Serotonin transporter CDNA

Isolated DNA encoding a serotonin transporter is disclosed. Also disclosed are vectors and host cells containing the aforesaid DNA, methods of using the same, purified protein by the same, and oligonucleotides and antibodies which bind thereto. Specific embodiments are cDNAs encoding rat and human serotonin transporter.

FIELD OF THE INVENTION 
This invention relates to a cDNA clone encoding a serotonin transporter 
protein, vectors containing this clone, host cells which express this 
clone, and methods of using the same. 
BACKGROUND OF THE INVENTION 
Selective antagonism of serotonin (5-hydroxytryptamine, 5HT) and 
noradrenaline (NA) transport by antidepressants is a key element to our 
current understanding of human behavioral disorders. See H. Ashton, Brain 
Systems, Disorders, and Psychotropic Drugs, 283-330 (Oxford University 
Press, New York, 1987). In several studies, 5HT uptake and/or transport 
sites have been found to be reduced in platelets of patients suffering 
from depression and reduced in post-mortem brain samples of depressed 
patients and suicide victims. See generally H. Meltzer, et al., Arch. Gen. 
Psychiat. 38, 1322-1326 (1981); B. Suranyi-Cadotte et al., Life Sci. 36, 
795-799; M. Briley et al., Science 209, 303-305; S. Paul et al., Arch. 
Gen. Psych. 38, 1315-1317 (1981); E. Perry et al., Brit. J. Psych. 142, 
188-192 (1983); M. Stanley et al., Science 216, 1337-1339 (1982). A better 
understanding of these transporter proteins would provide a better 
understanding of these behavioral disorders. 
Despite decades of study, only the more abundant amino acid 
neurotransmitter (Glu, GABA, Gly) transporters have been reconstituted 
after solubilization in an active state. See R. Radian et al., J. Biol. 
Chem. 25, 15437-15441 (1986); N. Danbolt et al., Biochemistry 29, 
6734-6740 (1990); B. Lopez-Corcuera & C. Aragon, Eur. J. Biochem. 181, 
519-524 (1989). Recently, GABA (rGAT1) and NA (hNAT) transporters have 
been cloned, revealing single, structurally-related polypeptides forming 
each carrier. See J. Guastella et al., Science 249, 1303-1306 (1990); H. 
Nelson et al., FEBS Lettr. 269, 181-184 (1990); T. Pacholczyk et al., 
Nature 350, 350-354 (1991). The inferred amino acid sequence of both GABA 
and NA transporters predicts .about.12 transmembrane domains, with one 
large extracellular loop bearing multiple sites for N-linked 
glycosylation. The structure of the putative seratonin transporter, 
however, has heretofore remained unknown. Accordingly, an object of the 
present invention is to provide a cDNA encoding a serotonin transporter 
and elucidate the structure thereof. 
SUMMARY OF THE INVENTION 
A first aspect of the present invention is isolated DNA encoding a 
serotonin transporter selected from the group consisting of: (a) isolated 
DNA which encodes rat serotonin transporter; (b) isolated DNA which 
hybridizes to isolated DNA of (a) above and which encodes a serotonin 
transporter; and (c) isolated DNA differing from the isolated DNAs of (a) 
and (b) above in codon sequence due to the degeneracy of the genetic code, 
and which encodes a serotonin transporter. In another respect, the present 
invention provides isolated DNA consisting essentially of isolated DNA 
encoding a serotonin transporter, preferably a mammalian serotonin 
transporter such as the human and rat serotonin transporters. Thus a 
specific embodiment of the foregoing is isolated DNA encoding a human 
serotonin transporter selected from the group consisting of: (a) isolated 
DNA which encodes the human serotonin transporter having the sequence 
given herein as SEQ ID NO:10; (b) isolated DNA which hybridizes to the 
isolated human DNA of (a) above and which encodes a human serotonin 
transporter; and (c) isolated human DNA differing from the isolated DNAs 
of (a) and (b) above in codon sequence due to the degeneracy of the 
genetic code, and which encodes a serotonin transporter. 
A second aspect of the present invention is a recombinant DNA sequence 
comprising vector DNA and a DNA encoding a serotonin transporter as given 
above. 
A third aspect of the present invention is a host cell containing a 
recombinant DNA sequence as given above. Host cells which express the 
serotonin transporter may be used in the assay procedure discussed below, 
either lysed to provide cell membranes or as whole cells. 
A fourth aspect of the present invention is an aqueous solution containing 
cell membranes, the cell membranes containing a serotonin transporter, 
wherein the cell membranes are free of other undesired neurotransmitter 
transporters such as the noradrenaline transporter. 
A fifth aspect of the present invention is an assay procedure comprising 
the steps of, first, providing an aqueous solution containing cell 
membranes as given above; then adding a test compound to the aqueous 
solution; and then monitoring the interaction of the test compound with 
the serotonin transporter (e.g., by (a) monitoring the transport of 
serotonin by the serotonin transporter; or (b) monitoring the binding of 
the test compound to the serotonin transporter). The cell membranes may be 
those of whole cells or lysed cells. The assay is useful for identifying 
serotonin transport inhibitors. 
A sixth aspect of the present invention is an oligonucleotide probe capable 
of selectively hybridizing to a DNA comprising a portion of a gene coding 
for a serotonin transporter. Preferably the probe does not hybridize to a 
gene coding for other neurotransmitter transporters such as the 
noradrenaline transporter. 
A seventh aspect of the present invention is isolated and purified 
serotonin transporter protein which is coded for by DNA selected from the 
group consisting of: (a) isolated DNA which encodes rat serotonin 
transporter; (b) isolated DNA which hybridizes to isolated DNA of (a) 
above and which encodes a serotonin transporter; and (c) isolated DNA 
differing from the isolated DNAs of (a) and (b) above in codon sequence 
due to the degeneracy of the genetic code, and which encodes a serotonin 
transporter. 
An eighth aspect of the present invention is antibodies (preferably 
monoclonal antibodies) which bind selectively to the serotonin transporter 
protein. 
The foregoing and other objects and aspects of the present invention will 
be made apparent from the drawings herein and the specification set forth 
below.

DETAILED DESCRIPTION OF THE INVENTION 
Amino acid sequences disclosed herein are presented in the amino to carboxy 
direction, from left to right. The amino and carboxy groups are not 
presented in the sequence. Nucleotide sequences are presented herein by 
single strand only, in the 5' to 3' direction, from left to right. 
Nucleotides and amino acids are represented herein in the manner 
recommended by the IU-IUB Biochemical Nomenclature Commission, or (for 
amino acids) by three letter code, in accordance with 37 CFR .sctn.1.822 
and established usage. See, e.g., PatentIn User Manual, 99-102 (Nov. 
1990)(U.S. Patent and Trademark Office, Office of the Assistant 
Commissioner for Patents, Washington, D.C. 20231); U.S. Pat. No. 4,871,670 
to Hudson et al. at Col. 3 lines 20-43 (applicants specifically intend 
that the disclosure of this and all other patent references cited herein 
be incorporated herein by reference). 
Serotonin transporters of the present invention include proteins homologous 
to, and having essentially the same biological properties as, the proteins 
coded for by the nucleotide sequence set forth as SEQ ID NO:6, SEQ ID NO:8 
or SEQ ID NO:10. This definition is intended to encompass natural allelic 
variations in the serotonin transporter sequence, but to exclude the 
noradrenaline transporter sequence. Cloned genes of the present invention 
may code for serotonin transporters of any species of origin, including 
mouse, rat, rabbit, cat, and human, but preferably code for receptors of 
mammalian origin. Thus, DNA sequences which hybridize to the sequences 
given in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 and which code for 
expression of a serotonin transporter are also an aspect of this 
invention. Conditions which will permit other DNA sequences which code for 
expression of a serotonin transporter to hybridize to the sequences given 
in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 can be determined in a routine 
manner. For example, hybridization of such sequences may be carried out 
under conditions of reduced stringency or even stringent conditions (e.g., 
conditions represented by a wash stringency of 0.3 Molar NaCl, 0.03M 
sodium citrate, 0.1% SDS at 60.degree. C. or even 70.degree. C.) to DNA 
encoding the rat or human serotonin transporter disclosed herein in a 
standard in situ hybridization assay. See J. Sambrook et al., Molecular 
Cloning, A Laboratory Manual, 2d Ed. (Cold Spring Harbor Laboratory 1989). 
In general, sequences which code for a serotonin transporter and hybridize 
to the DNA encoding the rat or human serotonin transporter disclosed 
herein will be at least 75% homologous, 85% homologous, or even 95% 
homologous or more with the sequence of the DNA encoding rat or human 
serotonin transporter disclosed herein. Determinations of homology are 
made with the two sequences (nucleic acid or amino acid) aligned for 
maximum matching. Gaps in either of the two sequences being matched are 
allowed in maximizing matching. Gap lengths of 10 or fewer are preferred, 
gap lengths of 5 or fewer are more preferred, and gap lengths of 2 or 
fewer still more preferred. 
Further, DNA sequences which code for polypeptides coded for by the 
sequence given in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or sequences 
which hybridize thereto and code for a serotonin transporter, but which 
differ in codon sequence from these due to the degeneracy of the genetic 
code, are also an aspect of this invention. The degeneracy of the genetic 
code, which allows different nucleic acid sequences to code for the same 
protein or peptide, is well known in the literature. See, e.g., U.S. Pat. 
No. 4,757,006 to Toole et al. at Col. 2, Table 1. 
The production of cloned genes, recombinant DNA, vectors, host cells, 
proteins and protein fragments by genetic engineering techniques is well 
known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6 line 3 
to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. at Col. 4 line 
38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling at Col. 3 line 
26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 to Wallner at Col. 6 
line 8 to Col. 8 line 59. 
DNA which encodes the serotonin transporter may be obtained, in view of the 
instant disclosure, by chemical synthesis, by screening reverse 
transcripts of mRNA from appropriate cells or cell line cultures, by 
screening genomic libraries from appropriate cells, or by combinations of 
these procedures, as illustrated below. Screening of mRNA or genomic DNA 
may be carried out with oligonucleotide probes generated from the 
serotonin transporter gene sequence information provided herein. Probes 
may be labeled with a detectable group such as a fluorescent group, a 
radioactive atom or a chemiluminescent group in accordance with known 
procedures and used in conventional hybridization assays, as described in 
greater detail in the Examples below. In the alternative, serotonin 
transporter gene sequences may be recovered by use of the polymerase chain 
reaction (PCR) procedure, with the PCR oligonucleotide primers being 
produced from the serotonin transporter nucleotide sequence provided 
herein (particularly from poorly conserved regions thereof). See U.S. Pat. 
No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis. 
The serotonin transporter may be synthesized in host cells transformed with 
vectors containing DNA encoding the serotonin transporter. A vector is a 
replicable DNA construct. Vectors are used herein either to amplify DNA 
encoding the serotonin transporter and/or to express DNA which encodes the 
serotonin transporter. An expression vector is a replicable DNA construct 
in which a DNA sequence encoding the serotonin transporter is operably 
linked to suitable control sequences capable of effecting the expression 
of the serotonin transporter in a suitable host. The need for such control 
sequences will vary depending upon the host selected and the 
transformation method chosen. Generally, control sequences include a 
transcriptional promoter, an optional operator sequence to control 
transcription, a sequence encoding suitable mRNA ribosomal binding sites, 
and sequences which control the termination of transcription and 
translation. Amplification vectors do not require expression control 
domains. All that is needed is the ability to replicate in a host, usually 
conferred by an origin of replication, and a selection gene to facilitate 
recognition of transformants. 
Vectors useful for practicing the present invention include plasmids, 
viruses (including phage), retroviruses, and integratable DNA fragments 
(i.e., fragments integratable into the host genome by homologous 
recombination). The vector replicates and functions independently of the 
host genome, or may, in some instances, integrate into the genome itself. 
Suitable vectors will contain replicon and control sequences which are 
derived from species compatible with the intended expression host. 
Transformed host cells are cells which have been transformed or 
transfected with the serotonin transporter vectors constructed using 
recombinant DNA techniques. Transformed host cells ordinarily express the 
serotonin transporter, but host cells transformed for purposes of cloning 
or amplifying the serotonin transporter DNA need not express the serotonin 
transporter. When expressed, the serotonin transporter will typically be 
located in the host cell membrane. 
DNA regions are operably linked when they are functionally related to each 
other. For example: a promoter is operably linked to a coding sequence if 
it controls the transcription of the sequence; a ribosome binding site is 
operably linked to a coding sequence if it is positioned so as to permit 
translation. Generally, operably linked means contiguous and, in the case 
of leader sequences, contiguous and in reading phase. 
Suitable host cells include prokaryotes, yeast cells or higher eukaryotic 
cells. Prokaryotes include gram negative or gram positive organisms, for 
example Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells 
include established cell lines of mammalian origin as described below. 
Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli 
X1776 (ATCC 31,537), and E. coli 294 (ATCC 31,446). Pseudomonas species, 
Bacillus species, and Serratia marcesans are also suitable. 
A broad variety of suitable microbial vectors are available. Generally, a 
microbial vector will contain an origin of replication recognized by the 
intended host, a promoter which will function in the host and a phenotypic 
selection gene such as a gene encoding proteins conferring antibiotic 
resistance or supplying an autotrophic requirement. Similar constructs 
will be manufactured for other hosts. E. coli is typically transformed 
using pBR322. See Bolivar et al., Gene 2, 95 (1977). pBR322 contains genes 
for ampicillin and tetracycline resistance and thus provides easy means 
for identifying transformed cells. 
Expression vectors should contain a promoter which is recognized by the 
host organism. This generally means a promoter obtained from the intended 
host. Promoters most commonly used in recombinant microbial expression 
vectors include the beta-lactamase (penicillinase) and lactose promoter 
systems (Chang et al., Nature 275, 615 (1978); and Goeddel et al., Nature 
281, 544 (1979)), a tryptophan (trp) promoter system (Goeddel et al., 
Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the 
tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 
(1983)). While these are commonly used, other microbial promoters are 
suitable. Details concerning nucleotide sequences of many have been 
published, enabling a skilled worker to operably ligate them to DNA 
encoding the serotonin transporter in plasmid or viral vectors (Siebenlist 
et al., Cell 20, 269 (1980)). The promoter and Shine-Dalgarno sequence 
(for prokaryotic host expression) are operably linked to the DNA encoding 
the serotonin transporter, i.e., they are positioned so as to promote 
transcription of the serotonin transporter messenger RNA from the DNA. 
Eukaryotic microbes such as yeast cultures may be transformed with suitable 
serotonin transporter-encoding vectors. See, e.g., U.S. Pat. No. 
4,745,057. Saccharomyces cerevisiae is the most commonly used among lower 
eukaryotic host microorganisms, although a number of other strains are 
commonly available. Yeast vectors may contain an origin of replication 
from the 2 micron yeast plasmid or an autonomously replicating sequence 
(ARS), a promoter, DNA encoding the serotonin transporter, sequences for 
polyadenylation and transcription termination, and a selection gene. An 
exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); 
Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 
(1980)). This plasmid contains the trp1 gene, which provides a selection 
marker for a mutant strain of yeast lacking the ability to grow in 
tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 
(1977)). The presence of the trp1 lesion in the yeast host cell genome 
then provides an effective environment for detecting transformation by 
growth in the absence of tryptophan. 
Suitable promoting sequences in yeast vectors include the promoters for 
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. 
Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. 
Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 
(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, 
hexokinase, pyruvate decarboxylase, phosphofructokinase, 
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, 
triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. 
Suitable vectors and promoters for use in yeast expression are further 
described in R. Hitzeman et al., EPO Publn. No. 73,657. 
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 the aforementioned 
metallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well as 
enzymes responsible for maltose and galactose utilization. In constructing 
suitable expression plasmids, the termination sequences associated with 
these genes may also be ligated into the expression vector 3' of the the 
serotonin transporter coding sequences to provide polyadenylation and 
termination of the mRNA. 
Cultures of cells derived from multicellular organisms are a desirable host 
for recombinant serotonin transporter synthesis. In principal, any higher 
eukaryotic cell culture is workable, whether from vertebrate or 
invertebrate culture, including insect cells. However, mammalian cells are 
preferred, as illustrated in the Examples. Propagation of such cells in 
cell culture has become a routine procedure. See Tissue Culture, Academic 
Press, Kruse and Patterson, editors (1973). Examples of useful host cell 
lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and 
WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such 
cells ordinarily include (if necessary) an origin of replication, a 
promoter located upstream from the gene to be expressed, along with a 
ribosome binding site, RNA splice site (if intron-containing genomic DNA 
is used), a polyadenylation site, and a transcriptional termination 
sequence. 
The transcriptional and translational control sequences in expression 
vectors to be used in transforming vertebrate cells are often provided by 
viral sources. For example, commonly used promoters are derived from 
polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat. 
No. 4,599,308. The early and late promoters are useful because both are 
obtained easily from the virus as a fragment which also contains the SV40 
viral origin of replication. See Fiers et al., Nature 273, 113 (1978). The 
vaccinia virus may be used as a vector, as described in the Examples. 
Further, the serotonin transporter promoter, control and/or signal 
sequences, may also be used, provided such control sequences are 
compatible with the host cell chosen. 
An origin of replication may be provided either by construction of the 
vector to include an exogenous origin, such as may be derived from SV40 or 
other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be 
provided by the host cell chromosomal replication mechanism. If the vector 
is integrated into the host cell chromosome, the latter may be sufficient. 
Rather than using vectors which contain viral origins of replication, one 
can transform mammalian cells by the method of cotransformation with a 
selectable marker and the serotonin transporter DNA. An example of a 
suitable selectable marker is dihydrofolate reductase (DHFR) or thymidine 
kinase. See U.S. Pat. No. 4,399,216. Such markers are proteins, generally 
enzymes, that enable the identification of transformant cells, i.e., cells 
which are competent to take up exogenous DNA. Generally, identification is 
by survival of transformants in culture medium that is toxic, or from 
which the cells cannot obtain critical nutrition without having taken up 
the marker protein. 
Host cells such as insect cells (e.g., cultured Spodoptera frugiperda 
cells) and expression vectors such as the baculovirus expression vector 
(e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni 
MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in 
carrying out the present invention, as described in U.S. Pat. Nos. 
4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus 
expression vector comprises a baculovirus genome containing the gene to be 
expressed inserted into the polyhedrin gene at a position ranging from the 
polyhedrin transcriptional start signal to the ATG start site and under 
the transcriptional control of a baculovirus polyhedrin promoter. 
Serotonin transporter made from cloned genes in accordance with the present 
invention may be used for screening compounds for their ability to 
interact with the serotonin transporter, such as for transporter 
inhibitory activity or competitive binding thereto, or for determining the 
amount of an inhibitory drug in a solution (e.g., blood plasma or serum). 
For example, host cells may be transformed with a vector of the present 
invention, serotonin transporter expressed in that host and the cells used 
whole to screen compounds for serotonin transporter inhibition activity. 
In another example, host cells may be transformed with a vector of the 
present invention, the serotonin transporter expressed in that host, the 
cells lysed, and the membranes from those cells used to screen compounds 
for competitive binding to the serotonin transporter with a labelled 
compound which binds to the serotonin transporter such as tritiated 
paroxetine or desimipramine. Assays in which such procedures may be 
carried out are well known, as illustrated by the Examples below. By 
selection of host cells which do not ordinarily express another 
transporter protein such as the noradrenaline transporter, GABA 
transporter, and/or dopamine transporter, preparations free of extraneous 
factors can be obtained. Further, the presence of a vesicular transport 
system for serotonin can be avoided by selecting as host cells cells which 
lack synaptic vesicles. Such assay systems have not heretofore been 
available. 
Cloned genes of the present invention, and oligonucleotides derived 
therefrom, are useful for screening for restriction fragment length 
polymorphism (RFLP) associated with certain disorders. 
Oligonucleotides of the present invention are useful as diagnostic tools 
for probing serotonin transporter gene expression in various tissues. For 
example, tissue can be probed in situ with oligonucleotide probes carrying 
detectable groups (i.e., "labelled") by conventional autoradiography 
techniques to investigate native expression of this transporter or 
pathological conditions relating thereto (e.g., human genetic disorders). 
This can be done routinely by temperature gradient electrophoresis. In 
addition, oligonucleotides of the present invention can be used to probe 
for other serotonin transporters subtypes or serotonin transporters in 
other species. Further, chromosomes can be probed to investigate the 
presence or absence of a serotonin transporter gene, and potential 
pathological conditions related thereto. 
A variety of detectable groups can be employed to label antibodies and 
probes as disclosed herein, and the term "labelled" is used herein to 
refer to the conjugating or covalent bonding of any suitable detectable 
group, including enzymes (e.g., horseradish peroxidase, 
.beta.-glucuronidase, alkaline phosphatase, and .beta.-D-galactosidase), 
fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., 
.sup.14 C, .sup.131 I, .sup.3 H, .sup.32 P, and .sup.35 S) to the compound 
being labelled. Techniques for labelling various compounds, including 
proteins, peptides, and antibodies, are well known. See, e.g., Morrison, 
Methods in Enzymology 32b, 103 (1974); Syvanen et al., J. Biol. Chem. 284, 
3762 (1973); Bolton and Hunter, Biochem. J. 133, 529 (1973). 
Antibodies which specifically bind to the serotonin transporter (i.e., 
antibodies which bind to a single antigenic site or epitope on the 
transporter) may be polyclonal or monoclonal in origin, but are preferably 
of monoclonal origin. Such antibodies are useful for the affinity 
purification of the serotonin transporter, and for the identification and 
assay of serotonin transporters in human tissue samples (e.g., post-mortem 
brain samples) or in peripheral platelet cells. The antibodies may be of 
any suitable species, such as rat, rabbit, or horse, but are generally of 
mammalian origin. The antibodies may be of any suitable immunoglobulin, 
such as IgG and IgM. Fragments of antibodies which retain the ability to 
specifically bind the serotonin transporter, such as F(ab').sub.2, F(ab'), 
and Fab fragments, are intended to be encompassed by the term "antibody" 
herein. The antibodies may be chimeric, as described by M. Walker et al., 
Molecular Immunol. 26, 403 (1989). Antibodies may be immobilized on a 
solid support of the type used as a packing in an affinity chromatography 
column, such as sepharose, silica, or glass beads, in accordance with 
known techniques. 
Monoclonal antibodies which bind to the serotonin transporter are made by 
culturing a cell or cell line capable of producing the antibody under 
conditions suitable for the production of the antibody (e.g., by 
maintaining the cell line in HAT media), and then collecting the antibody 
from the culture (e.g., by precipitation, ion exchange chromatography, 
affinity chromatography, or the like). The antibodies may be generated in 
a hybridoma cell line in the widely used procedure described by G. Kohler 
and C. Milstein, Nature 256, 495 (1975), or may be generated with a 
recombinant vector in a suitable host cell such as Escherichia coli in the 
manner described by W. Huse et al., Generation of a Large Combinatorial 
Library of the Immunoglobulin Repertoire in Phage Lambda, Science 246, 
1275 (1989). 
Isolated and purified serotonin transporter of the present invention is 
useful in the rational design of drugs which interact with this 
transporter, and is useful as an immunogen for the production of 
antibodies which bind to the serotonin transporter. The serotonin 
transporter may be purified from cell membranes or lysed cell fractions 
containing the transporter, as described above, in accordance with known 
procedures, including column chromatography (e.g., ion exchange, gel 
filtration, electrophoresis, affinity chromatography, etc.), optionally 
followed by crystallization. See generally Enzyme Purification and Related 
Techniques, Methods in Enzymology 22, 233-577 (1977). 
The present invention is explained in greater detail in the following 
non-limiting examples. In these examples, ".mu.g" means micrograms, "ng" 
means nanograms, ".mu.Ci" means microcuries, "ml" means milliliters, "SDS" 
means sodium dodecyl sulfate, "kb" means kilobase, "min" means minute, 
"hr" means hour, "mol" means mole ".mu.M" means microMolar, and 
temperatures are given in degrees Centigrade unless otherwise indicated. 
EXAMPLE 1 
Production of Rodent Brain rMB6-25 cDNA by PCR 
Using the polymerase chain reaction (PCR) (R. Saiki et al., Science 239, 
487-494 (1988)) with degenerate oligonucleotides (R. Rathe, J. Mol. Biol. 
183, 1-12 (1985)) derived from two highly conserved regions of recently 
cloned noradrenaline (hNAT) (T. Pacholczyk et al., Nature 350, 350-354 
(1991)) and gamma-aminobutyric acid (rGAT1)(J. Guastella et al., Science 
249, 1303-1306 (1990)) transporters, a large family of related gene 
products expressed in rodent brain were identified. 
PCR reaction products obtained from amplification of rodent and human cDNA, 
which were of a size (.about.700 base pairs (bp)) predicted by hNAT and 
rGAT1, were purified, subcloned, and sequenced. After sequence analysis, 8 
unique clones were identified. In pairwise sequence comparisons, most 
clones were equally similar to each other as to hNAT and rGAT1 with 
.about.50-60% identity. However, another group, comprised of clones rTB2-5 
and rMB6-25, were more closely related to hNAT, with 84% and 67% identity, 
respectively. Given the significant overlap in antagonist sensitivity 
among monoamine neurotransmitter transporters, see E. Richelson, Mayo 
Clin. Proc. 65, 1227-1236 (1990), we hypothesized that these two species 
could be partial clones encoding dopamine and serotonin (5HT) 
transporters. We accordingly focused our attention, in Example 2 below, on 
the size and regional distribution of rMB6-25 RNA in the rodent brain to 
determine the likely substrate for this transporter. 
EXAMPLE 2 
In Situ Hybridization Analysis of rMB6-25 cDNAs 
This Example shows that the rMB6-25 cDNA hybridizes to a single 3.7 kb RNA 
restricted to rat midbrain and brainstem, where it is highly enriched 
within the serotonergic raphe complex. 
For in situ hybridization experiments, synthetic [.sup.35 S]-labeled cRNA 
was synthesized with PCR fragment rMB6-25, cloned into the Xba1 and Xho1 
sites of pBluescript SKII(-), from either the T3 promoter after plasmid 
linearization with Xho1 (antisense cRNA) or from the T7 promoter after 
linearization with Xba1 (sense cRNA). cRNA synthesis and in situ 
hybridization to 4% paraformaldehyde-fixed rat brain sections was 
conducted in accordance with known techniques. See R. Fremeau et al., 
Proc. Natl. Acad. Sci. USA 88, 3772-3776 (1991). cDNA derived from PCR 
fragment rMB6-25 (100 ng) was radiolabeled with [.sup.32 P]-labeled dCTP 
(50 .mu.Ci) using random oligonucleotide primers and hybridized to a nylon 
(Zetaprobe, BioRad) transfer of total RNAs (20 .mu.g) derived from rat 
tissues and rat and human cell lines. Blots were prehybridized at 
42.degree. C. in 50% formamide, 5XSSPE, 5X Denhardt's, 10% dextran 
sulfate, 1% SDS, and 100 .mu.g/ml salmon sperm DNA for 2 hrs, probe added 
and hybridization continued for 14 hrs. Blot was rinsed with 2, 20 min 
22.degree. C. washes in 2XSSPE, 0.1% SDS, followed by a 1 hr rinse at 
65.degree. C. in 0.1X SSPE, 0.1% SDS, and then exposed to autoradiographic 
film with intensifying screen for 5 days. Positions of 18S (1950 kb) and 
28S (4700 kb) ribosomal RNAs are noted. All lanes were equivalently loaded 
based on even intensity of ribosomal RNAs. 
In situ hybridization analyses of endogenous RNA expression in 
slide-mounted sections of adult rat brain revealed a prominent and 
specific hybridization signal to radiolabeled antisense cRNA transcribed 
from rMB6-25 overlying dorsal and median subdivisions of the serotonergic 
midbrain raphe complex. See H. Steinbusch & R. Niewenhuys, in Chemical 
Neuroanatomy, 131-207 (P. Emson Ed., Raven Press, N.Y. 1983). Similarly, 
Northern hybridizations indicate the presence of a single 3.7 kb 
hybridizing RNA in rat midbrain and brainstem. Our inability to detect 
hybridization from total brain RNA underscores the restriction of gene 
expression for this putative transporter to cells of the mesencephalic and 
metencephalic raphe complex. Thus, the CNS distribution of hybridizing 
RNAs strongly suggests that rMB6-25 encodes a partial clone of the 5HT 
transporter. The adrenal RNA visualized in Northern analyses is unlikely 
to arise from cross-hybridization to the noradrenaline carrier as no RNAs 
were detected from the pheochromocytoma cells (PC12), derived from adrenal 
chromaffin cells, or human SK-N-SH neuroblastoma cells, both of which 
express high levels of the noradrenaline transporter. In this regard, 5HT 
has been detected in mast cells lining rat adrenal arterioles and in a 
population of medullary cells synthesizing adrenaline. See generally J. 
Hinson et al., J. Endocrinol. 121, 253-260 (1989); M. Holzworth & M. 
Brownfield, Neuroendocrinol. 41, 230-236 (1985); A. Verhofstad & G. 
Jonsson, Neuroscience 10, 1443-1453 (1983). The properties of the adrenal 
RNA (equivalent size, high-stringency hybridization) lead us to 
hypothesize that 3.7 kb mRNAs with high sequence correspondence to PCR 
clone rMB6-25 encode both brain and peripheral 5HT transporters. 
EXAMPLE 3 
Isolation of Rat 5HT Transporter cDNA BS4E-10 
This Example describes the isolation of a cDNA encoding the rat 5HT 
transporter. In brief, a synthetic antisense oligonucleotide corresponding 
to the poorly conserved amino acid sequence in the 5'end of PCR clone 
rMB6-25 was used to screen a rat brainstem cDNA library by plaque 
hybridization. See J. Sambrook et al., Molecular Cloning: A Laboratory 
Manual, 2d Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 
N.Y., 1989). One positive plaque from a total screen of 1.2.times.10.sup.6 
plaques was identified, purified, and the EcoR1 insert subcloned into 
pBluescript SKII.sup.- (Stratagene). 
The conserved amino acid sequences NVWRFPY (SEQ ID NO:1) and WIDAATQ (SEQ 
ID NO:2) of hNAT and rGAT1 were used to design degenerate inosine 
(I)-substituted oligonucleotides of sequence 5'CCGCTCGAGAA(C/T)GT 
(G/C)TGGCG(C/G)TT(C/T)CC(A/G/C/T)TA3' (SEQ ID NO:3) and 
5'GCTCTAGAGCTG(A/G)GTIGC(A/G)GC(A/G)TC(A/G)-A(T/G)CCA3' (SEQ ID NO:4), 
respectively (Underlined sequence indicates addition of 5' restriction 
sites for cloning). Oligonucleotides were combined with single-stranded, 
rat and human cDNAs, synthesized from poly(A).sup.+ RNA with random 
hexamer primers (Amersham), into PCR reactions conducted with Taq 
polymerase for 30 cycles of 94.degree.-1 min, 45.degree.-2 min, 
72.degree..sup.- 3 min, with the final extension lengthened to 15 min. 
Products of .about.700 bp, after phenol extraction and ethanol 
precipitation, were digested with EcoR1 to prevent recloning of rGAT1, 
which bears an EcoR1 site between the oligonucleotides utilized for 
amplification, and digested with Xba1 and Xho1 to produce staggered 
cloning ends. Samples were gel purified (GENECLEAN, Bio 101), and ligated 
into Xba1-Xho1 digested pBluescript SKII(.sup.-) (Stratagene). Partial 
sequencing of double-stranded plasmid clones was achieved by 
dideoxynucleotide chain termination using Sequenase (US Biochem). 
Utilizing an end-labeled oligonucleotide derived from the poorly conserved 
region of the 5'end of PCR clone rMB6-25 TIMAIFG (SEQ ID NO:5), we 
isolated a single positive plaque in a screen of 1.2.times.10.sup.6 
bacteriophage from a rat brainstem cDNA library prepared in .lambda.gt10 
(Clontech). The insert, designated BS4E-10, was liberated from purified 
bacteriophage with EcoR1 and ligated into EcoR1-digested pBluescript 
SKII(-). Dideoxynucleotide chain termination sequencing was achieved on 
both strands with Sequenase (US Biochem). Sequences obtained from two 
separate, partial cDNAs isolated from a rat midbrain cDNA library in a 
separate screen were also used to confirm the sequence and interpret 
compressions. MacVector DNA analysis software (IBI) was utilized for 
sequence assembly and analysis. 
The nucleotide and deduced amino acid sequence of the rat 5HT transporter 
(rSERT) encoded by BS4E-10 is given as SEQ ID NO:6 and SEQ ID NO:7, 
respectively. Sequences from bases 279-974 match those obtained from the 
partial cDNA clone rMB6-25. The sequence of BS4E-10 reveals an 1821 bp 
open reading frame (ORF) within a 2278 bp cDNA. The first ATG present in 
the cDNA begins at nucleotide 48 and was assigned as the initiation codon 
due to adherence to the initiation consensus sequence of Kozak, Nucleic 
Acids Res. 15, 8125-8148 (1987). The ORF predicts a protein of 607 amino 
acids with a relative molecular mass of 68,000 (M.sub.r 68K) and is 
distinguished by the presence of 11-12 regions of significantly extended 
hydrophobicity suitable for the formation of transmembrane (TM) domains. 
See J. Kyte & R. Doolittle, J. Molec. Biol. 157, 105-132 (1982). Two 
canonical sites for N-linked glycosylation are present on a large 
hydrophilic domain between putative TM domains 3 and 4, in a similar 
location to those observed for a predicted extracellular loop in the 
cloned noradrenaline and GABA transporters. See T. Pacholczyk et al., 
supra; J. Guastella et al., supra; H. Nelson et al., FEBS Lettr. 269, 
181-184 (1990). As with these carriers, the NH.sub.2 -terminus fails to 
score as a signal sequence for membrane insertion, suggesting its 
retention in the cytoplasm. See G. von Heijne, Eur. J. Biochem. 133, 17-21 
(1983). One consensus site for cAMP-dependent protein kinase 
phosphorylation (B. Kemp & R. Pearson, Trends. Biochem. Sci. 15, 342-346, 
(1990)) is present near the end of the NH.sub.2 -terminus. Interestingly, 
5HT transporters derived from a human placental choriocarcimoma cell line 
(JAR) exhibit cAMP-dependent regulation. See D. Cool et al., J. Biol. 
Chem. 266, 15750-15757 (1991). 
EXAMPLE 4 
Expression and Characterization of Rat 5HT 
Transporter cDNA BS4E-10 in HeLa Fibroblasts 
This example shows that transfection of a single 2.3 kb brainstem cDNA 
clone is sufficient to confer expression of a Na.sup.+ -dependent 5HT 
transporter (rSERT) upon nonneural cells, with transport selectively and 
potently antagonized by 5HT uptake-specific antidepressants, including 
paroxetine, citalopram, and fluoxetine. 
The cDNA (BS4E-10) insert was excised from .lambda.gt10 with EcoR1 and 
subcloned in pBluescript SKII(-) (Stratagene) placing the presumptive 
amino terminus (determined from PCR amplification and sequencing) 
immediately downstream of the T7 RNA polymerase promoter. Cells (10.sup.5 
/well) were infected with recombinant vaccinia virus strain VTF7-3 (T. 
Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126 (1986)), 
expressing T7 RNA polymerase as previously described (R. Blakely et al., 
Analyt. Biochem. 194, 302-308 (1991)), followed 30 min later by 
liposome-mediated (Lipofectin, BRL) transfection of the cDNA construct. 
Control transfections consisted of equivalent amounts of transfected 
vector alone. 5HT transport assays were conducted 8 hours after 
transfection as described for the analysis of the transfected NA carrier, 
utilizing 5-[1,2-.sup.3 H(N)]hydroxytryptamine creatine sulfate ([.sup.3 
H]5HT, 20 nM, Dupont/New England Nuclear) as substrate in 
Krebs-Ringers-Tris-Hepes (KRTH) uptake media. Assays were terminated and 
washed with cold KRTH, cells solubilized with 1% SDS and accumulated 
radioactivity determined by scintillation counting. Data presented as 
CPM/well represent mean .+-.SEM of triplicate experiments. 
Sodium-dependence was determined by isotonic substitution of assay NaCl 
with cholineCl. Inhibition assays, performed in duplicate or triplicate, 
were conducted .+-.increasing concentrations of selected substrates and 
antagonists of 5HT, norepinephrine, and dopamine transport. Nonspecific 
transport was assessed with a parallel transfection of pBluescript for 
each assay and values subtracted from signals obtained with BS4E-10 cDNA. 
Inhibition data are presented as a percentage of 5HT uptake obtained with 
labeled substrate alone. Errors associated with independent experiments 
were less than 10% of mean values plotted. Dopamine, noradrenaline, 
adrenaline, and histamine (K.sub.I &gt;10 .mu.M) were ineffective in blocking 
5HT transport induced by BS4E-10. Experiments with increasing 
concentrations of unlabeled 5HT yielded a Km of 1.5 .mu.M and a Vmax of 
6.7.times.10.sup.-18 mol/cell/min. 
FIG. 1a demonstrates that HeLa fibroblasts transfected with the BS4E-10 
cDNA, though not with the plasmid vector alone, express Na.sup.+ 
-dependent, 5HT uptake. 5HT transport was found to be saturable with 
substrate (data not shown), exhibiting an apparent Km of 1.5 .mu.M. 
Transport assays conducted in the presence of various uptake antagonists 
and substrates demonstrates a marked sensitivity of induced 5HT transport 
to tricyclic and heterocyclic antidepressants (FIG. 1b). The tertiary 
amine tricyclic antidepressants, amitriptyline and imipramine were 
significantly more potent antagonists (K.sub.I =16.9 and 18.7 nM, 
respectively) than their respective secondary amine congeners, 
nortriptyline and desipramine (K.sub.I =73.5 and 567 nM, respectively), 
giving a rank order potency of 
amitriptyline&gt;imipramine&gt;nortriptyline&gt;desipramine. In contrast, the 
cloned NA transporter exhibits a reverse rank order potency of 
desipramine&gt;nortriptyline&gt; imipramine&gt;amitriptyline. See T. Pacholczyk et 
al., supra. The order and magnitude of tricyclic potencies are generally 
equivalent to those obtained with serotonin transport studies in brain 
preparations. See R. Maxwell & H. White, in Handbook of 
Psychopharmacology, eds. L. L, Iversen, S. D, & Snyder, S. H., 83-155 
(Plenum Press, New York, 1978)As with brain preparations, halogenation of 
imipramine to chlorimipramine increases potency for transport in 
transfected cells by &gt;5 fold. Several of the nontricyclic antidepressants 
are considerably more selective for inhibition of endogenous 5HT over 
catecholamine transport, including fluoxetine, citalopram, and paroxetine. 
See L. Lemberger et al., Clin. Pharmacol. Ther. 23, 421-429 (1978); J. 
Hyttel et al., Psychopharmacology 51, 225-233 (1977); J. Buss Lassen, Eur. 
J. Pharmacol. 47, 351-358 (1978). In this regard, paroxetine has a K.sub.I 
of 0.39 nM for inhibition of 5HT transport activity after transfection 
with BS4E-10, nearly three orders of magnitude more potent than its 
inhibition of the cloned NA carrier. The nonselective monoamine transport 
antagonist cocaine (M. Ritz et al., Life Sci. 46, 635-645, (1990)) also 
blocked 5HT transport induced by the cloned cDNA, with predictably lower 
potency than observed for inhibition of cloned NA uptake. The selective DA 
and NA transport inhibitors, GBR12909 and mazindol, exhibited only weak 
potency for inhibition of 5HT uptake (K.sub.I =3.9 and 10.0 .mu.M, 
respectively). Thus, the activity of the protein encoded by the cloned 
cDNA, hereafter referred to as rSERT, bears marked similarity in 
pharmacologic properties to the rat brain 5HT transporter, possessing 
high-affinity sites for both tricyclic and (the more selective) 
heterocyclic antidepressant antagonists. 
EXAMPLE 5 
Further Sequencing of Rat 5HT Transporter cDNA BS4E-10 
On further sequencing a corrected sequence for rat 5HT transporter cDNA was 
obtained. While the sequence obtained in Example 3 was essentially correct 
and could be used as a probe to obtain rat 5HT transporter cDNA, further 
sequencing refined the knowledge of the actual sequence of rat 5HT 
transporter cDNA. 
The nucleotide and deduced amino acid sequence of the rat 5HT transporter 
(rSERT) encoded by BS4E-10 is given as SEQ ID NO:8 and SEQ ID NO:9, 
respectively. Sequences from bases 279-974 match those obtained from the 
partial cDNA clone rMB6-25. 
In an early sequence (Example 3) a gel compression was misread as two bases 
rather than one. The 120-CTGCAGTCCCCAGGCACAAG-140 should have been read as 
120-CTGCAGTCCCCAGCACAAG-139. With this alteration, it was clear that the 
true start for translation was present in a reading frame upstream of the 
start site indicated in Example 3, in a region that had been presumed to 
be a 5'noncoding sequence. The open reading frame encoding the transporter 
extends from base 116 to base 2005 of the revised cDNA, an open reading 
frame of 1890 bp. The ORF predicts a protein of 630 amino acids with a 
relative molecular mass of 70,000 (M.sub.r 70K). Expression of this 
construct in HeLa cells in parallel experiments with the original cDNA 
(Example 4) resulted in equivalent properties (data not shown). The 
differences in translation products of the two clones do not result in 
detectable differences in transport properties. The ability to screen for 
other DNA is not affected. 
EXAMPLE 6 
Structural Model of Rat 5 HT Transporter 
Alignment of amino acid sequences encoding rat 5HT (rSERT), human 
noradrenaline (hNAT), and rat GABA (rGAT1) transporters were produced by 
iterative use of the BESTFIT routine of the Wisconsin GCG software 
package. J. Devereux et al., Nucleic Acids Res. 12, 387-395 (1984). In 
brief, this analysis showed .about.31% of amino acid residues were 
absolutely conserved among all three carriers. A high degree of 
conservation among the three carriers between amino acids 76 and 98 was 
noted, where 17/23 residues are conserved. 
Comparison of the predicted amino acid sequences encoding rSERT, the human 
NA transporter (hNAT), and the rat GABA transporter (rGAT1) demonstrates 
striking sequence conservation. Although rSERT is more closely related to 
hNAT than to rGAT1, with 50% (vs 43%) absolutely conserved residues which 
rises to 72% (vs. 67%) similarity accepting conservative substitutions, 
.about.30% of all residues are absolutely conserved across the three 
carriers. Many of these absolutely conserved residues are positioned in or 
adjacent to the TM domains and are likely to be involved in determining 
critical aspects of secondary structure required for ion binding and/or 
substrate translocation. 
To gain insight into the amino acids likely to be involved in monoamine 
transporter-specific functions, such as binding of tricyclic 
antidepressants and cocaine, we determined the positions of absolutely 
conserved residues among rSERT and hNAT, but which were not conserved in 
rGAT1 as the latter transporter lacks sensitivity to these agents. 
Superimposed on a preliminary structural model of rSERT (FIG. 2), these 
residues cluster prominently in several putative transmembrane domains, 
particularly TM domains 5-7 (Compare with TM9 and 12). Interestingly, only 
one acidic residue (Asp 75, TM1) in the transmembrane domains is conserved 
between rSERT and hNAT, but absent from rGAT1. Most transport antagonists 
are believed to occupy sites overlapping the substrate binding site. See 
P. Andersen, Eur. J. Pharm. 166, 493-504 (1989); D. Graham et al., 
Biochem. Pharmacol. 38, 3819-3826 (1989); J. Marcusson et al., 
Psychopharmacology 99, 17-21 (1989). A negatively charged residue may be 
involved in the binding of polar amino groups of substrates and 
antagonists to monoamine transporters. See R. Maxwell & H. White, supra; 
B. Koe, J. Pharm. Exp. Ther. 199, 649-661 (1976). Thus we suggest that 
monoamine neurotransmitter transporters bind their substrates and 
antagonists in the plane of the membrane, possibly involving determinants 
of TM domains 1, 5-7. A similar extended intramembrane pocket has been 
proposed for G-protein coupled receptor binding of neurotransmitters and 
antagonists. See C. Strader et al., FASEB J. 3, 1825-1832 (1989); B. 
Kobilka et al., Science 240, 1310-1316 (1988). Outside of the 
aforementioned identities with noradrenaline and GABA transporters, no 
significant identities were obtained in sequence comparisons with other 
members of the GenBank data base including receptors, the Na.sup.+ 
/glucose and Na.sup.+ /proline transporters, or facilitated carriers. 
In summary, we have identified a single brain cDNA sufficient to form a 
fully functional 5HT transporter in nonneuronal cells. A higher resolution 
definition of the spatial organization of important residues in the 5HT 
transporter should assist synthetic approaches toward more selective 
therapeutic agents. In this regard, the presence of high-affinity 
tricyclic antidepressant binding sites on both rSERT and hNAT should 
permit rapid progress in the elucidation of key residues defining 
antagonist selectivity. Several selective serotonin (5HT) transport 
inhibitors are presently being prescribed for the clinical management of 
depression, obsessive-compulsive disorder, panic disorder, bulimia, and 
obesity. R. Fuller & D. Wong, Ann. NY. Acad. Sci. 600, 69-80 (1990). The 
cloning of rSERT provides an immediate tool for the direct study of 
transcriptional and posttranslational regulation of the 5HT transporter in 
animal models and provides a means for the identification of a human 
homolog, suitable for an assessment of potential 5HT transporter genetic 
disturbances underlying neuropsychiatric disorders. 
EXAMPLE 7 
Cloning of Human 5HT Transporter cDNAs 
This example describes the isolation of a cDNA encoding the human 5 HT 
transporter. The nucleotide and deduced amino acid sequence of the human 
5HT transporter (hSERT) is given as SEQ ID NO:10 and SEQ ID NO:11. 
Poly(A+)RNA, purified from a placental trophoblastic cell line (JAR; See 
Cool et al., J. Biol. Chem. 266, 15750-15757 (1991)) by the 
guanidium-isothocyanate/cesium chloride method of Chirgwin (See MacDonald 
et al., Meths. Enzymol. 152, 219-227 (1987)) was converted to single 
stranded cDNA (Superscript, Gibco-BRL) and subjected to polymerase-chain 
reaction (PCR; See Saiki et al., Science 238, 487-494 (1988); Hot-Tub DNA 
polymerase (Amersham) 30 cycles 94.degree. C.-1 min, 42.degree. C.-2 min, 
72.degree. C.-3 min, with 10 min extension times programmed on 1st and 
30th cycles). Amplifications were conducted with degenerate 
oligonucleotides (5'-CCGCTCGAGAA(C/T)GT(G/C) 
TGGCG(C/G)TT(C/T)CC(A/G/C/T)TA-3', (SEQ. ID No. 3) and 
5'-GCTCTAGAGCTG(A/G)GTIGC(A/G)GC(A/G)TC(A/G)A(T/G)CCA-3') (SEQ. ID No. 4) 
designed to encode highly conserved sequences of NE and GABA transporters 
and that had been previously employed for the identification of the rat 
brain 5HT transporter (see Example 3 above). Following direct subcloning 
of PCR fragments (TA vector, Invitrogen), dideoxynucleotide sequencing 
(Sequenase, United States Biochemical) was performed on plasmid DNA to 
identify partial human 5HT transporter candidates. A synthetic 21mer 
oligonucleotide (5'-AAAGGCAATGATGCAGATGGC-3'; SEQ ID NO:12), derived from 
the 5' end of the JAR cDNA, was 3' end labeled with .gamma.[.sup.32 P]ATP 
and polynucleotide kinase (See Sambrook et al., Molecular Cloning: A 
Laboratory Manual, 2d ed. (Cold Spring Harbor Laboratory Press, 1989), and 
used to screen a human placental cDNA library in .gamma.ZAPII (Stratagene) 
by MagnaGraph (MSI) filter hybridization at 57.degree. C. following 
manufacturer's protocols, substituting 0.5 mg/mL heparin sulfate to block 
nonspecific hybridization. 
Three hybridizing clones were identified in a screen of 1.6.times.10.sup.6 
plaques, and, following plaque rescreening, were obtained as individual 
plasmids by in vivo excision. Restriction analysis and sequencing revealed 
two of these clones to be homologous to rSERT and to be identical with 
each other except for the presence of distinct deletions in each cDNA. 
Initial sequence of one of these revealed an open reading frame in 
register with the amended sequence of the rat 5HT transporter with 
absolutely conserved amino and carboxy termini, and additional 5' and 3' 
noncoding sequences. Transfection of this cDNA into HeLa cells, however, 
failed to confer 5HT transport function, raising the possibility that a 
nonsense mutation, deletion or recombination had occurred during 
construction, amplification, or excision of the library. Full sequence of 
the cDNA revealed a 103 bp deletion, comprising amino acids 516-550 of the 
rat transporter. A second cDNA possessed the missing region, however, it 
lacked 168 bp of sequence possessed by clone 1 immediately 3' of the point 
where the deletion in clone 1 had occurred. Restriction mapping and direct 
sequencing demonstrated the two clones to be identical in regions of 
overlap except for these missing sequences. Therefore, we adopted a 
recombination PCR approach (See Kriegler, Gene Transfer and Expression: A 
Laboratory Manual, Stockton Press, NY (1990) to ligate in-frame the two 
pieces possessed uniquely by the two cDNAs, which was subsequently 
transferred back into the original clone at convenient restriction sites. 
The resultant cDNA was resequenced to confirm that the construction 
reproduced completely the sequence of both clones. 
EXAMPLE 8 
Expression of hSERT in Transfected Cells 
This example shows that the human cDNA identified in Example 7 encodes a 
high affinity, Na.sup.+ and Cl.sup.- dependent 5HT transporter. A 2158 bp 
EcoRI/ApaI fragment of the reconstructed cDNA, containing 72 bp of 5' 
noncoding and 196 bp of 3' noncoding sequence, was subcloned into 
pBluescript KSII- to place the translation initiation codon 3' to the 
plasmid-encoded T7 RNA polymerase promoter. Plasmid (1 .mu.g) was 
subsequently transfected into HeLa cells (100,000-200,000/well of a 24 
well plate) by liposome-mediated transfection (Lipofectin, GIBCO/BRL) 
previously infected with recombinant (VTF7-3) vaccinia virus encoding T7 
RNA polymerase at 10 pfu, See Blakely et al., Anal. Biochem. 194, 302-308 
(1991). Transport assays (15 min, 37.degree. C. unless indicated) with 20 
nM 5-[1,2-.sup.3 H(N)]hydroxytryptamine creatinine sulfate ([.sup.3 H]5HT, 
DuPont/NEN), 100 .mu.M pargyline and L-ascorbate, were performed 8-12 hrs 
following transfection in Krebs/Ringers/HEPES (KRH) buffer (120 mM NaCl, 
4.7 mM KCl, 2.2 mM CaCl.sub.2, 1.2 mM MgSO.sub.4, 1.2 mM KH2PO.sub.4, 10 
mM HEPES, pH 7.4) as previously described (see Blakely et al., Nature, 
354, 66-70 (1991)). Nonspecific [.sup.3 H]5HT transport was assessed in 
parallel transfections with the plasmid vector and subtracted from the 
data. Sodium dependence of 5HT transport was assessed by isotonic 
substitution of NaCl with choline CL, while Cl- dependence was assessed in 
media substituted with Nagluconate, Kgluconate, and CaNO.sub.3. Substrate 
K.sub.m and inhibitor K.sub.I values of antagonists were determined by 
nonlinear weighted least-square fits (Kaleidagraph) of 
concentration/uptake profiles performed in triplicate, adjusting for 
substrate concentration as provided by Cheng and Prusoff, Biochem. 
Pharmacol. 22, 3099-3108 (1973). Values are provided .+-.SEM. Paroxetine 
was a gift from Beecham Pharmaceuticals, fluoxetine from Eli-Lilly Co., 
and RTI-55 (3.beta.-[4-iodophenyl]tropan-2.beta.-carboxylic acid methyl 
ester tartrate) from F.Ivy Carrol. Nomifensine was obtained from Research 
Biochemicals Inc. All other compounds were obtained from Sigma. 
cDNA transfected cells, but not control cells transfected with the vector 
alone, rapidly accumulate 5HT in a Na.sup.+ -dependent manner to a level 
similar to that observed with parallel rSERT transfections (data not 
shown). Transport is abolished (0.3.+-.0.01% of control levels) when 
identical incubations were conducted in Cl-free media, verifying a 
requirement of induced 5HT transport on both extracellular Na.sup.+ and 
Cl.sup.-. Assays conducted with increasing concentrations of unlabeled 5HT 
confirmed saturability with respect to substrate (data not shown), with a 
single, high-affinity (Km=463 nM) interaction observed following 
Eadie-Hofstee data transformation. Uptake of radiolabeled 5HT is potently 
antagonized by well characterized 5HT transporter antagonists (data not 
shown). Thus the 5HT transport-selective antagonist paroxetine, but not 
the NE transport-selective antagonist nomifensine, potently inhibits 5HT 
uptake in transfected HeLa cells. Similarly, the tertiary amine tricyclic 
antidepressants, imipramine and amitriptyline are more potent than the 
secondary amine tricyclic desipramine, in contrast to their rank order 
potency for inhibition of NE transport (Desip&gt;&gt;Imip&gt;Amitrip). The 
nonselective monoamine transport antagonists cocaine and amphetamine block 
5HT transport at low micromolar concentrations, with the cocaine analogue 
RTI-55 exhibiting increased potency over cocaine, as also described for 
the rat brain DA transporter (See Boja et al., Eur. J. Pharm. 194, 133-134 
(1991)). The biogenic amines norepinephrine, dopamine and histamine are 
only weak inhibitors of induced 5HT uptake, with K.sub. I values &lt;10 
.mu.M. Thus, the identified human cDNA encodes a high-affinity, Na.sup.+ 
and Cl.sup.- -dependent, 5HT transporter, with antagonist specificities 
established in native placental, platelet, and brain membrane preparations 
and is hereafter referred to as hSERT. 
In the 2508 bp cDNA sequence of the largest hSERT cDNA an ORF of 1890 bp is 
present, encoding a polypeptide of 630 amino acids, identical in length to 
the corrected amino acid sequence of rSERT (see Example 5). The predicted 
start of translation possesses a good consensus for translation initiation 
(AAACATGG) following Kozak, Cell 44, 283-292 (1986). The encoded protein 
is predicted to have a core size of 70,320 (Mr) and an isoelectric point 
of 5.8. As expected for members of the GABA/NE transporter gene family 
(see Blakely, Curr. Op. Psych. 5, 69-73 (1992)), 12 regions of marked 
hydrophobicity (See Kyte and Doolittle, J. Molec. Biol. 157, 105-132 
(1982)) are present in perfect register with those identified in the rat 
transporter. The absence of a hydrophobic membrane insertion sequence (see 
Von Heijne, Eur. J. Biochem. 133, 17-21 (1983)) in the protein's amino 
terminus and a folding model to accommodate 12 TM domains places both 
amino and carboxy termini in the cytoplasm, as modeled for rSERT (see 
Example 6). The proteins encoded by hSERT and rSERT possess 92% amino acid 
identity, with differences largely restricted to the amino-terminus where 
20 out of 52 differences occur. Within the hSERT TM domains, only domains 
4, 9, and 12 exhibit multiple (and nonconservative) amino acid changes 
amino acid substitutions relative to rSERT. Like the rat 5HT transporter, 
the large hydrophilic loop between TM3 and TM4 possesses two canonical 
sites for N-linked glycosylation. Several recognition sites for protein 
kinase A (PKA-motif R/K-XX-S/T) and protein kinase C (PKC-motif S/T-X-R/K) 
are found in hSERT (see Kennely and Krebs, J. Biol. Chem. 266, 15555,15558 
(1991)), 5 of which are conserved with rSERT and 4 of these (Ser.sup.8, 
Ser.sup.13, Ser.sup.277, Thr.sup.603) lie in presumptive cytoplasmic 
domains. Interestingly, sequence identity between hSERT and rSERT is not 
confined to protein coding sequences, as the preceding 72 bp of 5' 
noncoding sequence and the 406 bp of 3' noncoding sequence exhibit 
conspicuous stretches of alignment, with 72% and 55% overall identity, 
respectively. In comparisons of hSERT amino acid sequence with other human 
and rodent members of the Na+/Cl-cotransporter gene family, hSERT is most 
closely related to the human norepinephrine transporter (48% AA identity) 
with which it shares antagonism by tricyclic antidepressants, and the rat 
dopamine transporter (44% AA identity), which, like the norepinephrine 
transporter, also binds cocaine. Other family members exhibit 35-39% 
identity. 
EXAMPLE 9 
Tissue and Chromosomal Localization of hSERT Gene 
This example demonstrates the pattern of 5HT transporter expression in 
human tissue. RNA distribution and heterogeneity were evaluated by 
hybridization of blotted human poly(A+) RNAs (Clontech) using 
random-primed hSERT cDNA as probe. Labeling and hybridizations were 
conducted with the Megaprime hybridization system (Amersham) following 
manufacturer's protocols except for the addition of two high-stringency 
washes at 65.degree. C. in 0.1X SSPE, 0.5% SDS. Following stripping of the 
blot, similar hybridizations were conducted with random-primed human 
.beta.-actin cDNA to insure for equivalent RNA loading and transfer. 
Somatic cell hybrid analysis was performed with both rat and human 5HT 
transporter cDNAs. A mapping panel consisting of 17 mouse-human (NA09925 - 
NA 09938, NA09940, NA10324, and NA10567) and 2 Chinese hamster--human 
(NA10611 and GM07298) hybrids was obtained from the National Institute of 
General Medical Services Mutant Cell Repository (NIGMS). Characterization 
and human chromosome content in these hybrids are described in detail in 
the NIGMS catalogue. Southern hybridization was performed as previously 
described (see Yang-Feng et al., Am. J. Hum. Genet. 37, 1117-1128 (1985)). 
For in situ hybridization, cDNA probe was nick-translated with [3H]dATP 
and [3H]dCTP to a specific activity of 3.times.10.sup.7 CPM/.mu.g. 
Hybridization to human metaphases, post-hybridization and emulsion 
autoradiography were carried out as previously described (see Yang-Feng et 
al., Am. J. Hum. Genet. 37, 1117-1128 (1985)). Chromosomes were G-banded 
using Wright's stain for silver grain analysis. 
RNA hybridizations were performed with the hSERT cDNA probe at high 
stringency. Three hybridizing RNAs of 6.8 kb, 4.9 kb, and 3.0 kb were 
detected in poly(A+) RNA from human placenta which contains 
syncititotrophoblasts known to exhibit antidepressant- and 
cocaine-sensitive 5HT transport (See Balkovitz et al., J. Biol. Chem. 264, 
2195-2198 (1989) and Cool et al., Biochemistry 29, 1818-1822 (1990)) but 
not in human skeletal muscle, liver, heart, and kidney, tissues lacking 
the 5HT carrier. Multiple hybridizing RNAs are also observed in human 
lung, wherein endothelial cells express an imipramine-sensitive 5HT 
transporter (see Lee and Fanburg, Am. J. Physiol. 250, C761-C765 (1986)). 
Control hybridization with a .beta.-actin cDNA confirmed RNA integrity and 
loading equivalence (data not shown). Interestingly, hybridization of 
total human brain poly(A+) RNA failed to detect 5HT transporter 
transcripts, likely a result of small quantities of midbrain and brainstem 
RNA in the commercial preparations that we utilized for hybridizations. 
Similar findings, however, are observed in the rat where midbrain and 
brainstem dissections are required to obtain enriched RNA suitable for 
visualization of 5HT transporter mRNA. Additional hybridizations conducted 
with human brainstem and JAR RNA revealed a single major band in brainstem 
comigrating with the 4.0 kB species visualized in placenta and lung, while 
JAR cells exhibited the placental hybridization pattern (data not shown). 
Southern blot analysis of 19 human and rodent somatic cell hybrids mapped 
the 5HT transporter gene to human chromosome 17. rSERT cDNA probe detected 
five mouse EcoRI fragments of 23, 6.7 5.8, 4.2, and 2.9 kB, two hamster 
hybridizing bands of 14 and 8.6 kB, and a 15 kB human fragment. The human 
cDNA probe detected two mouse, hamster and human specific fragments of 6.6 
and 5.8 kB, 14 and 8.3 kB, and 15 and 5.3 kB, respectively (data not 
shown). Both human fragments were found to specifically segregate with 
chromosome 17. In situ hybridization revealed specific labeling at region 
q11-q12 of chromosome 17. Of 137 grains in 100 cells analyzed, 23 were 
located at 17q11-q12. No other chromosomal site was labeled above 
background. As only 90 bp precedes the single internal EcoRI site of our 
hSERT cDNA probe, the two large hybridizing EcoRI fragments likely arise 
from a hSERT gene interrupted by one or more introns. 
The foregoing examples are illustrative of the present invention, and are 
not to be construed as limiting thereof. The invention is defined by the 
following claims, with equivalents of the claims to be included therein. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 12 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AsnVal TrpArgPheProTyr 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TrpIleAspAlaAlaThrGln 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CCGCTCGAGAAYGTSTGGCGSTTYCCNTA 29 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GCTCTAGAGCTGRGTNGCRGCRTCRAKCCA 30 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ThrIleMetAlaIlePheGly 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2278 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(viii) POSITION IN GENOME: 
(C) UNITS: 2278 basepairs 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 48..1868 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TCAGAAAGTGCTGTCAGAGTGTAAGGACAGAGAGGACTGTCAAGAAAATGGTGTTC56 
MetValPhe 
TACAGAAGGGTGTCCCCACCACAGCGGACAGGGCAGAGCCTAGCCAAA104 
TyrArgArgValSerProProGlnArgThrGlyGlnSerLeuAlaLys 
51015 
TATCCAATG GGTACTCTGCAGTCCCCAGGCACAAGTGCAGGGGACGAA152 
TyrProMetGlyThrLeuGlnSerProGlyThrSerAlaGlyAspGlu 
20253035 
GCTTC ACACTCGATCCCAGCTGCCACCACCACCCTGGTGGCTGAGATT200 
AlaSerHisSerIleProAlaAlaThrThrThrLeuValAlaGluIle 
404550 
CGCC AAGGGGAGCGGGAGACCTGGGGCAAGAAGATGGATTTCCTCCTG248 
ArgGlnGlyGluArgGluThrTrpGlyLysLysMetAspPheLeuLeu 
556065 
TCCGTC ATTGGCTATGCCGTGGACCTGGGCAACATCTGGCGGTTTCCT296 
SerValIleGlyTyrAlaValAspLeuGlyAsnIleTrpArgPhePro 
707580 
TACATATGCTAC CAGAATGGCGGAGGGGCCTTCCTCCTCCCTTATACC344 
TyrIleCysTyrGlnAsnGlyGlyGlyAlaPheLeuLeuProTyrThr 
859095 
ATCATGGCCATTTTCGGGGG GATCCCGCTCTTTTACATGGAGCTCGCA392 
IleMetAlaIlePheGlyGlyIleProLeuPheTyrMetGluLeuAla 
100105110115 
CTGGGCCAGTACCACC GAAACGGGTGCATTTCCATATGGAGGAAGATC440 
LeuGlyGlnTyrHisArgAsnGlyCysIleSerIleTrpArgLysIle 
120125130 
TGCCCGATTTTCAAA GGCATTGGTTACGCCATCTGCATCATCGCCTTT488 
CysProIlePheLysGlyIleGlyTyrAlaIleCysIleIleAlaPhe 
135140145 
TACATCGCCTCCTACTAC AACACCATCATAGCCTGGGCGCTCTACTAC536 
TyrIleAlaSerTyrTyrAsnThrIleIleAlaTrpAlaLeuTyrTyr 
150155160 
CTCATCTCCTCCCTCACGGACCG GCTGCCCTGGACCAGCTGCACGAAC584 
LeuIleSerSerLeuThrAspArgLeuProTrpThrSerCysThrAsn 
165170175 
TCCTGGAACACTGGCAACTGCACCAACTACT TCGCCCAGGACAACATC632 
SerTrpAsnThrGlyAsnCysThrAsnTyrPheAlaGlnAspAsnIle 
180185190195 
ACCTGGACGCTGCATTCCACGTCCCCC GCTGAGGAGTTCTACTTGCGC680 
ThrTrpThrLeuHisSerThrSerProAlaGluGluPheTyrLeuArg 
200205210 
CATGTCCTGCAGATCCACCAGTCTAAG GGACTCCAGGACCTGGGCACC728 
HisValLeuGlnIleHisGlnSerLysGlyLeuGlnAspLeuGlyThr 
215220225 
ATCAGCTGGCAGCTGACTCTCTGCATCGT GCTCATCTTCACCGTAATC776 
IleSerTrpGlnLeuThrLeuCysIleValLeuIlePheThrValIle 
230235240 
TACTTTAGCATCTGGAAAGGCGTCAAAACATCTG GCAAGGTGGTGTGG824 
TyrPheSerIleTrpLysGlyValLysThrSerGlyLysValValTrp 
245250255 
GTGACAGCCACCTTCCCATACATTGTCCTCTCTGTCCTGCTG GTGAGG872 
ValThrAlaThrPheProTyrIleValLeuSerValLeuLeuValArg 
260265270275 
GGGGCCACCCTTCCTGGAGCCTGGAGAGGGGTCGTCTTC TACTTGAAA920 
GlyAlaThrLeuProGlyAlaTrpArgGlyValValPheTyrLeuLys 
280285290 
CCCAACTGGCAGAAACTCTTGGAGACAGGGGTGTGGGT AGATGCCGCC968 
ProAsnTrpGlnLysLeuLeuGluThrGlyValTrpValAspAlaAla 
295300305 
GCTCAGATCTTCTTCTCTCTTGGCCCGGGCTTTGGGGTTC TCCTGGCT1016 
AlaGlnIlePhePheSerLeuGlyProGlyPheGlyValLeuLeuAla 
310315320 
TTTGCTAGCTACAACAAGTTCAACAACAACTGTTACCAAGATGCC CTG1064 
PheAlaSerTyrAsnLysPheAsnAsnAsnCysTyrGlnAspAlaLeu 
325330335 
GTGACCAGTGTGGTGAACTGCATGACAAGCTTCGTCTCTGGCTTCGTC111 2 
ValThrSerValValAsnCysMetThrSerPheValSerGlyPheVal 
340345350355 
ATCTTCACGGTGCTTGGCTACATGGCGGAGATGAGGAATGAAGATGTG 1160 
IlePheThrValLeuGlyTyrMetAlaGluMetArgAsnGluAspVal 
360365370 
TCAGAGGTGGCCAAAGACGCAGGCCCCAGCCTCCTCTTCATCACGTAT 1208 
SerGluValAlaLysAspAlaGlyProSerLeuLeuPheIleThrTyr 
375380385 
GCAGAGGCAATAGCCAACATGCCAGCATCCACGTTCTTTGCCATCATC 1256 
AlaGluAlaIleAlaAsnMetProAlaSerThrPhePheAlaIleIle 
390395400 
TTCTTCCTCATGTTAATCACGCTGGGATTGGACAGCACGTTCGCAGGC1304 
P hePheLeuMetLeuIleThrLeuGlyLeuAspSerThrPheAlaGly 
405410415 
CTGGAAGGTGTGATCACAGCTGTGCTGGATGAGTTCCCTCACATCTGG1352 
LeuGluGly ValIleThrAlaValLeuAspGluPheProHisIleTrp 
420425430435 
GCCAAGCGCAGGGAATGGTTCGTGCTCATCGTGGTCATCACGTGCGTC1400 
AlaLys ArgArgGluTrpPheValLeuIleValValIleThrCysVal 
440445450 
TTGGGATCCCTGCTCACACTGACGTCAGGAGGGGCATACGTGGTGACT1448 
LeuGl ySerLeuLeuThrLeuThrSerGlyGlyAlaTyrValValThr 
455460465 
CTGCTGGAGGAGTATGCCACGGGGCCAGCAGTGCTCACCGTGGCCCTC1496 
LeuLeuG luGluTyrAlaThrGlyProAlaValLeuThrValAlaLeu 
470475480 
ATCGAGGCCGTCGCCGTGTCTTGGTTCTATGGAATCACTCAGTTCTGC1544 
IleGluAlaVal AlaValSerTrpPheTyrGlyIleThrGlnPheCys 
485490495 
AGCGATGTGAAGGAGATGCTGGGCTTCAGCCCGGGATGGTTTTGGAGG1592 
SerAspValLysGluMetLeu GlyPheSerProGlyTrpPheTrpArg 
500505510515 
ATCTGCTGGGTGGCCATCAGCCCTCTGTTTCTCCTGTTCATCATTTGC1640 
IleCysTrpValAlaIl eSerProLeuPheLeuLeuPheIleIleCys 
520525530 
AGTTTTCTGATGAGCCCACCCCAGCTACGGCTTTTCCAATACAACTAT1688 
SerPheLeuMetSerP roProGlnLeuArgLeuPheGlnTyrAsnTyr 
535540545 
CCCCACTGGAGTATCGTCTTGGGCTACTGCATAGGGATGTCGTCCGTC1736 
ProHisTrpSerIleVal LeuGlyTyrCysIleGlyMetSerSerVal 
550555560 
ATCTGCATCCCTACCTATATCATTTATCGGCTGATCAGCACTCCGGGG1784 
IleCysIleProThrTyrIleIle TyrArgLeuIleSerThrProGly 
565570575 
ACACTTAAGGAGCGCATTATTAAAAGTATCACTCCTGAAACACCCACA1832 
ThrLeuLysGluArgIleIleLysSerIleTh rProGluThrProThr 
580585590595 
GAAATCCCGTGTGGGGACATCCGCATGAATGCTGTGTAACACACCC1878 
GluIleProCysGlyAspIleArgMetA snAlaVal 
600605 
TGGGAGAGGACACCTCTTCCCAGCCACCTCTCTCAGCTCTGAAAAGCCCCACTGGACTCC1938 
TCCCCTCTAAGCCAAGCCTGATGAAGACACGGTCCTAACCACTATGGTGCCCAGACTCTT199 8 
GTGGATTCCGACCACTTCTTTCCGTGGACTCTCAGACATGCTACCACATTCGATGGTGAC2058 
ACCACTGAGCTGGCCTCTTGGACACGTCAGGGAGTGGAAGGAGGGATGAACGCCACCCAG2118 
TCATCAGCTAGCTTCAGGTTTAGAATTAGGTCTGTGAGAGTC TGTATCATGTTTTTGGTA2178 
AGATCATACTACCCCGCATCTGTTAGCTTCTAAAGCCTTCAATGTTCATGAATACATAAA2238 
CCACCTAAGAGAAAACAGAGATGTCTTGCTAGCCATATAT2278 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 607 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetValPheTyrArgArgValSerProProGlnArgThrGlyGlnSer 
1510 15 
LeuAlaLysTyrProMetGlyThrLeuGlnSerProGlyThrSerAla 
202530 
GlyAspGluAlaSerHisSerIleProAlaAlaThrThrThrLeuVa l 
354045 
AlaGluIleArgGlnGlyGluArgGluThrTrpGlyLysLysMetAsp 
505560 
PheLeuLeuSerValIleGly TyrAlaValAspLeuGlyAsnIleTrp 
65707580 
ArgPheProTyrIleCysTyrGlnAsnGlyGlyGlyAlaPheLeuLeu 
85 9095 
ProTyrThrIleMetAlaIlePheGlyGlyIleProLeuPheTyrMet 
100105110 
GluLeuAlaLeuGlyGlnTyrHisArgAsnGlyCys IleSerIleTrp 
115120125 
ArgLysIleCysProIlePheLysGlyIleGlyTyrAlaIleCysIle 
130135140 
IleAlaPheT yrIleAlaSerTyrTyrAsnThrIleIleAlaTrpAla 
145150155160 
LeuTyrTyrLeuIleSerSerLeuThrAspArgLeuProTrpThrSer 
1 65170175 
CysThrAsnSerTrpAsnThrGlyAsnCysThrAsnTyrPheAlaGln 
180185190 
AspAsnIleThrTrpThrLeuHis SerThrSerProAlaGluGluPhe 
195200205 
TyrLeuArgHisValLeuGlnIleHisGlnSerLysGlyLeuGlnAsp 
210215220 
LeuGlyThrIleSerTrpGlnLeuThrLeuCysIleValLeuIlePhe 
225230235240 
ThrValIleTyrPheSerIleTrpLysGlyValLysThrSerGlyLys 
245250255 
ValValTrpValThrAlaThrPheProTyrIleValLeuSerValLeu 
260265270 
LeuValArgGlyA laThrLeuProGlyAlaTrpArgGlyValValPhe 
275280285 
TyrLeuLysProAsnTrpGlnLysLeuLeuGluThrGlyValTrpVal 
290295 300 
AspAlaAlaAlaGlnIlePhePheSerLeuGlyProGlyPheGlyVal 
305310315320 
LeuLeuAlaPheAlaSerTyrAsnLysPheAsnAsnAsn CysTyrGln 
325330335 
AspAlaLeuValThrSerValValAsnCysMetThrSerPheValSer 
340345350 
Gl yPheValIlePheThrValLeuGlyTyrMetAlaGluMetArgAsn 
355360365 
GluAspValSerGluValAlaLysAspAlaGlyProSerLeuLeuPhe 
370 375380 
IleThrTyrAlaGluAlaIleAlaAsnMetProAlaSerThrPhePhe 
385390395400 
AlaIleIlePhePheLeuMetLeuIleT hrLeuGlyLeuAspSerThr 
405410415 
PheAlaGlyLeuGluGlyValIleThrAlaValLeuAspGluPhePro 
420425 430 
HisIleTrpAlaLysArgArgGluTrpPheValLeuIleValValIle 
435440445 
ThrCysValLeuGlySerLeuLeuThrLeuThrSerGlyGlyAlaTyr 
450455460 
ValValThrLeuLeuGluGluTyrAlaThrGlyProAlaValLeuThr 
465470475480 
ValAlaLeuIleGluAl aValAlaValSerTrpPheTyrGlyIleThr 
485490495 
GlnPheCysSerAspValLysGluMetLeuGlyPheSerProGlyTrp 
500 505510 
PheTrpArgIleCysTrpValAlaIleSerProLeuPheLeuLeuPhe 
515520525 
IleIleCysSerPheLeuMetSerProProGlnLeuArgL euPheGln 
530535540 
TyrAsnTyrProHisTrpSerIleValLeuGlyTyrCysIleGlyMet 
545550555560 
SerSer ValIleCysIleProThrTyrIleIleTyrArgLeuIleSer 
565570575 
ThrProGlyThrLeuLysGluArgIleIleLysSerIleThrProGlu 
580 585590 
ThrProThrGluIleProCysGlyAspIleArgMetAsnAlaVal 
595600605 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2415 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 116..2005 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CCCCCTCGAGCTTTCCGTCTTGTCCCCATAACCCGAGAGGAGATTCAAACCAAGAACCAA 60 
GAGCTAGCCTGGGTCCTCGGCAGATGGGAATCCGCATCACTTACTGACCAGCAGCATG118 
Met 
1 
GAGACCACACCCTTGAATTCACAGAAAGTGCTGTCAGAGTGTAAGGAC166 
GluThrThrProLeuAsnSerGlnLysValLeuSerGluCysLysAsp 
510 15 
AGAGAGGACTGTCAAGAAAATGGTGTTCTACAGAAGGGTGTCCCCACC214 
ArgGluAspCysGlnGluAsnGlyValLeuGlnLysGlyValProThr 
202530 
ACAGCGGACAGGGCAGAGCCTAGCCAAATATCCAATGGGTACTCTGCA262 
ThrAlaAspArgAlaGluProSerGlnIleSerAsnGlyTyrSerAla 
354045 
GTCCCCA GCACAAGTGCAGGGGACGAAGCTTCACACTCGATCCCAGCT310 
ValProSerThrSerAlaGlyAspGluAlaSerHisSerIleProAla 
50556065 
GCC ACCACCACCCTGGTGGCTGAGATTCGCCAAGGGGAGCGGGAGACC358 
AlaThrThrThrLeuValAlaGluIleArgGlnGlyGluArgGluThr 
707580 
TGG GGCAAGAAGATGGATTTCCTCCTGTCCGTCATTGGCTATGCCGTG406 
TrpGlyLysLysMetAspPheLeuLeuSerValIleGlyTyrAlaVal 
859095 
GACCT GGGCAACATCTGGCGGTTTCCTTACATATGCTACCAGAATGGC454 
AspLeuGlyAsnIleTrpArgPheProTyrIleCysTyrGlnAsnGly 
100105110 
GGAGGGGCCT TCCTCCTCCCTTATACCATCATGGCCATTTTCGGGGGG502 
GlyGlyAlaPheLeuLeuProTyrThrIleMetAlaIlePheGlyGly 
115120125 
ATCCCGCTCTTTTACATG GAGCTCGCACTGGGCCAGTACCACCGAAAC550 
IleProLeuPheTyrMetGluLeuAlaLeuGlyGlnTyrHisArgAsn 
130135140145 
GGGTGCATTTCCATA TGGAGGAAGATCTGCCCGATTTTCAAAGGCATT598 
GlyCysIleSerIleTrpArgLysIleCysProIlePheLysGlyIle 
150155160 
GGTTACGCCATCTG CATCATCGCCTTTTACATCGCCTCCTACTACAAC646 
GlyTyrAlaIleCysIleIleAlaPheTyrIleAlaSerTyrTyrAsn 
165170175 
ACCATCATAGCCTGGG CGCTCTACTACCTCATCTCCTCCCTCACGGAC694 
ThrIleIleAlaTrpAlaLeuTyrTyrLeuIleSerSerLeuThrAsp 
180185190 
CGGCTGCCCTGGACCAGCTGC ACGAACTCCTGGAACACTGGCAACTGC742 
ArgLeuProTrpThrSerCysThrAsnSerTrpAsnThrGlyAsnCys 
195200205 
ACCAACTACTTCGCCCAGGACAACATCACC TGGACGCTGCATTCCACG790 
ThrAsnTyrPheAlaGlnAspAsnIleThrTrpThrLeuHisSerThr 
210215220225 
TCCCCCGCTGAGGAGTTCTACTTGCG CCATGTCCTGCAGATCCACCAG838 
SerProAlaGluGluPheTyrLeuArgHisValLeuGlnIleHisGln 
230235240 
TCTAAGGGACTCCAGGACCTGGGCA CCATCAGCTGGCAGCTGACTCTC886 
SerLysGlyLeuGlnAspLeuGlyThrIleSerTrpGlnLeuThrLeu 
245250255 
TGCATCGTGCTCATCTTCACCGTAATC TACTTTAGCATCTGGAAAGGC934 
CysIleValLeuIlePheThrValIleTyrPheSerIleTrpLysGly 
260265270 
GTCAAAACATCTGGCAAGGTGGTGTGGGTGACA GCCACCTTCCCATAC982 
ValLysThrSerGlyLysValValTrpValThrAlaThrPheProTyr 
275280285 
ATTGTCCTCTCTGTCCTGCTGGTGAGGGGGGCCACCCTTCC TGGAGCC1030 
IleValLeuSerValLeuLeuValArgGlyAlaThrLeuProGlyAla 
290295300305 
TGGAGAGGGGTCGTCTTCTACTTGAAACCCAACTGGC AGAAACTCTTG1078 
TrpArgGlyValValPheTyrLeuLysProAsnTrpGlnLysLeuLeu 
310315320 
GAGACAGGGGTGTGGGTAGATGCCGCCGCTCAGATC TTCTTCTCTCTT1126 
GluThrGlyValTrpValAspAlaAlaAlaGlnIlePhePheSerLeu 
325330335 
GGCCCGGGCTTTGGGGTTCTCCTGGCTTTTGCTAGCTAC AACAAGTTC1174 
GlyProGlyPheGlyValLeuLeuAlaPheAlaSerTyrAsnLysPhe 
340345350 
AACAACAACTGTTACCAAGATGCCCTGGTGACCAGTGTGGTGAA CTGC1222 
AsnAsnAsnCysTyrGlnAspAlaLeuValThrSerValValAsnCys 
355360365 
ATGACAAGCTTCGTCTCTGGCTTCGTCATCTTCACGGTGCTTGGCTAC1 270 
MetThrSerPheValSerGlyPheValIlePheThrValLeuGlyTyr 
370375380385 
ATGGCGGAGATGAGGAATGAAGATGTGTCAGAGGTGGCCAAAGACGCA 1318 
MetAlaGluMetArgAsnGluAspValSerGluValAlaLysAspAla 
390395400 
GGCCCCAGCCTCCTCTTCATCACGTATGCAGAGGCAATAGCCAACATG 1366 
GlyProSerLeuLeuPheIleThrTyrAlaGluAlaIleAlaAsnMet 
405410415 
CCAGCATCCACGTTCTTTGCCATCATCTTCTTCCTCATGTTAATCACG 1414 
ProAlaSerThrPhePheAlaIleIlePhePheLeuMetLeuIleThr 
420425430 
CTGGGATTGGACAGCACGTTCGCAGGCCTGGAAGGTGTGATCACAGCT1462 
LeuGlyLeuAspSerThrPheAlaGlyLeuGluGlyValIleThrAla 
435440445 
GTGCTGGATGAGTTCCCTCACATCTGGGCCAAGCGCAGGGAATGGTTC1510 
ValLeuAs pGluPheProHisIleTrpAlaLysArgArgGluTrpPhe 
450455460465 
GTGCTCATCGTGGTCATCACGTGCGTCTTGGGATCCCTGCTCACACTG1558 
ValL euIleValValIleThrCysValLeuGlySerLeuLeuThrLeu 
470475480 
ACGTCAGGAGGGGCATACGTGGTGACTCTGCTGGAGGAGTATGCCACG1606 
Thr SerGlyGlyAlaTyrValValThrLeuLeuGluGluTyrAlaThr 
485490495 
GGGCCAGCAGTGCTCACCGTGGCCCTCATCGAGGCCGTCGCCGTGTCT1654 
GlyPro AlaValLeuThrValAlaLeuIleGluAlaValAlaValSer 
500505510 
TGGTTCTATGGAATCACTCAGTTCTGCAGCGATGTGAAGGAGATGCTG1702 
TrpPheTyrGl yIleThrGlnPheCysSerAspValLysGluMetLeu 
515520525 
GGCTTCAGCCCGGGATGGTTTTGGAGGATCTGCTGGGTGGCCATCAGC1750 
GlyPheSerProGlyTrpP heTrpArgIleCysTrpValAlaIleSer 
530535540545 
CCTCTGTTTCTCCTGTTCATCATTTGCAGTTTTCTGATGAGCCCACCC1798 
ProLeuPheLeuLeu PheIleIleCysSerPheLeuMetSerProPro 
550555560 
CAGCTACGGCTTTTCCAATACAACTATCCCCACTGGAGTATCGTCTTG1846 
GlnLeuArgLeuPhe GlnTyrAsnTyrProHisTrpSerIleValLeu 
565570575 
GGCTACTGCATAGGGATGTCGTCCGTCATCTGCATCCCTACCTATATC1894 
GlyTyrCysIleGlyMe tSerSerValIleCysIleProThrTyrIle 
580585590 
ATTTATCGGCTGATCAGCACTCCGGGGACACTTAAGGAGCGCATTATT1942 
IleTyrArgLeuIleSerThrP roGlyThrLeuLysGluArgIleIle 
595600605 
AAAAGTATCACTCCTGAAACACCCACAGAAATCCCGTGTGGGGACATC1990 
LysSerIleThrProGluThrProThrGlu IleProCysGlyAspIle 
610615620625 
CGCATGAATGCTGTGTAACACACCCTGGGAGAGGACACCTCTTCCCAGCCACCTC2045 
ArgMetAsnAlaVal 
630 
TCTCAGCTCTGAAAAGCCCCACTGGACTCCTCCCCTCTAAGCCAAGCCTGATGAAGACAC2105 
GGTCCTAACCACTATGGTGCCCAGACTCTTGTGGATTCCGACCACTTCTTTCCGTGGACT2165 
CTCAGACATGCTACCACATTCGATGGTGACACCACTGAG CTGGCCTCTTGGACACGTCAG2225 
GGAGTGGAAGGAGGGATGAACGCCACCCAGTCATCAGCTAGCTTCAGGTTTAGAATTAGG2285 
TCTGTGAGAGTCTGTATCATGTTTTTGGTAAGATCATACTACCCCGCATCTGTTAGCTTC2345 
TAAAGCCTTCAATGTT CATGAATACATAAACCACCTAAGAGAAAACAGAGATGTCTTGCT2405 
AGCCATATAT2415 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 630 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetGluThrThrProLeuAsnSerGlnLysValLeuSerGluCysLys 
151015 
AspArgGluAspCysGlnGluAsn GlyValLeuGlnLysGlyValPro 
202530 
ThrThrAlaAspArgAlaGluProSerGlnIleSerAsnGlyTyrSer 
3540 45 
AlaValProSerThrSerAlaGlyAspGluAlaSerHisSerIlePro 
505560 
AlaAlaThrThrThrLeuValAlaGluIleArgGlnGlyGluArgGlu 
65 707580 
ThrTrpGlyLysLysMetAspPheLeuLeuSerValIleGlyTyrAla 
859095 
ValAspLeuGly AsnIleTrpArgPheProTyrIleCysTyrGlnAsn 
100105110 
GlyGlyGlyAlaPheLeuLeuProTyrThrIleMetAlaIlePheGly 
115 120125 
GlyIleProLeuPheTyrMetGluLeuAlaLeuGlyGlnTyrHisArg 
130135140 
AsnGlyCysIleSerIleTrpArgLysIleCysProIlePheLys Gly 
145150155160 
IleGlyTyrAlaIleCysIleIleAlaPheTyrIleAlaSerTyrTyr 
165170175 
A snThrIleIleAlaTrpAlaLeuTyrTyrLeuIleSerSerLeuThr 
180185190 
AspArgLeuProTrpThrSerCysThrAsnSerTrpAsnThrGlyAsn 
195 200205 
CysThrAsnTyrPheAlaGlnAspAsnIleThrTrpThrLeuHisSer 
210215220 
ThrSerProAlaGluGluPheTyrLeuArgHis ValLeuGlnIleHis 
225230235240 
GlnSerLysGlyLeuGlnAspLeuGlyThrIleSerTrpGlnLeuThr 
245250 255 
LeuCysIleValLeuIlePheThrValIleTyrPheSerIleTrpLys 
260265270 
GlyValLysThrSerGlyLysValValTrpValThrAlaThrPhePro 
275280285 
TyrIleValLeuSerValLeuLeuValArgGlyAlaThrLeuProGly 
290295300 
AlaTrpArgGlyValValPheT yrLeuLysProAsnTrpGlnLysLeu 
305310315320 
LeuGluThrGlyValTrpValAspAlaAlaAlaGlnIlePhePheSer 
325 330335 
LeuGlyProGlyPheGlyValLeuLeuAlaPheAlaSerTyrAsnLys 
340345350 
PheAsnAsnAsnCysTyrGlnAspAlaLeuValThr SerValValAsn 
355360365 
CysMetThrSerPheValSerGlyPheValIlePheThrValLeuGly 
370375380 
TyrMetAlaGl uMetArgAsnGluAspValSerGluValAlaLysAsp 
385390395400 
AlaGlyProSerLeuLeuPheIleThrTyrAlaGluAlaIleAlaAsn 
40 5410415 
MetProAlaSerThrPhePheAlaIleIlePhePheLeuMetLeuIle 
420425430 
ThrLeuGlyLeuAspSerThrPheA laGlyLeuGluGlyValIleThr 
435440445 
AlaValLeuAspGluPheProHisIleTrpAlaLysArgArgGluTrp 
450455460 
PheValLeuIleValValIleThrCysValLeuGlySerLeuLeuThr 
465470475480 
LeuThrSerGlyGlyAlaTyrValValThrLeuLeuGluGluTyrAla 
485490495 
ThrGlyProAlaValLeuThrValAlaLeuIleGluAlaValAlaVal 
500505510 
SerTrpPheTyrGl yIleThrGlnPheCysSerAspValLysGluMet 
515520525 
LeuGlyPheSerProGlyTrpPheTrpArgIleCysTrpValAlaIle 
530535 540 
SerProLeuPheLeuLeuPheIleIleCysSerPheLeuMetSerPro 
545550555560 
ProGlnLeuArgLeuPheGlnTyrAsnTyrProHisTrpS erIleVal 
565570575 
LeuGlyTyrCysIleGlyMetSerSerValIleCysIleProThrTyr 
580585590 
Ile IleTyrArgLeuIleSerThrProGlyThrLeuLysGluArgIle 
595600605 
IleLysSerIleThrProGluThrProThrGluIleProCysGlyAsp 
610 615620 
IleArgMetAsnAlaVal 
625630 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2508 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(viii) POSITION IN GENOME: 
(C) UNITS: 2278 basepairs 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 73..1962 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CAAATCCAAGCACCCAGAGATCAATTGGGATCCTTGGCAGATGGACATCAGTGTCATTTA60 
CTAACCAGCAGGATGGAGACGACGCCCTTGA ATTCTCAGAAGCAGCTA108 
MetGluThrThrProLeuAsnSerGlnLysGlnLeu 
1510 
TCAGCGTGTGAAGATGGAGAAGATTGTCAGGAAAA CGGAGTTCTACAG156 
SerAlaCysGluAspGlyGluAspCysGlnGluAsnGlyValLeuGln 
152025 
AAGGTTGTTCCCACCCCAGGGGACAAAGTGGAGTCCGGGC AAATATCC204 
LysValValProThrProGlyAspLysValGluSerGlyGlnIleSer 
303540 
AATGGGTACTCAGCAGTTCCAAGTCCTGGTGCGGGAGATGACACACGG 252 
AsnGlyTyrSerAlaValProSerProGlyAlaGlyAspAspThrArg 
45505560 
CACTCTATCCCAGCGACCACCACCACCCTAGTGGCTGAGCTTCAT CAA300 
HisSerIleProAlaThrThrThrThrLeuValAlaGluLeuHisGln 
657075 
GGGGAACGGGAGACCTGGGGCAAGAAGGTGGATTTCCTTCTCTC AGTG348 
GlyGluArgGluThrTrpGlyLysLysValAspPheLeuLeuSerVal 
808590 
ATTGGCTATGCTGTGGACCTGGGCAATGTCTGGCGCTTCCCCTACA TA396 
IleGlyTyrAlaValAspLeuGlyAsnValTrpArgPheProTyrIle 
95100105 
TGTTACCAGAATGGAGGGGGGGCATTCCTCCTCCCCTACACCATCATG 444 
CysTyrGlnAsnGlyGlyGlyAlaPheLeuLeuProTyrThrIleMet 
110115120 
GCCATTTTTGGGGGAATCCCGCTCTTTTACATGGAGCTCGCACTGGGA492 
AlaI lePheGlyGlyIleProLeuPheTyrMetGluLeuAlaLeuGly 
125130135140 
CAGTACCACCGAAATGGATGCATTTCAATATGGAGGAAAATCTGCCCG540 
GlnTyrHisArgAsnGlyCysIleSerIleTrpArgLysIleCysPro 
145150155 
ATTTTCAAAGGGATTGGTTATGCCATCTGCATCATTGCCTTTTACATT588 
IlePheLysGlyIleGlyTyrAlaIleCysIleIleAlaPheTyrIle 
160165170 
GCTTCCTACTACAACACCATCATGGCCTGGGCGCTATACTACCTCATC636 
Al aSerTyrTyrAsnThrIleMetAlaTrpAlaLeuTyrTyrLeuIle 
175180185 
TCCTCCTTCACGGACCAGCTGCCCTGGACCAGCTGCAAGAACTCCTGG684 
SerSerP heThrAspGlnLeuProTrpThrSerCysLysAsnSerTrp 
190195200 
AACACTGGCAACTGCACCAATTACTTCTCCGAGGACAACATCACCTGG732 
AsnThrGlyAsnCys ThrAsnTyrPheSerGluAspAsnIleThrTrp 
205210215220 
ACCCTCCATTCCACGTCCCCTGCTGAAGAATTTTACACGCGCCACGTC780 
ThrLeuHisSer ThrSerProAlaGluGluPheTyrThrArgHisVal 
225230235 
CTGCAGATCCACCGGTCTAAGGGGCTCCAGGACCTGGGGGGCATCAGC828 
LeuGlnIleHi sArgSerLysGlyLeuGlnAspLeuGlyGlyIleSer 
240245250 
TGGCAGCTGGCCCTCTGCATCATGCTGATCTTCACTGTTATCTACTTC876 
TrpGlnLeuAlaL euCysIleMetLeuIlePheThrValIleTyrPhe 
255260265 
AGCATCTGGAAAGGCGTCAAGACCTCTGGCAAGGTGGTGTGGGTGACA924 
SerIleTrpLysGlyVal LysThrSerGlyLysValValTrpValThr 
270275280 
GCCACCTTCCCTTATATCATCCTTTCTGTCCTGCTGGTGAGGGGTGCC972 
AlaThrPheProTyrIleIleLeuSer ValLeuLeuValArgGlyAla 
285290295300 
ACCCTCCCTGGAGCCTGGAGGGGTGTTCTCTTCTACTTGAAACCCAAT1020 
ThrLeuProGlyAlaTrpArgGl yValLeuPheTyrLeuLysProAsn 
305310315 
TGGCAGAAACTCCTGGAGACAGGGGTGTGGATAGATGCAGCCGCTCAG1068 
TrpGlnLysLeuLeuGluThrG lyValTrpIleAspAlaAlaAlaGln 
320325330 
ATCTTCTTCTCTCTTGGTCCGGGCTTTGGGGTCCTGCTGGCTTTTGCT1116 
IlePhePheSerLeuGlyProGly PheGlyValLeuLeuAlaPheAla 
335340345 
AGCTACAACAAGTTCAACAACAACTGCTACCAAGATGCCCTGGTGACC1164 
SerTyrAsnLysPheAsnAsnAsnCysTyr GlnAspAlaLeuValThr 
350355360 
AGCGTGGTGAACTGCATGACGAGCTTCGTTTCGGGATTTGTCATCTTC1212 
SerValValAsnCysMetThrSerPheValSerGlyPh eValIlePhe 
365370375380 
ACAGTGCTCGGTTACATGGCTGAGATGAGGAATGAAGATGTGTCTGAG1260 
ThrValLeuGlyTyrMetAlaGluMetArgAsnG luAspValSerGlu 
385390395 
GTGGCCAAAGACGCAGGTCCCAGCCTCCTCTTCATCACGTATGCAGAA1308 
ValAlaLysAspAlaGlyProSerLeuLeuPhe IleThrTyrAlaGlu 
400405410 
GCGATAGCCAACATGCCAGCGTCCACTTTCTTTGCCATCATCTTCTTT1356 
AlaIleAlaAsnMetProAlaSerThrPhePheAla IleIlePhePhe 
415420425 
CTGATGTTAATCACGCTGGGCTTGGACAGCACGTTTGCAGGCTTGGAG1404 
LeuMetLeuIleThrLeuGlyLeuAspSerThrPheAlaGl yLeuGlu 
430435440 
GGGGTGATCACGGCTGTGCTGGATGAGTTCCCACACGTCTGGGCCAAG1452 
GlyValIleThrAlaValLeuAspGluPheProHisValTrpAlaLys 
445450455460 
CGCCGGGAGCGGTTCGTGCTCGCCGTGGTCATCACCTGCTTCTTTGGA1500 
ArgArgGluArgPheValLeuAlaValValIleThrCysPhePhe Gly 
465470475 
TCCCTGGTCACCCTGACTTTTGGAGGGGCCTACGTGGTGAAGCTGCTG1548 
SerLeuValThrLeuThrPheGlyGlyAlaTyrValValLysLeu Leu 
480485490 
GAGGAGTATGCCACGGGGCCCGCAGTGCTCACTGTCGCGCTGATCGAA1596 
GluGluTyrAlaThrGlyProAlaValLeuThrValAlaLeuIleGl u 
495500505 
GCAGTCGCTGTGTCTTGGTTCTATGGCATCACTCAGTTCTGCAGGGAC1644 
AlaValAlaValSerTrpPheTyrGlyIleThrGlnPheCysArgAsp 
510515520 
GTGAAGGAAATGCTCGGCTTCAGCCCGGGGTGGTTCTGGAGGATCTGC1692 
ValLysGluMetLeuGlyPheSerProGlyTrpPheTrpArgIleCys 
525 530535540 
TGGGTGGCCATCAGCCCTCTGTTTCTCCTGTTCATCATTTGCAGTTTT1740 
TrpValAlaIleSerProLeuPheLeuLeuPheIleIleCysSerPhe 
545550555 
CTGATGAGCCCGCCACAACTACGACTTTTCCAATATAATTATCCTTAC1788 
LeuMetSerProProGlnLeuArgLeuPheGlnTyrAsnTyrProTyr 
560565570 
TGGAGTATCATCTTGGGTTACTGCATAGGAACCTCATCTTTCATTTGC1836 
TrpSerIleIleLeuGlyTyrCysIleGlyThrSerSerPheIleCys 
575 580585 
ATCCCCACATATATAGCTTATCGGTTGATCATCACTCCAGGGACATTT1884 
IleProThrTyrIleAlaTyrArgLeuIleIleThrProGlyThrPhe 
590 595600 
AAAGAGCGTATTATTAAAAGTATTACCCCAGAAACACCAACAGAAATT1932 
LysGluArgIleIleLysSerIleThrProGluThrProThrGluIle 
605610 615620 
CCTTGTGGGGACATCCGCTTGAATGCTGTGTAACACACTCACCGAGAGGA1982 
ProCysGlyAspIleArgLeuAsnAlaVal 
625630 
AAAAGGC TTCTCCACAACCTCCTCCTCCAGTTCTGATGAGGCACGCCTGCCTTCTCCCCT2042 
CCAAGTGAATGAGTTTCCAGCTAAGCCTGATGATGGAAGGGCCTTCTCCACAGGGACACA2102 
GTCTGGTGCCCAGACTCAAGGCCTCCAGCCACTTATTTCCATGGATTCCCCT GGACATAT2162 
TCCCATGGTAGACTGTGACACAGCTGAGCTGGCCTATTTTGGACGTGTGAGGATGTGGAT2222 
GGAGGTGATGAAAACCACCCTATCATCAGTTAGGATTAGGTTTAGAATCAAGTCTGTGAA2282 
AGTCTCCTGTATCATTTCTTGGTATGATCA TTGGTATCTGATATCTGTTTGCTTCTAAAG2342 
GTTTCACTGTTCATGAATACGTAAACTGCGTAGGAGAGAACAGGGATGCTATCTCGCTAG2402 
CCATATATTTTCTGAGTAGCATATAGAATTTTATTGCTGGAATCTACTAGAACCTTCTAA2462 
TCCATGT GCTGCTGTGGCATCAGGAAAGGAAGATGTAAGAAGCTAA2508 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 630 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetGluThrThrProLeu AsnSerGlnLysGlnLeuSerAlaCysGlu 
151015 
AspGlyGluAspCysGlnGluAsnGlyValLeuGlnLysValValPro 
20 2530 
ThrProGlyAspLysValGluSerGlyGlnIleSerAsnGlyTyrSer 
354045 
AlaValProSerProGlyAlaGlyAspAspThrArgHisSe rIlePro 
505560 
AlaThrThrThrThrLeuValAlaGluLeuHisGlnGlyGluArgGlu 
65707580 
ThrTrp GlyLysLysValAspPheLeuLeuSerValIleGlyTyrAla 
859095 
ValAspLeuGlyAsnValTrpArgPheProTyrIleCysTyrGlnAsn 
100 105110 
GlyGlyGlyAlaPheLeuLeuProTyrThrIleMetAlaIlePheGly 
115120125 
GlyIleProLeuPheTyrMetGluLeuAla LeuGlyGlnTyrHisArg 
130135140 
AsnGlyCysIleSerIleTrpArgLysIleCysProIlePheLysGly 
145150155 160 
IleGlyTyrAlaIleCysIleIleAlaPheTyrIleAlaSerTyrTyr 
165170175 
AsnThrIleMetAlaTrpAlaLeuTyrTyrLeuIleSerSerPheThr 
180185190 
AspGlnLeuProTrpThrSerCysLysAsnSerTrpAsnThrGlyAsn 
195200205 
CysThrAsnTyrPheSer GluAspAsnIleThrTrpThrLeuHisSer 
210215220 
ThrSerProAlaGluGluPheTyrThrArgHisValLeuGlnIleHis 
225230235 240 
ArgSerLysGlyLeuGlnAspLeuGlyGlyIleSerTrpGlnLeuAla 
245250255 
LeuCysIleMetLeuIlePheThrValIleTyrPheSer IleTrpLys 
260265270 
GlyValLysThrSerGlyLysValValTrpValThrAlaThrPhePro 
275280285 
TyrIleI leLeuSerValLeuLeuValArgGlyAlaThrLeuProGly 
290295300 
AlaTrpArgGlyValLeuPheTyrLeuLysProAsnTrpGlnLysLeu 
305310 315320 
LeuGluThrGlyValTrpIleAspAlaAlaAlaGlnIlePhePheSer 
325330335 
LeuGlyProGlyPheGlyValLeuLeu AlaPheAlaSerTyrAsnLys 
340345350 
PheAsnAsnAsnCysTyrGlnAspAlaLeuValThrSerValValAsn 
355360 365 
CysMetThrSerPheValSerGlyPheValIlePheThrValLeuGly 
370375380 
TyrMetAlaGluMetArgAsnGluAspValSerGluValAlaLysAsp 
385 390395400 
AlaGlyProSerLeuLeuPheIleThrTyrAlaGluAlaIleAlaAsn 
405410415 
MetProAlaSerThrP hePheAlaIleIlePhePheLeuMetLeuIle 
420425430 
ThrLeuGlyLeuAspSerThrPheAlaGlyLeuGluGlyValIleThr 
435440 445 
AlaValLeuAspGluPheProHisValTrpAlaLysArgArgGluArg 
450455460 
PheValLeuAlaValValIleThrCysPhePheGlySerLeuValThr 
465470475480 
LeuThrPheGlyGlyAlaTyrValValLysLeuLeuGluGluTyrAla 
485490495 
ThrGl yProAlaValLeuThrValAlaLeuIleGluAlaValAlaVal 
500505510 
SerTrpPheTyrGlyIleThrGlnPheCysArgAspValLysGluMet 
515 520525 
LeuGlyPheSerProGlyTrpPheTrpArgIleCysTrpValAlaIle 
530535540 
SerProLeuPheLeuLeuPheIleIleCysSerPheL euMetSerPro 
545550555560 
ProGlnLeuArgLeuPheGlnTyrAsnTyrProTyrTrpSerIleIle 
565570 575 
LeuGlyTyrCysIleGlyThrSerSerPheIleCysIleProThrTyr 
580585590 
IleAlaTyrArgLeuIleIleThrProGlyThrPheLysGluArgIle 
595600605 
IleLysSerIleThrProGluThrProThrGluIleProCysGlyAsp 
610615620 
IleArgLeuAsnAlaVal 
625 630 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AAAGGCAATGATGCAGATGGC 21