3 and 5 (SNST1-top; SGLT1-bottom). The alignment was made using the 
widely available Genetics Computer Group program, GAP (Devereux et al., 
Nucleic Acids Res. 12:387-395 (1984). Lines in FIG. 3 denote identical 
amino acids, and colons show chemically similar amino acids. The hybrid 
transporter, SNST1c, used for expression experiments, contained amino 
acids 1-79 of SGLT1 attached to amino acids 80-872 of SNST1. The three 
predicted N-glycosylation sites (at N.sup.250, N.sup.306, N.sup.399), 
conserved residues D.sup.25, G.sup.40, R.sup.300, "SOB motif" (-GLY - - - 
ALA-X-X-X-X-LEU-X-X-X-GLY-ARG-) described by Deguchi et al., J, Biol. 
Chem. 265:21704-21708 (1990), and G.sup.380, A.sup.416, L.sup.421, 
G.sup.425, R.sup.426, are indicated. 
There is 61% identity and 80% similarity between the two sequences, 
including regions of striking identity, particularly toward the 
amino-terminus. SNST1 contains three putative N-linked glycosylation 
sites, two of which are shared with SGLT1 and a third that may be in a 
transmembrane helix. A single residue, Asn.sup.248, is N-glycosylated in 
SGLT1 (Hediger, et al, Biophys. Biochim. Acta, 1064:360-364 (1991) and 
this may be the case for SNST1 as the extent of glycosylation seen after 
in vitro translation (increase of 6 kDa in the presence of microsomes) 
appears to be the same for both transporters. The hydropathy plots of 
SNST1 and SGLT1 are also very similar, and a model of the predicted 
secondary structure of SNST1, a membrane protein with 12 transmembrane 
domains, is shown in FIG. 5. The predicted structure was based on 
Kyte-Doolittle analysis (Kyte and Doolittle, J. Mol. Biol. 147:105 
(1982)). Intracellular residues are shown in FIG. 5 below, and 
extracellular residues are shown above the putative transmembrane helices. 
Residues identical with SGLT1 are filled circles in the figure. SNST1 is 
ten amino acids longer than SGLT1 and most of the additional amino acids 
have been inserted into the large extracellular loop between transmembrane 
helices 11 and 12. 
SNST1 is intermediate in sequence homology between the mammalian SGLT1-type 
Na+/glucose cotransporters, which share more than 80% of their residues, 
and the bacterial Na+/proline (PutP) (Nakao et al, Mol. Gen. Genet, 
208:70-75, (1987)) and Na+/pantothenate (PanF) transporters (Jackowski and 
Alix, Bacteriol, 172:3842-3848, (1990), which have about 25% identity with 
the SGLT1s. Many of the residues conserved between the mammalian SGLT1s 
and bacterial PutP and PanF are also conserved in SNST1. These include 
Gly.sup.43 and Arg.sup.300, which are implicated in Na+ binding (Yamato et 
al, J. Biol. Chem., 265:2450-2455, (1990)), and the SOB motif (Deguchi et 
al., supra) (FIG. 2). Finally, Asp.sup.28, the residue that is mutated to 
asparagine in the SGLT1 of patients with glucose-galactose malabsorption 
syndrome (Turke et al., Nature 350:354-356 (1990), is conserved in SNST1 
at position 25. 
The sequences of SNST1 and SGLT1 show remarkable similarity at their 5' 
ends (FIG. 3), so that only portions of the N-terminus and first 
transmembrane helix of SNST1 were changed (the new segment contained 51 
identical and 11 conserved amino acids out of a total of 79). This 
approach may prove useful to those wishing to express a 5'-untruncated 
cDNA or to increase the expression of a poorly-expressing cDNA. 
A positive signal was observed with a pool of nucleosides (adenosine, 
uridine, guanosine, and cytidine) but no signals were seen with other 
substrates (.alpha.-MDG, myoinositol, betaine, thiamine, cholate 
pyruvate). In the presence of nucleosides, water-injected oocytes 
transported 297.+-.99 pmol .sup.22 Na.sup.+ /oocyte/hr while 
SNST1c-injected oocytes transported 752.+-.162 pmol .sup.22 Na/oocyte/hr 
(n=8 oocytes). Control experiments showed that the Na+/glucose 
cotransporter did not transport nucleosides (water-injected:248.+-.56, 
SGLT1-injected 309.+-.78, SNST1c-injected=722.+-.108 fmol .sup.3 
H-uridine/oocyte/hr, n=5 oocytes) and, conversely, oocytes injected with 
SNST1c RNA do not transport sugars (water:0.4.+-.0.1, SGLT1:70.+-.19, 
SGLT1c:0.5.+-.0.1 pmol .sup.14 C-.alpha.MDG/oocyte/hr, n=6 oocytes). This 
suggests that the first 79 amino acids of these transporters do not 
determine sugar or nucleoside specificity. 
Oocytes injected with SNST1c RNA transported [.sup.3 H]-uridine in the 
presence of Na.sup.+, and transport was inhibited when Na+ was replaced by 
choline or Li.sup.+ (FIG. 4A). There also appeared to be Na.sup.+ 
/nucleoside cotransport in the oocyte membrane, and this transport 
differed from SNST1c in substrate specificity (e.g., dideoxycytidine did 
not inhibit the oocyte transporter but did inhibit SNST1c). The transport 
of uridine by SNST1c was inhibited by a variety of nucleosides including 
uridine, 2-deoxyuridine, adenosine, guanosine and cytidine, as well as 
dideoxycytidine, a drug currently in clinical trials for use in AIDS 
therapy (FIG. 4B). This broad substrate specificity is characteristic of 
Na.sup.+ /uridine cotransport in brush border membranes from rat (Lee et 
al., Am. J. Physiol. 258:F1203-F1210 (1990)) and rabbit (Williams et al., 
Biochem. 284:223-231 (1989) kidney. 
Although not being bound by any particular theory of the mechanism of 
action of SNST1, the physiological function of SNST1 in the kidney may be 
in the reabsorption of nucleosides from the glomerular filtrate by the 
proximal tubule, as Na.sup.+ -dependent nucleoside transport has been 
reported for the cortical brush border, but not basolateral, membrane 
(Williams et al., supra). Rabbit intestinal brush border membrane vesicles 
also exhibit Na.sup.+ /nucleoside cotransport (Jarvis, Biochim. Biophys. 
Acta. 979:132-138 (1989)), but this appears to be a different gene product 
because SNST1 mRNA was not found in the intestinal mucosa (FIG. 1). SNST1 
was abundantly expressed in the heart and so it may play a role in the 
physiological action of adenosine, which regulates cardiac contractility 
(Belardinelli et al., Prog. Cardiovasc. Diseases 32:73-97 (1989)). 
EXAMPLE 2 
This example describes the identification and use of specific inhibitors of 
SNST1. 
Phloridzin, a specific inhibitor of Na.sup.+ /glucose cotransport also 
inhibits Na.sup.+ /nucleoside cotransport in the kidney brush border (Lee 
et al. Am. J. Physiol. 258:F1203-F1210 (1990)), which may reflect the 
evolutionary relationship between the two transport systems. 
Uptake of radiolabeled nucleosides into Xenopus oocytes was measured in the 
presence or absence of Na.sup.+ as described above in Example 1, and in 
the presence and absence of 1 mM phloridzin (Sigma, St. Louis, Mo.). The 
expressed Na.sup.+ /nucleoside cotransporter was 87% (n=2) inhibited by 1 
mM phloridzin. 
Although not wishing to be bound by any theory, phloridzin may inhibit 
SNST1 in the same manner as it inhibits SGLT1, i.e. the sugar residue 
competes for the nucleoside active site. 
Phloridzin analogs such as phloretin-ribosides are expected to be more 
effective inhibitors of nucleoside transport because ribose should 
interact more specifically with the nucleoside active site. The position 
of the hydroxyl residues on the A and B rings of the phloretin moiety of 
phloridzin affects the potency of the inhibitor on glucose transport. For 
example the 4' glycoside is inactive and there is evidence that the 4' 
hydroxyl group is important in binding to the glucose transport protein 
(Diedrich et al., (1990), supra). Various phloridzin analogs are tested 
for inhibition of SNST1 using the procedure described above for testing 
inhibition by phloridzin. In these experiments, to investigate the 
parameters of inhibition, nucleoside concentration is varied by 
maintaining concentration of the phloridzin analog constant, and vice 
versa. 
As will be apparent to those skilled in the art to which the invention 
pertains, the present invention may be embodied in forms other than those 
specifically disclosed above without departing from the spirit or 
essential characteristics of the invention. The particular embodiments of 
the invention described above, are, therefore, to be considered as 
illustrative and not restrictive. The scope of the present invention is as 
set forth in the appended claims rather than being limited to the examples 
contained in the foregoing description. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2238 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Oryctolagus cuniculus 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 7..2022 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GTACGAATGGAGGAACACATGGAGGCAGGCTCCAGACTGGGGCTGGGG48 
MetGluGluHisMetGlu AlaGlySerArgLeuGlyLeuGly 
1510 
GACCACGGGGCTCTCATCGACAATCCTGCTGACATCGCGGTCATTGCT96 
AspHisGlyAlaLeuIleAspAsnProAlaAsp IleAlaValIleAla 
15202530 
GCTTATTTCCTGCTGGTCATTGGTGTCGGCTTGTGGTCCATGTGCAGA144 
AlaTyrPheLeuLeuValIleGlyValGly LeuTrpSerMetCysArg 
354045 
ACCAACAGAGGCACCGTGGGTGGCTACTTCCTGGCAGGACGAAGCATG192 
ThrAsnArgGlyThrValGlyGlyTyrPh eLeuAlaGlyArgSerMet 
505560 
GTGTGGTGGCCGGTTGGGGCCTCTCTCTTTGCTAGCAATATCGGCAGT240 
ValTrpTrpProValGlyAlaSerLeuPheA laSerAsnIleGlySer 
657075 
GGCCACTTTGTGGGCCTGGCGGGGACCGGTGCTGCAAACGGCTTGGCT288 
GlyHisPheValGlyLeuAlaGlyThrGlyAlaAla AsnGlyLeuAla 
808590 
GTGGCTGGATTTGAGTGGAATGCGCTGTTCGTGGTGCTGCTCCTGGGT336 
ValAlaGlyPheGluTrpAsnAlaLeuPheValValLeuLeuLeu Gly 
95100105110 
TGGCTGTTCGCGCCGGTGTACCTGACCGCAGGCGTCATTACGATGCCG384 
TrpLeuPheAlaProValTyrLeuThrAlaGlyValIleTh rMetPro 
115120125 
CAGTACCTGCGCAAGCGCTTCGGCGGCCATCGGATCCGCCTCTACTTG432 
GlnTyrLeuArgLysArgPheGlyGlyHisArgIleArgL euTyrLeu 
130135140 
TCCGTGCTCTCGCTTTTTCTGTACATCTTCACCAAGATCTCGGTGGAC480 
SerValLeuSerLeuPheLeuTyrIlePheThrLysIleSer ValAsp 
145150155 
ATGTTCTCCGGGGCGGTGTTTATTCAGCAGGCTCTAGGCTGGAATATT528 
MetPheSerGlyAlaValPheIleGlnGlnAlaLeuGlyTrpAsnIle 
160165170 
TACGCTTCGGTCATCGCGCTCCTGGGCATCACCATGGTTTACACCGTG576 
TyrAlaSerValIleAlaLeuLeuGlyIleThrMetValTyrThrVal 
175 180185190 
ACAGGAGGGCTGGCAGCGCTGATGTACACAGACACAGTGCAGACCTTT624 
ThrGlyGlyLeuAlaAlaLeuMetTyrThrAspThrValGlnThrPhe 
195200205 
GTCATCATCGCGGGGGCCTTCATCCTCACCGGTTACGCCTTCCACGAG672 
ValIleIleAlaGlyAlaPheIleLeuThrGlyTyrAlaPheHisGlu 
210215220 
GTGGGCGGGTATTCCGGGCTCTTCGACAAATACATGGGAGCGATGACT720 
ValGlyGlyTyrSerGlyLeuPheAspLysTyrMetGlyAlaMetThr 
225230235 
TCGCTGACGGTGTCCGAGGACCCGGCTGTGGGCAACATCTCCAGCTCC768 
SerLeuThrValSerGluAspProAlaValGlyAsnIleSerSerSer 
240 245250 
TGCTACCGACCCCGGCCTGACTCCTATCATCTGCTCCGGGACCCTGTG816 
CysTyrArgProArgProAspSerTyrHisLeuLeuArgAspProVal 
255260 265270 
ACGGGGGACCTACCATGGCCCGCGCTGCTCCTGGGGCTCACCATCGTC864 
ThrGlyAspLeuProTrpProAlaLeuLeuLeuGlyLeuThrIleVal 
275 280285 
TCGGGCTGGTACTGGTGCAGTGACCAGGTCATAGTACAGCGCTGCCTG912 
SerGlyTrpTyrTrpCysSerAspGlnValIleValGlnArgCysLeu 
290 295300 
GCCGGGAGGAACCTGACCCACATCAAGGCAGGCTGCATCTTGTGTGGC960 
AlaGlyArgAsnLeuThrHisIleLysAlaGlyCysIleLeuCysGly 
305 310315 
TACCTGAAGCTGACGCCCATGTTCCTCATGGTCATGCCAGGAATGATC1008 
TyrLeuLysLeuThrProMetPheLeuMetValMetProGlyMetIle 
320325 330 
AGCCGCATCCTTTACCCTGACGAGGTGGCGTGCGTGGCGCCTGAGGTG1056 
SerArgIleLeuTyrProAspGluValAlaCysValAlaProGluVal 
335340 345350 
TGTAAGCGCGTGTGTGGCACGGAAGTGGGCTGCTCCAACATCGCCTAT1104 
CysLysArgValCysGlyThrGluValGlyCysSerAsnIleAlaTyr 
355 360365 
CCGCGGCTCGTTGTGAAGCTCATGCCCAACGGTCTGCGCGGACTCATG1152 
ProArgLeuValValLysLeuMetProAsnGlyLeuArgGlyLeuMet 
3703 75380 
CTGGCGGTCATGTTGGCCGCGCTCATGTCTTCGCTGGCCTCCATCTTC1200 
LeuAlaValMetLeuAlaAlaLeuMetSerSerLeuAlaSerIlePhe 
385390 395 
AACAGCAGCAGCACTCTCTTCACCATGGACATCTACACGCTGCGGCCC1248 
AsnSerSerSerThrLeuPheThrMetAspIleTyrThrLeuArgPro 
400405 410 
CGCGCCGGCGAAGGCGAGCTGCTGCTAGTAGGACGGCTCTGGGTGGTG1296 
ArgAlaGlyGluGlyGluLeuLeuLeuValGlyArgLeuTrpValVal 
415420425 430 
TTCATCGTGGCGGTGTCGGTGGCCTGGCTACCTGTGGTGCAGGCGGCA1344 
PheIleValAlaValSerValAlaTrpLeuProValValGlnAlaAla 
435440 445 
CAGGGCGGGCAGCTCTTCGATTACATCCAGTCCGTTTCCAGCTACTTG1392 
GlnGlyGlyGlnLeuPheAspTyrIleGlnSerValSerSerTyrLeu 
450455 460 
GCCCCGCCTGTGTCTGCAGTCTTCGTCGTGGCGCTCTTCGTGCCGCGC1440 
AlaProProValSerAlaValPheValValAlaLeuPheValProArg 
465470475 
GTTAATGAGAAGGGCGCCTTCTGGGGACTGATAGGGGGCCTGCTAATG1488 
ValAsnGluLysGlyAlaPheTrpGlyLeuIleGlyGlyLeuLeuMet 
480485490 
GGCCTG GCACGCCTTATTCCCGAGTTCTCCTTCGGCACGGGCAGCTGC1536 
GlyLeuAlaArgLeuIleProGluPheSerPheGlyThrGlySerCys 
495500505510 
GTG CGACCCTCTGCTTGCCCGGCATTCCTGTGTCGGGTGCACTACCTC1584 
ValArgProSerAlaCysProAlaPheLeuCysArgValHisTyrLeu 
515520525 
TA CTTCGCCATTGTGCTCTTCTTCTGCTCTGGCCTCCTCATCATCATC1632 
TyrPheAlaIleValLeuPhePheCysSerGlyLeuLeuIleIleIle 
530535540 
GTCT CCTTGTGCACTGCACCCATCCCACGCAAGCACCTCCACCGCCTG1680 
ValSerLeuCysThrAlaProIleProArgLysHisLeuHisArgLeu 
545550555 
GTTTTCAGT CTCCGGCACAGCAAGGAGGAACGGGAAGACCTGGATGCT1728 
ValPheSerLeuArgHisSerLysGluGluArgGluAspLeuAspAla 
560565570 
GACGAGCTGGAAGCCCCG GCCTCTCCCCCTGTCCAGAATGGGCGCCCA1776 
AspGluLeuGluAlaProAlaSerProProValGlnAsnGlyArgPro 
575580585590 
GAGCACGCAGTGGA GATGGAAGAGCCCCAGGCCCCGGGCCCAGGCCTG1824 
GluHisAlaValGluMetGluGluProGlnAlaProGlyProGlyLeu 
595600605 
TTCCGCCAGTGCT TGCTGTGGTTCTGTGGAATGAACAGGGGCAGGGCA1872 
PheArgGlnCysLeuLeuTrpPheCysGlyMetAsnArgGlyArgAla 
610615620 
GGTGGCCCCGCACCC CCTACCCAGGAGGAGGAGGCTGCAGCGGCCAGG1920 
GlyGlyProAlaProProThrGlnGluGluGluAlaAlaAlaAlaArg 
625630635 
CGGCTGGAGGACATCAACGAG GACCCGCGCTGGTCCCGGGTGGTCAAC1968 
ArgLeuGluAspIleAsnGluAspProArgTrpSerArgValValAsn 
640645650 
CTCAATGCCCTGCTCATGATGGCCGTGGC CATGTTTTTCTGGGGCTTT2016 
LeuAsnAlaLeuLeuMetMetAlaValAlaMetPhePheTrpGlyPhe 
655660665670 
TATGCCTAGGGCCGACTGTGTTGGGCATC ACGAGCCACAGGTCAGGACAGGGCTGG2072 
TyrAla 
CCGCACAATGAGCAGGGATCAGGAGCCTGCAGCGGTCCCCGGAAAGGGGGAAGGGGCAGG2132 
AGTGGTATGGGAAGGCCCAGTCCATTTGATTGGCAGTCACTTGCACGAGGCCTCAGCCAA2192 
GCTGCCCTAACGTTTCCCTCAGCAAAAATAAAGCAGCCGTTCCCCC2238 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 672 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetGluGluHis MetGluAlaGlySerArgLeuGlyLeuGlyAspHis 
151015 
GlyAlaLeuIleAspAsnProAlaAspIleAlaValIleAlaAlaTyr 
20 2530 
PheLeuLeuValIleGlyValGlyLeuTrpSerMetCysArgThrAsn 
354045 
ArgGlyThrValGlyGlyTyrPheLeuAlaGlyAr gSerMetValTrp 
505560 
TrpProValGlyAlaSerLeuPheAlaSerAsnIleGlySerGlyHis 
65707580 
PheValGlyLeuAlaGlyThrGlyAlaAlaAsnGlyLeuAlaValAla 
859095 
GlyPheGluTrpAsnAlaLeuPheValValLeuLeuLeuGlyTrpLeu 
100105110 
PheAlaProValTyrLeuThrAlaGlyValIleThrMetProGlnTyr 
115120125 
LeuArgLysArgPheGlyGlyHis ArgIleArgLeuTyrLeuSerVal 
130135140 
LeuSerLeuPheLeuTyrIlePheThrLysIleSerValAspMetPhe 
145150155 160 
SerGlyAlaValPheIleGlnGlnAlaLeuGlyTrpAsnIleTyrAla 
165170175 
SerValIleAlaLeuLeuGlyIleThrMetValTyrThrValTh rGly 
180185190 
GlyLeuAlaAlaLeuMetTyrThrAspThrValGlnThrPheValIle 
195200205 
IleAlaGlyAla PheIleLeuThrGlyTyrAlaPheHisGluValGly 
210215220 
GlyTyrSerGlyLeuPheAspLysTyrMetGlyAlaMetThrSerLeu 
225230 235240 
ThrValSerGluAspProAlaValGlyAsnIleSerSerSerCysTyr 
245250255 
ArgProArgProAspSerTyrHisLeuLeuArg AspProValThrGly 
260265270 
AspLeuProTrpProAlaLeuLeuLeuGlyLeuThrIleValSerGly 
275280285 
T rpTyrTrpCysSerAspGlnValIleValGlnArgCysLeuAlaGly 
290295300 
ArgAsnLeuThrHisIleLysAlaGlyCysIleLeuCysGlyTyrLeu 
3053 10315320 
LysLeuThrProMetPheLeuMetValMetProGlyMetIleSerArg 
325330335 
IleLeuTyrProAspGluVal AlaCysValAlaProGluValCysLys 
340345350 
ArgValCysGlyThrGluValGlyCysSerAsnIleAlaTyrProArg 
355360 365 
LeuValValLysLeuMetProAsnGlyLeuArgGlyLeuMetLeuAla 
370375380 
ValMetLeuAlaAlaLeuMetSerSerLeuAlaSerIlePheAsnSer 
385 390395400 
SerSerThrLeuPheThrMetAspIleTyrThrLeuArgProArgAla 
405410415 
GlyGluGlyG luLeuLeuLeuValGlyArgLeuTrpValValPheIle 
420425430 
ValAlaValSerValAlaTrpLeuProValValGlnAlaAlaGlnGly 
435 440445 
GlyGlnLeuPheAspTyrIleGlnSerValSerSerTyrLeuAlaPro 
450455460 
ProValSerAlaValPheValValAlaLeuPheValProArg ValAsn 
465470475480 
GluLysGlyAlaPheTrpGlyLeuIleGlyGlyLeuLeuMetGlyLeu 
485490495 
AlaArgLeuIleProGluPheSerPheGlyThrGlySerCysValArg 
500505510 
ProSerAlaCysProAlaPheLeuCysArgValHisTyrLeuTyrPhe 
51 5520525 
AlaIleValLeuPhePheCysSerGlyLeuLeuIleIleIleValSer 
530535540 
LeuCysThrAlaProIleProArgLysHisL euHisArgLeuValPhe 
545550555560 
SerLeuArgHisSerLysGluGluArgGluAspLeuAspAlaAspGlu 
565570 575 
LeuGluAlaProAlaSerProProValGlnAsnGlyArgProGluHis 
580585590 
AlaValGluMetGluGluProGlnAlaProGlyProGlyLeuPhe Arg 
595600605 
GlnCysLeuLeuTrpPheCysGlyMetAsnArgGlyArgAlaGlyGly 
610615620 
ProAlaProProThrGlnGl uGluGluAlaAlaAlaAlaArgArgLeu 
625630635640 
GluAspIleAsnGluAspProArgTrpSerArgValValAsnLeuAsn 
645 650655 
AlaLeuLeuMetMetAlaValAlaMetPhePheTrpGlyPheTyrAla 
660665670 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 672 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Oryctolagus cuniculus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetGluGluHisMetGluAlaGlySerArgLeuGlyLeuGlyAspHis 
15 1015 
GlyAlaLeuIleAspAsnProAlaAspIleAlaValIleAlaAlaTyr 
202530 
PheLeuLeuValIleGlyValGlyLeuTrpSer MetCysArgThrAsn 
354045 
ArgGlyThrValGlyGlyTyrPheLeuAlaGlyArgSerMetValTrp 
505560 
TrpProVal GlyAlaSerLeuPheAlaSerAsnIleGlySerGlyHis 
65707580 
PheValGlyLeuAlaGlyThrGlyAlaAlaAsnGlyLeuAlaValAla 
8 59095 
GlyPheGluTrpAsnAlaLeuPheValValLeuLeuLeuGlyTrpLeu 
100105110 
PheAlaProValTyrLeuThrAlaG lyValIleThrMetProGlnTyr 
115120125 
LeuArgLysArgPheGlyGlyHisArgIleArgLeuTyrLeuSerVal 
130135140 
LeuSerLeuPheLeuTyrIlePheThrLysIleSerValAspMetPhe 
145150155160 
SerGlyAlaValPheIleGlnGlnAlaLeuGlyTrpAsnIleTyrAla 
165170175 
SerValIleAlaLeuLeuGlyIleThrMetValTyrThrValThrGly 
180185190 
GlyLeuAlaAlaLe uMetTyrThrAspThrValGlnThrPheValIle 
195200205 
IleAlaGlyAlaPheIleLeuThrGlyTyrAlaPheHisGluValGly 
210215 220 
GlyTyrSerGlyLeuPheAspLysTyrMetGlyAlaMetThrSerLeu 
225230235240 
ThrValSerGluAspProAlaValGlyAsnIleSerSerS erCysTyr 
245250255 
ArgProArgProAspSerTyrHisLeuLeuArgAspProValThrGly 
260265270 
Asp LeuProTrpProAlaLeuLeuLeuGlyLeuThrIleValSerGly 
275280285 
TrpTyrTrpCysSerAspGlnValIleValGlnArgCysLeuAlaGly 
290 295300 
ArgAsnLeuThrHisIleLysAlaGlyCysIleLeuCysGlyTyrLeu 
305310315320 
LysLeuThrProMetPheLeuMetValMe tProGlyMetIleSerArg 
325330335 
IleLeuTyrProAspGluValAlaCysValAlaProGluValCysLys 
340345 350 
ArgValCysGlyThrGluValGlyCysSerAsnIleAlaTyrProArg 
355360365 
LeuValValLysLeuMetProAsnGlyLeuArgGlyLeuMetLeuAla 
370375380 
ValMetLeuAlaAlaLeuMetSerSerLeuAlaSerIlePheAsnSer 
385390395400 
SerSerThrLeuPheThr MetAspIleTyrThrLeuArgProArgAla 
405410415 
GlyGluGlyGluLeuLeuLeuValGlyArgLeuTrpValValPheIle 
420 425430 
ValAlaValSerValAlaTrpLeuProValValGlnAlaAlaGlnGly 
435440445 
GlyGlnLeuPheAspTyrIleGlnSerValSerSerTyrLe uAlaPro 
450455460 
ProValSerAlaValPheValValAlaLeuPheValProArgValAsn 
465470475480 
GluLys GlyAlaPheTrpGlyLeuIleGlyGlyLeuLeuMetGlyLeu 
485490495 
AlaArgLeuIleProGluPheSerPheGlyThrGlySerCysValArg 
500 505510 
ProSerAlaCysProAlaPheLeuCysArgValHisTyrLeuTyrPhe 
515520525 
AlaIleValLeuPhePheCysSerGlyLeu LeuIleIleIleValSer 
530535540 
LeuCysThrAlaProIleProArgLysHisLeuHisArgLeuValPhe 
545550555 560 
SerLeuArgHisSerLysGluGluArgGluAspLeuAspAlaAspGlu 
565570575 
LeuGluAlaProAlaSerProProValGlnAsnGlyArgProGluHis 
580585590 
AlaValGluMetGluGluProGlnAlaProGlyProGlyLeuPheArg 
595600605 
GlnCysLeuLeuTrpPhe CysGlyMetAsnArgGlyArgAlaGlyGly 
610615620 
ProAlaProProThrGlnGluGluGluAlaAlaAlaAlaArgArgLeu 
625630635 640 
GluAspIleAsnGluAspProArgTrpSerArgValValAsnLeuAsn 
645650655 
AlaLeuLeuMetMetAlaValAlaMetPhePheTrpGly PheTyrAla 
660665670 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 662 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Oryctolagus cuniculus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetAspSerSerThrLeuSerProLeuThrThrSerThrAlaAlaPro 
151015 
LeuGluSerTyrGluArgIleArgAsnAlaAlaAspI leSerValIle 
202530 
ValIleTyrPheLeuValValMetAlaValGlyLeuTrpAlaMetPhe 
354045 
SerThrA snArgGlyThrValGlyGlyPhePheLeuAlaGlyArgSer 
505560 
MetValTrpTrpProIleGlyAlaSerLeuPheAlaSerAsnIleGly 
6570 7580 
SerGlyHisPheValGlyLeuAlaGlyThrGlyAlaAlaSerGlyIle 
859095 
AlaThrGlyGlyPheGluTrpAsnAlaLeu IleMetValValValLeu 
100105110 
GlyTrpValPheValProIleTyrIleArgAlaGlyValValThrMet 
115120125 
ProGluTyrLeuGlnLysArgPheGlyGlyLysArgIleGlnIleTyr 
130135140 
LeuSerIleLeuSerLeuLeuLeuTyrIlePheThrLysIleSerAla 
145 150155160 
AspIlePheSerGlyAlaIlePheIleGlnLeuThrLeuGlyLeuAsp 
165170175 
IleTyrValAlaIleIle IleLeuLeuValIleThrGlyLeuTyrThr 
180185190 
IleThrGlyGlyLeuAlaAlaValIleTyrThrAspThrLeuGlnThr 
195200 205 
AlaIleMetMetValGlySerValIleLeuThrGlyPheAlaPheHis 
210215220 
GluValGlyGlyTyrGluAlaPheThrGluLysTyrMetArgAlaIle 
22 5230235240 
ProSerGlnIleSerTyrGlyAsnThrSerIleProGlnLysCysTyr 
245250255 
ThrProA rgGluAspAlaPheHisIlePheArgAspAlaIleThrGly 
260265270 
AspIleProTrpProGlyLeuValPheGlyMetSerIleLeuThrLeu 
275 280285 
TrpTyrTrpCysThrAspGlnValIleValGlnArgCysLeuSerAla 
290295300 
LysAsnLeuSerHisValLysAlaGlyCysIleLeuCys GlyTyrLeu 
305310315320 
LysValMetProMetPheLeuIleValMetMetGlyMetValSerArg 
325330 335 
IleLeuTyrThrAspLysValAlaCysValValProSerGluCysGlu 
340345350 
ArgTyrCysGlyThrArgValGlyCysThrAsnIleAlaPheProThr 
355360365 
LeuValValGluLeuMetProAsnGlyLeuArgGlyLeuMetLeuSer 
370375380 
ValMetMetAlaSerLeuMetSerSerL euThrSerIlePheAsnSer 
385390395400 
AlaSerThrLeuPheThrMetAspIleTyrThrLysIleArgLysLys 
405410 415 
AlaSerGluLysGluLeuMetIleAlaGlyArgLeuPheMetLeuPhe 
420425430 
LeuIleGlyIleSerIleAlaTrpValProIleValGlnSer AlaGln 
435440445 
SerGlyGlnLeuPheAspTyrIleGlnSerIleThrSerTyrLeuGly 
450455460 
ProProIleAlaAlaVa lPheLeuLeuAlaIlePheTrpLysArgVal 
465470475480 
AsnGluProGlyAlaPheTrpGlyLeuValLeuGlyPheLeuIleGly 
485 490495 
IleSerArgMetIleThrGluPheAlaTyrGlyThrGlySerCysMet 
500505510 
GluProSerAsnCysProThrIleIleCysG lyValHisTyrLeuTyr 
515520525 
PheAlaIleIleLeuPheValIleSerIleIleThrValValValVal 
530535540 
SerLeu PheThrLysProIleProAspValHisLeuTyrArgLeuCys 
545550555560 
TrpSerLeuArgAsnSerLysGluGluArgIleAspLeuAspAlaGly 
565570575 
GluGluAspIleGlnGluAlaProGluGluAlaThrAspThrGluVal 
580585590 
ProLysLysLysLysGlyPh ePheArgArgAlaTyrAspLeuPheCys 
595600605 
GlyLeuAspGlnAspLysGlyProLysMetThrLysGluGluGluAla 
610615 620 
AlaMetLysLeuLysLeuThrAspThrSerGluHisProLeuTrpArg 
625630635640 
ThrValValAsnIleAsnGlyValIleLeuLeuAlaValAlaValP he 
645650655 
CysTyrAlaTyrPheAla 
660