Purified DNA polymerase from bacillus stearothermophilus ATCC 12980

Composition and methods for the expression of recombinant DNA polymerase enzymes derived from Bacillus stearothermophilus. The present invention also concerns methods for purifying recombinant Bst DNA polymerase enzymes compositions containing the purified enzymes in a form suitable for conducting biochemical reactions, and methods for using the purified enzymes.

FIELD OF THE INVENTION 
The invention relates to purified thermostable DNA polymerase enzymes 
derived from the Gram-positive bacterium Bacillus stearothermophilus. 
These enzymes are useful in biochemical procedures requiring the 
template-directed synthesis of a nucleic acid strand, such as sequencing 
and nucleic acid amplification procedures. The invention also relates to 
methods of making and using these enzymes. 
BACKGROUND OF THE INVENTION 
DNA polymerase enzymes are naturally-occurring intracellular enzymes, and 
are used by a cell to replicate a nucleic acid strand using a template 
molecule to manufacture a complementary nucleic acid strand. Enzymes 
having DNA polymerase activity catalyze the formation of a bond between 
the 3' hydroxyl group at the growing end of a nucleic acid primer and the 
5' phosphate group of a nucleotide triphosphate. These nucleotide 
triphosphates are usually selected from deoxyadenosine triphosphate (A), 
deoxythymidine triphosphate (T), deoxycytidine triphosphate (C) and 
deoxyguanosine triphosphate (G). However, DNA polymerases may incorporate 
modified or altered versions of these nucleotides. The order in which the 
nucleotides are added is dictated by base pairing to a DNA template 
strand; such base pairing is accomplished through "canonical" 
hydrogen-bonding (hydrogen-bonding between A and T nucleotides and G and C 
nucleotides of opposing DNA strands), although non-canonical base pairing, 
such as G:U base pairing, is known in the art. See e.g., Adams et al., The 
Biochemistry of the Nucleic Acids 14-32 (11th ed. 1992). 
The in-vitro use of enzymes having DNA polymerase activity has in recent 
years become more common in a variety of biochemical applications 
including cDNA synthesis and DNA sequencing reactions (see Sambrook e al., 
(2nd ed. Cold Spring Harbor Laboratory Press, 1989) hereby incorporated by 
reference herein), and amplification of nucleic acids by methods such as 
the polymerase chain reaction (PCR) (Mullis et al., U.S. Pat. Nos. 
4,683,195, 4,683,202, and 4,800,159, hereby incorporated by reference 
herein) and RNA transcription-mediated amplification methods (e.a., Kacian 
et al., PCT Publication No. WO91/01384 which enjoys common ownership with 
the present application and is hereby incorporated by reference herein). 
Methods such as PCR make use of cycles of primer extension through the use 
of a DNA polymerase activity, followed by thermal denaturation of the 
resulting double-stranded nucleic acid in order to provide a new template 
for another round of primer annealing and extension. Because the high 
temperatures necessary for strand denaturation result in the irreversible 
inactivations of many DNA polymerases, the discovery and use of DNA 
polymerases able to remain active at temperatures above about 37.degree. 
C. to 42.degree. C. (thermostable DNA polymerase enzymes) provides an 
advantage in cost and labor efficiency. Thermostable DNA polymerases have 
been discovered in a number of thermophilic organisms including, but not 
limited to Thermus aquaticus, Thermus thermophilus, and species of the 
Bacillus, Thermococcus, Sulfobus, Pyrococcus genera. DNA polymerases can 
be purified directly from these thermophilic organisms. However, 
substantial increases in the yield of DNA polymerase can be obtained by 
first cloning the gene encoding the enzyme in a multicopy expression 
vector by recombinant DNA technology methods, inserting the vector into a 
host cell strain capable of expressing the enzyme, culturing the 
vector-containing host cells, then extracting the DNA polymerase from a 
host cell strain which has expressed the enzyme. 
The bacterial DNA polymerases that have been characterized to date have 
certain patterns of similarities and differences which has led some to 
divide these enzymes into two groups: those whose genes contain 
introns--intervening non-coding nucleotide sequences--(Class B DNA 
polymerases), and those whose DNA polymerase genes are roughly similar to 
that of E. coli DNA polymerase I and do not contain introns (Class A DNA 
polymerases). 
By "non-coding" is meant that the nucleotides comprising both nucleic acid 
strands in such sequences do not contain 3-nucleotide codons that encode 
and correspond to amino acid residues of the mature protein. Introns are 
most often found in the genes of eukaryotic higher organisms but have also 
been found in lower organisms such as archaebacteria. 
Several Class A and Class B thermostable DNA polymerases derived from 
thermophilic organisms have been cloned and expressed. Among the class A 
enzymes: Lawyer, et al., J. Biol. Chem. 264:6427-6437 (1989) and Gelfund 
et al, U.S. Pat. No. 5,079,352, report the cloning and expression of a 
full length thermostable DNA polymerase derived from Thermus aquaticus 
(Taq). Lawyer et al., in PCR Methods and Applications, 2:275-287 (1993), 
and Barnes, PCT Publication No. WO92/06188 (1992), disclose the cloning 
and expression of truncated versions of the same DNA polymerase, while 
Sullivan, EPO Publication No. 0482714A1 (1992), reports cloning a mutated 
version of the Taq DNA polymerase. Asakura et al., J. Ferment. Bioeng. 
(Japan), 74:265-269 (1993) have reportedly cloned and expressed a DNA 
polymerase from Thermus thermophilus. Gelfund et al., PCT Publication No. 
WO92/06202 (1992), have disclosed a purified thermostable DNA polymerase 
from Thermosipho africanus. A thermostable DNA polymerase from Thermus 
flavus was reported by Akhmetzjanov and Vakhitov, Nucleic Acids Res., 
20:5839 (1992). Uemori et al., J. Biochem. 113:401-410 (1993) and EPO 
Publication No. 0517418A2 (1992) have reported cloning and expressing a 
DNA polymerase from the thermophilic bacterium Bacillus caldotenax. Ishino 
et al., Japanese Patent Application No. HEI 4[1992]-131400 (publication 
date Nov. 19, 1993) report cloning a DNA polymerase from Bacillus 
stearothermophilus. 
Among the Class B enzymes: A recombinant thermostable DNA polymerase from 
Thermococcus litoralis was reported by Comb et al., EPO Publication No. 0 
455 430 A3 (1991), Comb et al., EPO Publication No. 0547920A2 (1993), and 
Perler et al., Proc. Natl. Acad. Sci. (USA), 89:5577-5581 (1992). A cloned 
thermostable DNA polymerase from Sulfolobus solofatarius is disclosed in 
Pisani et al., Nucleic Acids Res. 20:2711-2716 (1992) and in PCT 
Publication WO93/25691 (1993). The thermostable enzyme of Pyrococcus 
furiosus is disclosed in Uemori et al., Nucleic Acids Res., 21:259-265 
(1993), while a recombinant DNA polymerase was derived from Pyrococcus sp. 
as disclosed in Comb et al., EPO Publication No. 0547359A1 (1993). 
By "thermostable" is meant that the enzyme remains has an optimal 
temperature of activity at a temperature greater than about 37.degree. C. 
to 42.degree. C. Preferrably, the enzymes of the present invention have an 
optimal temperature for activity of between about 50.degree. C. and 
75.degree. C.; most preferably between 55.degree. C. and 70.degree. C., 
and most preferably between 60.degree. C. and 65.degree. C. 
Many thermostable DNA polymerases possess activities additional to a DNA 
polymerase activity; these may include a 5'-3' exonuclease activity and/or 
a 3'-5' exonuclease activity. The activities of 5'-3' and 3'-5' 
exonucleases are well known to those of ordinary skill in the art. The 
3'-5' exonuclease activity improves the accuracy of the newly-synthesized 
strand by removing incorrect bases that may have been incorporated; DNA 
polymerases in which such activity is low or absent, reportedly including 
Taq DNA polymerase, (see Lawyer et al., J. Biol Chem. 264:6427-6437), are 
prone to errors in the incorporation of nucleotide residues into the 
primer extension strand. In applications such as nucleic acid 
amplification procedures in which the replication of DNA is often 
geometric in relation to the number of primer extension cycles, such 
errors can lead to serious artifactual problems such as sequence 
heterogeneity of the nucleic acid amplification product (amplicon). Thus, 
a 3'-5' exonuclease activity is a desired characteristic of a thermostable 
DNA polymerase used for such purposes. 
By contrast, the 5'-3' exonuclease activity often present in DNA polymerase 
enzymes is often undesired in a particular application since it may digest 
nucleic acids, including primers, that have an unprotected 5' end. Thus, a 
thermostable DNA polymerase with an attenuated 5'-3' exonuclease activity, 
or in which such activity is absent, is also a desired characteristic of 
an enzyme for biochemical applications. Various DNA polymerase enzymes 
have been described where a modification has been introduced in a DNA 
polymerase which accomplishes this object. For example, the Klenow 
fragment of E. coli DNA polymerase I can be produced as a proteolytic 
fragment of the holoenzyme in which the domain of the protein controlling 
the 5'-3' exonuclease activity has been removed. The Klenow fragment still 
retains the polymerase activity and the 3'-5' exonuclease activity. 
Barnes, supra, and Gelfund et al., U.S. Pat. No. 5,079,352 have produced 
5'-3' exonuclease-deficient recombinant Taq DNA polymerases. Ishino et 
al., EPO Publication No. 0517418A2, have produced a 5'-3' 
exonuclease-deficient DNA polymerase derived from Bacillus caldotenax. 
Preparation of antisera or moloclonal antibodies to particular DNA 
polymerase enzymes has been described and is well known in the art. For 
example, Hu et al., J. Virol. 60:267-274 (1986) report specific 
immunoprecipiation of cloned reverse transcriptase and fusion proteins 
from Moloney Murine Leukemia Virus expressed in E. coli by recovering 
PAGE-separated MMLV reverse transcriptase from the gel, immunizing rabbits 
with the purified protein, and recovering the antisera. Livingston et al., 
Virology 50:388-395 (1972) disclose affinity chromatography of Avian Type 
C Viral transcriptase using antibodies able to differentiate between viral 
and host cell DNA polymerases. Spadari and Weissbach, J. Biol. Chem. 
249:5809-5815 (1974) report that HeLa-derived DNA polymerase is not 
inhibited by antisera prepared against reverse transcriptases obtained 
from either the Mason-Pfizer monkey virus, the Wooley monkey virus, or the 
Rauscher murine leukemia virus. These publications are hereby incorporated 
herein by reference. 
SUMMARY OF THE INVENTION 
The present invention provides recombinant and/or purified thermostable DNA 
polymerase enzymes from Bacillus stearothermophilus (Bst). One or more of 
the enzymes of the present invention may be produced and purified from a 
culture of Bacillus stearothermophilus or the genes encoding these enzymes 
may be cloned into a suitable expression vector, expressed in a 
heterologous host and purified. Among the DNA polymerase enzymes disclosed 
herein are mutated or truncated forms of the native enzyme which contain 
deletions in the 5'-3' exonuclease domain of the enzyme and/or its 
corresonding gene. 
These enzymes may be used in nucleic acid amplification methods and other 
biochemical protocols that require a DNA polymerase activity. Furthermore, 
because the enzymes provided herein are thermostable, they are suitable 
for use in biochemical applications using higher temperatures than many 
other DNA polymerase enzymes, such as the Klenow fragment from E. coli DNA 
polymerase I. As permitted by the particular biochemical application, the 
enzymes provided herein may be used in an unpurified form. Alternatively, 
these enzymes may be purified prior to use. 
Accordingly, the present invention also provides methods for the 
purification and use of Bst DNA polymerase enzymes. A preferred method of 
purification of the Bst DNA polymerases comprises two anion-exchange steps 
and phosphocellulose chromatography. Preferred chromatography conditions 
are described herein. However, it will be appreciated that variation of 
these conditions or their order would be apparent to one of skill in the 
art in light of the present disclosure. 
Additionally, the present invention provides compositions comprising DNA 
fragments containing the genes encoding the enzymes of the present 
invention, vectors containing these genes, and methods of producing these 
recombinant enzymes. 
The invention also encompasses a stable enzyme formulation comprising one 
or more of the DNA polymerase enzymes of the present invention in a buffer 
containing stabilizing agents. 
Both the full length Bst polymerase enzyme and the variants thereof 
described and claimed herein may be cloned as a single uninterrupted gene 
on a multicopy vector in an E. coli host strain without being lethal to 
the host cell or under the control of a strong repressor. Moreover, the 
Bst polymerases may be expressed constitutively within the E. coli host 
cell; inducible expression of these enzymes, while possible, is not 
necessary to obtain a high yield of active enzyme.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention relates to purified DNA polymerase enzymes derived 
from Bacillus stearothermophilus, DNA fragments encoding said enzymes for 
expression in a heterologous host cell, and methods of their production, 
purification and use. These enzymes are useful in biochemical 
applications, such as nucleic acid sequencing and amplification, including 
transcription based amplification systems. Preferably, the enzymes of the 
present invention are optimally active at temperatures above about 
37.degree. C. to 42.degree. C., and are thus suitable for biochemical 
applications that require a relatively high temperature of reaction. Most 
preferably, the enzymes of the present invention are optimally active at a 
temperature of about 60.degree. C. to 65.degree. C. 
The enzymes of the present invention have an amino acid sequence that bears 
some resemblance to DNA polymerase enzymes of the Class A designation, of 
which the non-thermostable E. coli DNA polymerase I is a member. A 
comparison of the amino acid sequences of the Class A DNA polymerases 
reveals regions of relative sequence homology seperated by a number of 
reasonably well-defined "variable" regions. By variable regions is meant 
that a comparison of the amino acid sequences in these regions reveals 
that about 10% or more of the contiguous amino acid residues within a 
given region of the compared DNA polymerase sequences are different. For 
this purpose, a region is defined as 20 or more contiguous amino acid 
residues. 
Likewise, a comparison of the nucleotide sequences of the genes encoding 
the Class A DNA polymerases reveal regions in which the nucleotide 
sequences are highly conserved between species, and other, variable 
regions. Because of the degeneracy of the genetic code, the amount of 
nucleotide sequence variability may be greater than the amount of amino 
acid variability between the corresponding proteins, expressed as a 
percentage. Alternatively, because each amino acid is encoded by three 
nucleotide residues and a change in one of them may result in a codon 
corresponding to a different amino acid, the amount of nucleotide sequence 
variability in the genes encoding these enzymes may be less than that of 
the corresponding amino acid sequence on a percentage basis. 
Expression of recombinant proteins in RNase-deficient cells and the use of 
tetracycline-resistance as a selectable marker gene have been described in 
published European Patent Application 688,870. 
DEFINITIONS 
As used herein the following terms have the indicated meanings unless 
expressly indicated otherwise. 
By "selectable marker gene" is meant a DNA fragment encoding a gene which, 
when carried and expressed by a host cell, is capable of conferring a 
growth advantage to that host cell as compared to cells not containing the 
selectable marker gene when both are grown in a culture media of a given 
composition. For example, the gene encoding .beta.-lactamase will confer 
resistance to ampicillin on host cells containing this gene, whereas cells 
not containing the gene will be sensitive to ampicillin; thus only cells 
expressing the gene for .beta.-lactamase will grow in media containing 
ampicillin. Similarly, cells unable to synthesize an essential amino acid 
will not grow in media not containing that amino acid, whereas cells 
containing a gene allowing the cell to make the essential amino acid will 
grow in the same media. 
A selectable marker gene may be covalently linked, for example in a plasmid 
or expression vector, to one or more other gene or genetic element as a 
means of identifying cells containing both the selectable gene and the 
"silent" gene(s) and/or genetic element(s). 
By a "purified" nucleic acid or protein is meant a nucleic acid or protein 
subjected to at least one step which removes cellular components such as 
carbohydrates, lipids, unwanted nucleic acids, or unwanted proteins from 
the indicated nucleic acid or protein. 
By "upstream" is meant to the 5' side of a given locus on a nucleic acid 
strand, or in the case of a double stranded nucleic acid molecule, to the 
5' side of a particular locus with respect to the direction of gene 
transcription in that region of the nucleic acid molecule. 
By "downstream" is meant to the 3' side of a given locus on a nucleic acid 
strand, or in the case of a double stranded nucleic acid molecule, to the 
3' side of a particular locus with respect to the direction of gene 
transcription in that region of the nucleic acid molecule. 
By "T.sub.m " is meant the temperature at which 50% of a population of a 
double-stranded nucleic acid molecules, or nucleic acid molecules having a 
double-stranded region, become single-stranded or thermally denatured. 
By "recombinant" is meant that a nucleic acid molecule or protein is at 
least partially the result of in vitro biochemical techniques. A 
"recombinant DNA molecule" is thus a non-naturally occurring molecule. 
Such recombinant molecules include, but are not limited to molecules which 
comprise restriction endonuclease fragments, in vitro nucleic acid 
ligation products, in vitro exonuclease fragments, and expression vectors 
comprising heterologous genetic elements such as one or more of the 
following: promoters, repressor genes, selectable marker genes, 
temperature-sensitive DNA replication elements, structural genes, and the 
like. 
"Recombinant" proteins or enzymes are those not found in nature. These 
include purified protein preparations and proteins produced from 
recombinant DNA molecules. The latter proteins are usually expressed in a 
heterologous host cell, i.e., one not native to the protein or enzyme in 
question. However, the gene encoding a recombinant protein may reside on 
an expression vector contained within a host cell of the same species as 
the organism from which the protein in question was derived. 
By "truncated" is meant a smaller version of the gene or protein in 
question; with respect to the primary nucleotide or amino acid sequence, a 
truncated form of a reference nucleic acid or protein is one that lacks 
one or more nucleotides or amino acids as compared to the reference 
molecule. 
By "substantial sequence homology" is meant that a first nucleic acid or 
protein molecule has a recognizably non-random similarity to a second 
reference nucleic acid or protein over at least about 89% of its 
nucleotide or amino acid sequence respectively. 
By a nucleic acid or protein "domain" is meant at least one definite region 
of contiguous nucleotide or amino acid residues. 
By "origin of replication" is meant a specific region of DNA at which 
primer production and initiation of DNA polymerase activity begins. In 
this specification, the term is used to mean a nucleic acid element 
present on a DNA expression vector that allows the expression vector to 
increase in copy number within a given host cell. 
By "promoter" is meant a genetic element comprising a specific region of 
DNA at which an RNA polymerase enzyme can bind and begin transcription of 
a DNA template, thus providing the first step of translating the genetic 
information contained in the sequence of a nucleic acid into the 
production of a protein of an amino acid sequence corresponding to that 
nucleic acid sequence. 
By "expression", "gene expression" or "protein expression" is meant the 
production of protein from information contained within a gene by a host 
organism. 
By "transformation" is meant a biochemical method of inducing a host cell 
to internalize a nucleic acid molecule. Such nucleic acid molecules are 
usually genetic elements comprising at least an origin of replication, a 
selectable marker gene, and a promoter for expression of the selectable 
marker gene within the host cell. 
By "heterologous" is meant not of the same species. Thus, an enzyme 
expressed in a heterologous host cell is produced in a host cell of a 
different species than the one from which the enzyme was originally 
derived. 
By "gene" is meant a nucleic acid region having a nucleotide sequence that 
encodes an expressible protein or polypeptide. A gene may comprise one or 
more "coding sequences" containing codons that correspond to amino acid 
residues of the expressed protein; the gene may also comprise, but need 
not comprise, one or more "non-coding" nucleotide sequence regions that do 
not contain codons corresponding to amino acid residues of the expressed 
protein. 
MATERIALS AND METHODS 
Sources of Bacterial Strains, Plasmids and Enzymes 
The Bacillus stearothermophilus (Bst) ATCC type strain number 12980 was 
obtained from the American Type Culture Collection, Rockville, Md. Three 
strains of the bacterium Escherichia coli were used as host cells for 
cloning and expression of the Bst DNA polymerase enzymes of the present 
invention: E. coli strains XL1-Blue MRF' and JM109 were obtained from 
Stratagene Cloning Systems (San Diego, Calif.), and E. coli strain 1200 
(CGSC strain #4449) was obtained from the E. coli Genetic Stock Center 
(Yale University, New Haven, Conn.). 
Plasmid vector pUC 18 was obtained from Life Technologies Inc. 
(Gaithersburg, Md.), and vector pGem 3Z was obtained from Promega Corp. 
(Madison, Wis.). All restriction endonucleases and nucleic acid modifying 
enzymes, such as T4 DNA ligases, the Klenow fragment from E. coli DNA 
polymerase I, thermostable DNA polymerase, and polynucleotide kinase were 
obtained from commercial suppliers and were used in accordance with the 
manufaturer's instructions unless stated otherwise. 
Bacterial Cultures 
Bacillus stearothermophilus and E. coli cultures were grown in 1% (w/v) 
tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) sodium chloride (LB 
broth) or on Petri plates of the same solution containing 1.3% (w/v) agar 
(LB agar). When required and as indicated in the following disclosure, 
ampicillin was used at a concentration of 100 .mu.g/ml, tetracycline at a 
concentration of 12 .mu.g/ml, isopropylthio-.beta.-galactoside (IPTG) at a 
concentration of 0.5 mM, and 
5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside (X-gal) at 50 .mu.g/ml. B. 
stearothermophilus cultures were incubated at 55.degree. C. and E. coli 
cultures were incubated at 37.degree. C., both with shaking to aerate. 
DNA Preparations 
Plasmid DNA preparations were done by one of two methods as indicated in 
the following disclosure. The first is a standard boiling method for 
plasmid minipreparations as described in Sambrook et al., supra at page 
1.29, previously incorporated by reference herein. The second method 
utilized the Qiagen Plasmid Kit available from Qiagen Inc. (Chatsworth, 
Calif.) which was used for preparing purified DNA. This method makes use 
of a proprietary anion exchange resin and a series of proprietary elution 
buffers to prepare plasmid DNA without the need for CsCl gradients. The 
method is described in the Oiagen Plasmid Handbook for Plasmid Midi Kit 
and Plasmid Maxi Kit .COPYRGT.1992 Diagen GmbH, Qiagen, Inc. For 
preparations of B. stearothermophilus genomic DNA, overnight cultures of 
cells were centrifuged and the pellet was resuspended in 1/50 the original 
volume of 10 mM Tris-HCl, 100 mM NaCl and 5 mM ethylenediamine tetraacetic 
acid (EDTA) (pH 7.0). Lysozyme was added to a final concentration of 2 
mg/ml, and the suspension was incubated at 37.degree. C. for 20 minutes. 
Nine volumes of a solution containing 10 mM Tris-Cl (pH 8.0), 250 mM NaCl, 
1.2% (v/v) Triton X-100, 100 .mu.g/ml RNase A, 12 mM EDTA and 0.5 M 
guanidine-HCl were added to the cell suspension and the mixture was 
incubated on ice for 20 minutes. The mixture was made 2 mg/ml in 
proteinase K and incubated at 50.degree. C. for 2 hours with gentle 
shaking. The solution was then centrifuged at 15-20,000.times.g for 10 
minutes and the supernatant decanted off. Bst genomic DNA was then 
prepared using a variation of the Qiagen method described above for the 
recovery of plasmid DNA; other methods of preparing genomic DNA from 
cleared cell lysates are well known to those of ordinary skill in the art. 
Probe Labeling 
Single-stranded DNA oligomer probes were labeled by one of two methods as 
indicated in the following disclosure. The first method was by utilizing 
T4 polynucleotide kinase to label the 5' end of an oligonucleotide with 
radioactive .sup.32 P, as exemplified in Sambrook et al., supra, at page 
10.60, previously incorporated by reference. Other methods of labeling 
probes with radioactive atoms are well known to those of ordinary skill in 
the art. This protocol was used to label oligonucleotides 16, 24 and 25. 
The second labeling method utilized was the LIGHTSMITH.TM. 
chemiluminescent system (high stringency) obtained from Promega Corp., 
which was used to label oligonucleotides 15, 21 and 20 (SEQ ID NO:13, SEQ 
ID NO:9 and SEQ ID NO:11, respectively). This method makes use of 
non-radioactive labels and is thus generally more convenient than using 
.sup.32 P or other radionuclides for detection. However, oligonucleotides 
15, 20 and 21 may easily be labelled with radioactive atoms as described 
above with no loss in detection ability. 
Gel Electrophoresis and Gel Isolation of DNA Fragments 
Unless indicated otherwise, agarose gels were 1% (w/v) agarose (Life 
Technologies Inc.). The agarose gels were run in 1.times. TAE buffer (40 
mM Tris base (pH 8.0), 20 mM sodium acetate, 2 mM EDTA) containing 2 
.mu.g/ml ethidium bromide. To gel purify DNA fragments, agarose gel slices 
containing the desired fragments were excised and frozen on dry ice. The 
gel slices were then thawed, crushed and extracted with TAE-saturated 
phenol. Following a brief centrifugation, the aqueous phases were 
collected and extracted with a solution of 50%. (v/v) TE-(10 mM Tris (pH 
8.0) and 1 mM EDTA) saturated phenol, 49% (v/v) chloroform and 1% (v/v/) 
isoamyl alcohol. This was followed by extraction of the aqueous phase with 
a solution of 24:1 (v/v) chloroform:isoamyl alcohol. To ethanol 
precipitate the nucleic acids, the aqueous phases were then collected, 
given 1/10 volume of 3 M sodium acetate and 2 1/2 volumes of ethanol, and 
centrifuged. The pellets were dissolved in an appropriate volume of TAE 
buffer. 
Southern Blot, Hybridization, Wash and Detection Methods 
DNA fragments were separated on 1% (w/v) agarose gels and transferred by 
capillary action in 20.times.SSC (3 M sodium chloride, 0.3 M sodium 
citrate) to Nytran (+) nylon membranes (Schleicher & Schuell, Inc., Keene, 
N.H.) by the method of Southern as described in Sambrook et al., supra, at 
page 9.38, previously incorporated by reference herein. The membranes were 
air dried and baked at 80.degree. C. in a vacuum oven for 2 hours prior to 
hybridization. 
Membranes to be hybridized with the .sup.32 P labeled probes were first 
pre-hybridized at 37.degree. C. for approximately 2 hours in 6.times.SSPE 
(20.times.SSPE=3.0 M NaCl, 0.2 M NaH.sub.2 PO.sub.4 (pH 7.4), 0.02 M EDTA) 
(Life Technologies Inc.), 5.times. Denhardt's solution (0.1% (w/v) of each 
of the following: bovine serum albumin, ficoll and polyvinylpyrrolidone), 
1% (w/v) SDS (sodium dodecyl sulfate), 100 .mu.g/ml sonicated denatured 
salmon sperm DNA and formamide (25% (v/v) for oligomer 16 and 20% (v/v) 
for oligomers 24 and 25). The membranes were then incubated overnight at 
37.degree. C. in a hybridization solution made as above except with 1 
.times. (rather than 5.times.) Denhardt's solution and with the addition 
of 1.times.10.sup.6 counts per minute (CPM)/ml of the labeled probe. The 
membranes were then sequentially washed at room temperature in aqueous 
solutions of 5.times.SSC and 0.1% SDS, 1.times.SSC and 0.5% SDS, and 
0.2.times.SSC and 0.5% SDS. Membranes incubated with labeled 
oligonucleotides 24 and 25 were additionally washed with a solution of 
0.1.times.SSC and 0.1% (w/v) SDS. Following the wash steps, the membranes 
were dried and allowed to expose X-ray film using intensifier screens at 
-80.degree. C. for 3 hours. 
Membranes to be hybridized with oligomers 15, 21 and 20 were processed 
according to the manufacturer's "high stringency" protocol (Promega, 
Inc.). As stated above, the use of chemiluminescent probes was for 
convenience only; had the probes been .sup.32 P labelled, the Southern 
hybridization procedure could have been performed as described above. The 
hybridization and wash temperatures used were 56.degree. C. for oligomer 
15, 48.degree. C. for oligomer 21 and 51.degree. C. for oligomer 20. 
Sequencing Reactions 
Plasmid DNA preparations of clones pGem Bst 2.1 Sst and pGem Bst 5' end 
were used as the templates for sequencing the Bst DNA polymerase gene 
using the dideoxy chain-termination method. See e.g., Sanger et al., Proc. 
Nat. Acad. Sci. (USA) 74:5463-5467 (1977) hereby incorporated by reference 
herein. Four .mu.g of DNA were used with 1 pmol of primer in each 
reaction. Sequencing was done using a Sequenase.TM. kit (version 2.0) 
obtained from United States Biochemical Co. according to the 
manufacturer's protocol. In regions of the nucleic acid strand which were 
difficult to sequence a variety of techniques known to those of skill in 
the art were used to minimize inter- and intramolecular reannealing in the 
sequencing reactions and the polyacrylamide gel. The most successful 
technique for resolving hard to read regions of the nucleotide sequences 
was the inclusion of 40% (v/v) formamide in the sequencing gel. Variations 
of the dideoxy sequencing method are well known to those of ordinary skill 
in the art, as are other nucleic acid sequencing methods such as the 
method of Maxam and Gilbert, Methods in Enzymology 65:497-559 (1980) 
hereby incorporated by reference herein. 
Bst DNA Polymerase Activity Assays 
Bst DNA polymerase activity was determined by a cDNA synthesis reaction 
using a synthetic single-stranded template and primer complementary to a 
portion of the template. Detection of the cDNA strand was accomplished by 
hybridizing the polymerase product with an acridinium ester-labeled probe 
designed to be complementary to the cDNA strand. The labeled 
double-stranded hybrid was detected using the hybridization protection 
assay (HPA) as described in Arnold et al., Clin. Chem. 35:1588-1594 (1989) 
and Arnold et al., U.S. Pat. No. 5,283,174, the latter of which enjoys 
common ownership with the present application and both of which are hereby 
incorporated by reference herein. The sample suspected of containing Bst 
DNA polymerase was incubated in a reaction mixture containing 50 mM Tris 
(pH 7.5), 25 mM KCl, 4 mM MgCl.sub.2, 2 mM spermidine, 0.2 mM each dNTP at 
60.degree. C. for 8 minutes with 20 fmol of an 86 base pair synthetic DNA 
template derived from bacteriophage T7 gene 10 plus 30 pmol of a 23 base 
primer complementary to the 3' end of the template strand. The reaction 
mixture was incubated at 95.degree. C. for 3 minutes to denature the DNA 
strands, then incubated at 60.degree. C. for 10 minutes with 1.5 pmol of 
the acridinium ester labeled detection probe. Unhybridized probe was 
hydrolysed at 60.degree. C. for 7 minutes with an alkaline borate buffer 
and the remaining chemiluminescence, contributed by the hybridized labeled 
probe, was measured in a LEADER 1.TM. luminometer, (Gen-Probe 
Incorporated, San Diego, Calif.), after injection of a dilute solution of 
H.sub.2 O.sub.2 and a solution of sodium hydroxide. 
Primer and Probe Design 
Several DNA polymerase genes have been cloned and sequenced, and alignment 
of these sequences reveals numerous areas in which the nucleotide 
sequences of the DNA polymerase genes are somewhat conserved between 
species. See e.g., Delarue, Protein Engineering 3:461-4670 (1990). The 
published Bca sequence (see Uemori et al., J. Biochem. 113:401-410 (1993)) 
was used as a basis for designing primers and probes to some of these 
conserved regions using the Bca nucleotide sequence as a starting point; 
the Bca DNA polymerase nucleotide sequence contained in this publication 
is hereby incorporated by reference herein. The nucleotide sequences of 
the primers and probes used in the course of the present invention are 
shown in FIG. 1. Mismatches between the Bca DNA polymerase sequence and 
these primers and probes are present in some cases. These primers and 
probes were purposely designed with mismatches for one of two reasons. 
First, a mismatch was sometimes designed in order to create a codon, based 
on an analysis of codon usage in various B. stearothermophilus genes 
encoding proteins of known sequence, thought to be preferred by B. 
stearothermophilus over the codon present in the Bca DNA polymerase gene. 
The second reason that a mismatch between the Bca DNA polymerase 
nucleotide sequence and the primers described herein was designed was to 
better match an interspecies consensus of the nucleotides present in that 
relative position, as deduced from alignments of other DNA polymerases. 
Occasionally, a T was used in the Bst primers and probes in place of a C 
in the Bca DNA polymerase sequence since a G/T mismatch is relatively 
stable and therefore the oligonucleotides would be better able to 
hybridize to different targets. 
Purification of Bst Polymerase Enzymes 
Bacterial host cells containing genes encoding Bst polymerase enzymes were 
grown in liquid culture for sixteen hours with shaking, as described 
above. The preferred host cell strain was E. coli strain 1200. After 
sixteen hours at 37.degree. C., the cell cultures were centrifuged at 
9000.times.g for 10 minutes, and the cell pellets were washed once with 20 
mM Tris HCl (pH 7.5) containing 0.1 mM EDTA. Fifty grams of cell pellets 
were suspended in ten volumes of lysis buffer (25 mM Tris HCl, 10 mM EDTA, 
5 mM DTT, 1% (v/v) Triton X-100, 10 mM NaCl, 10% (v/v) glycerol and 1 mM 
phenylmethylsulfonyl fluoride (PMSF)). The cell suspension was then passed 
twice through a Gaulin cell homogenizer at 8000 psi to lyse the cells. The 
cell lysate was then centrifuged at 12,000 .times.g for 15 minutes and the 
supernatant collected and stored at -70.degree. C. 
Chromotography was performed at 25.degree. C. Two hundred fifty ml of the 
cell lysate was applied to a 190 .times.26 mm column of Poros-HQ anion 
exchange resin (PerSeptive Biosystems, Cambridge, Mass.) The column was 
washed with 160 ml of a solution containing 20 mM Tris-HCl (pH 8.0) and 
0.1 mM EDTA (Buffer A). The bound proteins were eluted with a 500 ml 
linear gradient from 0 to 0.5 M NaCl in Buffer A at a flow rate of 5 
ml/minute. DNA polymerase activity eluted at an ionic strength 
corresponding to a salt concentration of between 0.1 and 0.2 M NaCl. Ten 
ml fractions were collected. In some cases, active fractions were pooled 
and passed through a second Poros-HQ column under similar conditions. 
The pooled anion exchange fractions, in a volume of 40 ml, were diluted 
with 3 volumes of buffer A and applied to a 190.times.26 mm 
phosphocellulose P-11 column equilibrated in Buffer A containing 50 mM 
NaCl. The column was washed with 200 ml of the same buffer. The bound 
proteins were eluted in a linear gradient of 0.1 M to 0.7 M NaCl in Buffer 
A at a flow rate of 3 ml/minute. The DNA polymerase activity eluted from 
this column at an ionic strength corresponding to a salt concentration of 
about 0.25-0.30 M NaCl. Fractions of 10 ml were collected. 
The pooled active fractions from the phosphocellulose step were dialyzed 
twice against 1 liter of Buffer A at 25.degree. C. and applied to an 
250.times.10 mm SynChropak AX-300 anion exchange HPLC column 
pre-equilibrated in Buffer A (Rainin Corp., Emeryville, Calif.) at a flow 
rate of 2.4 ml/minute. Samples were in Buffer A. Bound proteins were 
eluted with a fifty ml linear gradient from 100 mM to 700 mM NaCl in 
Buffer A at 2.4 ml/minute. Bst DNA polymerase activity eluted at an ionic 
strength corresponding to a salt concentration of between about 0.2 and 
0.4 M NaCl. 
In some cases, the purified full-length Bst polymerase was further treated 
with a protease to generate an active truncated form of the enzyme. In 
such cases, purified Bst polymerase (0.33 mg/ml) was treated with 
subtilisin in Buffer A at a 1:200 (w/w) ratio of protease to Bst 
polymerase at 25.degree. C. The reaction mixture was incubated at 
25.degree. C. for 40 minutes, and the reaction was terminated with the 
addition of PMSF to a final concentration of 1 mM. The active proteolytic 
fragment of Bst DNA polymerase was purified using a 60.times.10 mm column 
of hydroxyapatite (HA) (Bio Gel-HT, BioRad Laboratories, Richmond, Calif.) 
according to the method of Jacobsen et al., Eur. J. Biochem. 45:623 (1974) 
the disclosure of which is hereby incorporated by reference herein. The HA 
column was equilibrated in 20 mM sodium phosphate (pH 7.0), and Bst 
polymerase was eluted with a linear gradient from 20 to 350 mM sodium 
phosphate (pH 7.0) at a flow rate of 1 ml/minute. The active protein 
eluted at an ionic strength corresponding to about 0.3 M sodium phosphate. 
The active fractions were pooled. 
FIG. 14 is a photograph of an SDS-PAGE gel containing a crude bacterial 
lysate, purified Bst 1, purified Bst 3, a purified preparation of the 
naturally-occurring breakdown product of Bst 3, and Bst 4 (described 
further below). 
The purification scheme described above resulted in the preparation of 
highly purified Bst polymerase enzymes as determined by SDS-PAGE followed 
by staining with Coomassie Brilliant Blue. However, variations based on 
this scheme or alteration of the order of the steps outlined above will be 
readily apparent to one of ordinary skill in the art in light of the 
present specification. 
EXAMPLES 
The examples which follow are intended to illustrate various embodiments of 
the present invention in order to allow one of ordinary skill in the art 
to make and use the methods and compositions of the present invention. 
However, it will be appreciated that variations in the nucleotide 
sequences of the nucleic acids described herein or in the amino acid 
sequences of the proteins described herein, or both, may exist due to 
variation between different strains of Bacillus stearothermolphilus, or 
due to spontaneous mutations arising as the result of genetic drift. 
Furthermore, the nucleotide and/or amino acid sequences disclosed herein 
may easily be modified by genetic and biochemical techniques to produce 
derivative proteins having DNA polymerase activities. The resulting 
protein will have substantially the same amino acid sequence as the Bst 
polymerase enzymes disclosed herein, and may exhibit a higher or lower 
level of DNA polymerase activity. The activity of any such derivative may 
be detected or measured as described above. 
Thus, the scope of the present invention is not to be limited solely to the 
embodiments which follow, said scope to be determined solely by the claims 
which follow this disclosure. 
Example 1 
Identification of the Genomic Bst DNA Polymerase Gene 
Amplicons 885 and 764 
The location of PCR amplicons and primers used to generate these amplicons 
are shown in FIG. 2 relative to the Bst DNA polymerase gene. The 
polymerase chain reaction (PCR) is a proprietary method of conducting 
nucleic acid amplification, and is patented under the following U.S. 
patents: Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 
4,800,159, assigned to Hoffman-La Roche, Inc., Nutley, N.J. Amplicon 885 
was produced by PCR amplification of Bst genomic DNA. Amplicon 764 was 
generated using amplicon 885 as a substrate using a second set of primers 
to nucleotide sequences within amplicon 885. 
Oligonucleotides 16 and 25, shown in FIG. 1 (SEQ ID NOS: 1 and 3, 
respectively) were used as primers in a PCR reaction using genomic Bst DNA 
as the template. The PCR reaction mixtures contained 50 pmoles of each 
primer, 0.5 .mu.g template DNA and 5 units of Thermus thermophilus DNA 
polymerase in 100 .mu.l of 10 mM Tris-HCl (pH 8.3), 50 mM potassium 
chloride, 1.5 mM magnesium chloride, and 0.2 mM each of DATP, dCTP, dGTP 
and dTTP. The reaction mixtures were overlaid with 100 .mu.l of silicone 
oil and incubated in an thermocycler apparatus at 95.degree. C. for 1.5 
minutes followed by thirty cycles of 95.degree. C. 0.5 min, 50.degree. C. 
2.5 min and 72.degree. C. 1.5 min. A second set of reactions was done as 
above except dimethylsulfoxide (DMSO) was added to a final concentration 
of 13.3% (v/v) to reduce the T.sub.m of the primers by approximately 
8.degree. C. The effective annealing temperature of the resulting reaction 
was 58.degree. C. (see Chester and Marshak, Anal. Biochem. 209:284-290 
(1993)). Separate reactions were run with no Bst template DNA added as 
negative controls. 
Oligomers 17 and 24, also shown in FIG. 1 (SEQ ID NO: 5 and 7, 
respectively) were used as primers in secondary PCR reactions. Aliquots of 
2 .mu.l containing the amplicons from each of the primary reaction 
mixtures described above were used as templates. All reaction conditions 
were the same as in the primary reactions; amplicons generated at the 
50.degree. C. annealing temperature in the primary amplifications were 
used at 50.degree. C. in the secondary reactions, and amplicons generated 
using 13.3% DMSO in the primary amplifications were similarly incubated 
with 13.3% DMSO in the secondary reactions. 
Aliquots of 16 .mu.l from each reaction mixture were subjected to 
electrophoresis on a 1.5% agarose gel and the gels were stained with 
ethidium bromide. Expected amplicon sizes were calculated based on the 
published Bca sequence. In each gel lane corresponding to a reaction 
mixture containing template DNA, a single band appeared having 
approximately the expected size: a band of approximately 885 base pairs in 
the reaction mixture using oligomers 16 and 25 as PCR primers, and a band 
of approximately 764 base pairs in the reaction mixture using oligomers 17 
and 24 as PCR primers. No amplicons were observed in the negative controls 
lacking template DNA. The gel was Southern blotted and probed with 
labelled oligonucleotide 20, shown in FIG. 1 (SEQ ID NO: 11) as described 
above. The primer extension products of both the primary and secondary PCR 
reactions were detected by labelled oligonucleotide 20. No hybridization 
was observed in the negative control reactions. 
Amplicon 1143 
Amplicon 1143 (also shown in FIG. 2) was produced by PCR amplification of 
Bst genomic DNA using the same conditions as in the primary amplifications 
above. The primers used in this reaction were oligonucleotides 20 and 21, 
shown in FIG. 1 (SEQ ID NOS: 11 and 9, respectively). An aliquot of this 
reaction mixture was subjected to electrophoresis on a 1% agarose gel and 
stained with ethidium bromide. A single amplicon of approximately the 
expected size of 1143 base pairs was observed, and no amplicon was 
observed in the negative controls. The gel was subjected to the Southern 
transfer method and the membrane probed with labelled oligonucleotide 16 
(FIG. 1) as described above. The primer extension product hybridized with 
labelled oligonucleotide 16 (SEQ ID NO:1). No hybridization was observed 
with the negative control reactions. 
Cloning of amplicons 885 and 1143 
Amplicons 885 and 1143 were gel isolated as described above. The purified 
amplicons were incubated with T4 DNA polymerase (Stratagene Cloning 
Systems) to assure that the ends were blunt. The amplicons were incubated 
at 11.degree. C. for 20 minutes with 5 units of T4 DNA polymerase in a 50 
.mu.l reaction containing 10 mM Tris-HCl (pH 7.9), 10 mM magnesium 
chloride, 50 mM sodium chloride, 1 mM dithiothreitol, 100 .mu.g/ml 
acetylated bovine serum albumin (BSA) (New England Biolabs) and 0.1 mM 
each of DATP, dCTP, dGTP and dTTP. Following the reaction, the amplicons 
were diluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) and 
extracted with solutions of phenol/chloroform/isoamyl alcohol and 
chloroform/isoamyl alcohol as described above. The primer extension 
products were co-precipitated in ethanol with 0.15 .mu.g plasmid pGem3Z 
which had been digested with Sma I and again extracted using the same two 
solutions as above. The precipitated nucleic acids were resuspended and 
incubated overnight at room temperature in a 10 .mu.l total volume 
containing 50 mM Tris-HCl (pH 7.6), 10 mM magnesium chloride, 1 mM ATP, 1 
mM dithiothreitol, 5% polyethylene glycol-8000, and 10 units T4 DNA 
ligase. Eight units of Sma I were also added to this reaction to prevent 
religation of the vector. 
The resulting circularized amplicon-containing plasmids were used to 
transform XL1-Blue MRF' cells. The transformed cells were plated on LB 
agar plates containing ampicillin, IPTG and X-gal. White colonies, 
indicating the presence of DNA inserts, were selected and grown in LB 
broth with ampicillin. Plasmid minipreparations were made according to the 
standard boiling procedure (see e.g., Sambrook, supra, previously 
incorporated by reference) and the isolates were analyzed using 
restriction endonuclease digestions of the clones. 
Detection of the 885 amplicon insert was accomplished by digesting each 
plasmid miniprep with Eco RI plus Hind III. The digests were subjected to 
electrophoresis on a 1% agarose gel and Southern blotted as described 
above. The Southern blots were hybridized with labelled oligonucleotide 
20. The probe detected faint low molecular weight bands. Since the 
sequence of oligonucleotide 20 was expected to be near the end of the 
amplicon (see FIGS. 2 and 3), it appeared likely that its corresponding 
sequence within the amplicon was located between the vector restriction 
site and an Eco RI or Hind III restriction site within the amplicon; 
oligonucleotide 16 (one of the primers) contained a known Eco RI site but 
would not generate such a small restriction fragment. Two isolates were 
tested further by performing both separate and combined Eco RI and Hind 
III digestions, as well as Sst I and Hind III digestions, followed by 
Southern blotting and hybridization with labelled oligonucleotide 20. The 
structure of the amplicon 885-containing clone was deduced from these 
experiments, and is shown in FIG. 3. This clone was named pGem Bst 885. 
Detection of the 1143 amplicon insert was performed as above by digesting 
each plasmid miniprep with Eco RI and Hind III followed by agarose gel 
electrophoresis. Inserts of the predicted size were observed in several 
isolates as determined by ethidium bromide staining. After Southern blot 
hybridization analysis, the inserts were found to hybridize strongly with 
labelled oligonucleotide 16. One clone was selected as representative and 
the deduced restriction map is shown in FIG. 4. This clone was named pGem 
Bst 1143. 
Partial Sequencing of the Amplicon Clones 
Sequencing reactions were performed as described above using both pGem Bst 
885 and pGem Bst 1143 DNA samples. The primers used in both sets of 
reactions were the SP6 and T7 promoter primers available from Promega 
Corp. (SEQ ID NOS: 15 and 16, respectively). These primers were specific 
for the SP6 and T7 promoter regions in the pGem vector, and were useful 
for sequencing the Bst amplicon inserts in both directions. The resulting 
amplicon sequences were aligned with the known Bca polymerase gene 
sequence and were found to be approximately 88% homologous in the 
overlapping regions. In addition, the sequences present in the overlap 
region of the two amplicons were the same, indicating that they had arisen 
from the same gene. The evidence therefore indicated that the amplicons 
represented true fragments of the Bst DNA polymerase gene. 
The sequences obtained from the 885 and 1143 amplicon clones provided two 
pieces of information that would allow the isolation of gene fragments 
obtained from genomic Bst DNA. First, the sequence of the Bst polymerase 
gene in the regions corresponding to oligonucleotides 16, 17, 20 and 24 
indicated that these oligonucleotides would be suitable for use as probes 
of genomic Bst DNA in Southern blots. Second, two restriction endonuclease 
sites within the Bst DNA polymerase gene were identified: an Sst I 
restriction site at Bca coordinate 1516 and a Hind III restriction site at 
Bca coordinate 1687. These sites provide a strategy for isolating 
fragments of the Bst polymerase gene from the genomic DNA. 
Example 2 
Identification and Cloning of Bst DNA Polymerase 
Cloning the 3' End of the Gene 
Aliquots of genomic Bst DNA were digested with Sst I and subjected to 
electrophoresis on 1% agarose gels, and Southern blotted as described 
above. The transfer membranes were then probed separately with six 
different labelled oligonucleotides and autoradiographed as described 
above. As shown in FIG. 5, labelled oligonucleotides 16, 20, 24 and 25 
hybridized to an Sst I fragment approximately 2.1 kb in length. These 
oligonucleotides were designed based upon Bca DNA polymerase sequences 
near the 3' end of the gene. Two other oligonucleotides, 15 and 21, based 
upon Bca sequences toward the 5' end of the gene, did not hybridize to 
this Sst I fragment. These results indicated that the Sst I restriction 
site could be used to isolate a genomic DNA fragment containing the 31 end 
of the gene. 
Twenty five .mu.g of purified Bst genomic DNA was digested with Sst I and 
subjected to electrophoresis on a 1% agarose gel. Gel slices were excised 
in a region of the gel corresponding to approximately 2.1 kb. The DNA was 
purified from the gel slices as described above; approximately 0.45 .mu.g 
were recovered. 
Vector pGem 3Z was digested with Sst I and sequentially extracted with 
solutions of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl 
alcohol as described above. The gel purified 2.1 kb Sst I fragment was 
ethanol precipitated together with 0.23 .mu.g of the Sst I-digested pGem 
3Z vector. The precipitated DNA was redissolved and ligated at 16.degree. 
C. in a 15 .mu.l reaction overnight as described above. The ligation 
mixture was used to transform XL1-Blue MRF' cells and the transformed 
cells were plated onto LB agar plates containing ampicillin, IPTG and 
X-gal. White colonies, indicating insert DNA, were selected and grown 
overnight in 200 .mu.l LB broth cultures containing ampicillin in 
microtiter dishes. One hundred .mu.l aliquots of each culture were 
filtered onto a Schleicher & Schuell Nytran (+) membrane using a Bio Rad 
Bio-Dot microfiltration apparatus and washed with 200 .mu.l of 
10.times.SSC. The membrane was air dried for 5 minutes and then 
successively placed onto filter papers soaked with: 10% SDS for 3 minutes, 
0.5 M sodium hydroxide for 5 minutes, 1M Tris-HCl (pH 8.0) for 5 minutes 
and 0.7 M Tris-HCl (pH 8.0) containing 1.5 M sodium chloride for 5 
minutes. The filter was air dried, baked in a vacuum oven at 80.degree. C. 
for 2 hours and then hybridized with labelled oligonucleotide 20, as 
described above. 
A clone was identified which hybridized to oligonucleotide 20. This clone 
was cultured overnight at 37.degree. C. in LB broth containing ampicillin. 
Plasmid minipreparations were made as described above, and plasmid DNA was 
digested with Sst I and Hind III, both separately and together. The 
restriction digests were subjected to electrophoresis on a 1% agarose gel 
and stained with ethidium bromide. Two Sst I bands were observed at 
locations corresponding to approximately 2.1 kb and 2.7 kb, and virtually 
the same pattern was observed on gels loaded with plasmid DNA digested 
with Sst I plus Hind III. Plasmid DNA digested with Hind III alone gave 
rise to a large band upon electrophoresis of approximately 4.5 kb, and a 
very small band of approximately 0.1-0.2 kb. The gel was Southern blotted 
and allowed to hybridize with labelled oligonucleotide 25 as described 
above. The probe hybridized to the 2.1 kb Sst I bands in lanes 
corresponding to both the Sst I and Sst I plus Hind III restriction 
digestion reactions and to the 4.5 kb band in lanes corresponding to the 
Hind III digestion. 
To verify that the clone contained the expected 5' end of the 2.1 kb Sst I 
fragment, it was also probed with labelled oligonucleotide 16, which was 
expected to be complementary to a region of the Bst DNA polymerase gene 
very near the Sst I site. In this case, DNA dot blots were used to 
identify clones containing the desired nucleotide sequence, rather than a 
Southern hybridization procedure. One .mu.g aliquots of plasmids pGem 3Z, 
pGem Bst 1143 and the plasmid thought to contain the 2.1 kb Sst I fragment 
were denatured in 110 .mu.l 0.3 M sodium hydroxide at 65.degree. C. for 
one hour. One hundred and ten .mu.l of 2M ammonium acetate was added and 
the samples were filtered onto a Schleicher & Schuell Nytran (+) membrane 
using a Bio Rad Bio-Dot microfiltration apparatus as described above. The 
membrane was washed with 1 M ammonium acetate and baked in a vacuum oven 
at 80.degree. C. for 45 minutes. The membrane was then allowed to 
hybridize with oligonucloetide 16 as described above. Both the plasmid 
thought to contain the 2.I kb Sst I genomic fragment clone and the pGem 
Bst 1143 amplicon clone hybridized strongly with the labelled probe, 
indicating that the 5' end of the fragment was present in both plasmids. 
Preliminary sequencing reactions were done as described above, using the 
SP6 promoter primer (SEQ ID NO: 15). The resulting nucleotide sequence 
matched the sequence deduced from the amplicon clones pGem Bst 885 and 
pGem Bst 1143 and also confirmed the presence of the Hind III site in the 
genomic clone. 
Additional restriction endonuclease digestions of this plasmid with 
restriction endonuclease Sal I yielded two bands of approximately 3.1 and 
1.8 kb which indicated that a Sal I site was present in the 3' non-coding 
region downstream from the 3' end of the polymerase gene. This clone was 
named pGem Bst 2.1 Sst and is shown in FIG. 6. 
Cloning the 5' End of the Gene 
Because the 3' end clone pGem Bst 2.1 Sst contained a Hind III restriction 
site near the 5' end, the Hind III site was used to isolate a genomic Bst 
DNA fragment overlapping the 2.1 kb Sst I gene fragment of pGem Bst 2.1 
Sst. In order to accomplish this, Bst genomic DNA was digested with Hind 
III plus a panel of second enzymes to identify a fragment of at least 1.7 
kb, calculated to be large enough to contain the missing 5' portion of the 
DNA polymerase gene. 
Bst genomic DNA was digested with Hind III alone and with Hind III plus the 
following second enzymes: Bam HI, Eco RI, Kpn I, Sph I, Xba I and Xmn I. 
Three microgram aliquots of each reaction mixture were subjected to 
electrophoresis in duplicate 1% agarose gels. Each gel was then analyzed 
by Southern blot using labelled oligonucleotide 20 or 21 as a probe, as 
described above. Upon analysis, each of the duplicate membranes displayed 
identical hybridization patterns. In lanes corresponding to each 
restriction digest, except the Hind III plus Sph I and Hind III plus Xmn I 
samples, a single band of approximately 4 kb was seen, indicating that the 
closest Hind III site upstream from the previously determined Hind III 
site in the 3' fragment clone was 4 kb distant, and that there were no 
restriction sites for the second enzymes between these Hind III sites. The 
lanes corresponding to the Hind III plus Xmn I digests displayed a single 
band of approximately 1.4 kb, which would not be long enough to contain 
the entire 5' end of the gene, as predicted from the Bca nucleotide 
sequence. The lanes corresponding to the Hind III plus Sph I digests 
displayed a single band of approximately 2.8-3 kb. 
This 3 kb fragment was purified and cloned as follows. Bst genomic DNA was 
digested with Hind III plus Sph I at 37.degree. C. Vector pGem 3Z was also 
digested with the same enzymes. Both digests were subjected to 
electrophoresis on a 1% agarose gel and stained with ethidium bromide. The 
resulting vector fragment and the 3 kb Hind III/Sph I Bst genomic fragment 
were excised from the gel, and the DNA was gel purified as described 
above. Approximately 125 ng of the vector DNA were ethanol precipitated 
together with the 3 kb Hind III/Sph I fragment. The precipitated DNA was 
redissolved and allowed to ligate overnight at 16.degree. C. in a reaction 
mixture containing one unit of T4 DNA ligase in a total volume of 15 
.mu.l. The ligation reaction mixture was used to transform XL1-Blue MRF' 
cells and the transformed cells were plated onto LB agar plates containing 
ampicillin, IPTG and X-gal. White colonies, indicating DNA inserts, were 
selected and grown overnight in 200 .mu.l LB broth cultures containing 
ampicillin in microtiter dishes. One hundred .mu.l aliquots of each 
culture were filtered onto duplicate hybridization membranes as described 
above, air dried, baked in a vacuum oven at 80.degree. C. for 2 hours. The 
duplicate membranes were separately allowed to hybridize with labelled 
oligonucleotides 20 and 21. Samples obtained from three cultures showed 
some hybridization with each probe. These cultures were cultured overnight 
in LB broth containing ampicillin and plasmid minipreparations were made 
as described above. The resulting plasmid DNA from each sample was 
digested with Xmn I alone and with Hind III plus Sph I, subjected to 
electrophoresis on a 1% agarose gel and stained with ethidium bromide. 
Samples from two of the clones appeared to yield DNA bands of the expected 
size. (The Hind III plus Sph I reaction was expected to yield fragments of 
approximately 3 kb and 2.7 kb. These bands could not be resolved on the 
gel. The Xmn I reaction was expected to yield DNA fragments of 
approximately 3.4 kb and 2.4 kb). One of these clones was selected for 
further analysis. The plasmid DNA from this clone was digested with Hind 
III and subjected to electrophoresis, as described above. A single band of 
approximately 5.6 kb was present as predicted for the linear plasmid. The 
same plasmid DNA was also digested with Xmn I, Hind III plus Xmn I and 
Hind III plus Sph I, subjected to electrophoresis on triplicate 1% agarose 
gels, stained with ethidium bromide and transferred to hybridization 
membranes by the method of Southern, as described above. The triplicate 
membranes were then separately allowed to hybridize with labelled 
oligonucleotides 15, 21 and 20. A summary of the results obtained are 
indicated below. 
______________________________________ 
Observed 
Detected Detected Detected 
ethidium 
with with with 
stained 
labelled labelled labelled 
bands (kb) 
oligo 15 oligo 20 oligo 21 
______________________________________ 
Digested 3.2 3.2 3.2 
with Xmn I 
1.1 1.1 
0.9 
0.23 
Digested 1.8 
with Hind 1.4 1.4 1.4 
III + Xmn 1.1 1.1 
I 0.9 
0.23 
Digested 2.7 2.7 2.7 2.7 
with Hind (doublet) 
III + Sph 
______________________________________ 
These results indicated that this clone contained the 5' end of the Bst DNA 
polymerase gene. Preliminary sequencing reactions were done as described 
above, using the SP6 promoter primer of nucleotide sequence SEQ ID NO: 15. 
This promoter-primer primes a sequencing reaction beginning from outside 
the Bst DNA polymerase coding region and extending towards the 5' end of 
the gene. The results of the sequencing reaction showed that the 
nucleotide sequence of the DNA polymerase gene nearest the vector cloning 
site matched the sequence that had been previously obtained from the 5' 
end of the 3' gene fragment of pGem Bst 2.1 Sst. These data thereby 
indicated that the new 5' gene fragment clone overlapped the cloned 3' 
gene fragment insert. Additional restriction mapping of the new insert 
also revealed the presence of two Sal I sites: one approximately 0.2 kb 
upstream from the 5' end of the gene in the 5' flanking region, and one 
approximately 0.5 kb downstream from the 5' end of the gene, in the coding 
region. This new plasmid was named pGem Bst 5' end, and is shown in FIG. 
7. 
Example 3 
Construction of a Plasmid Containing the Full Length Bst DNA Polymerase 
Gene 
A plasmid containing a full length copy of the Bst DNA polymerase gene was 
constructed by combining segments of the 5' and 3' gene fragment clones 
pGem Bst 5' end and pGem Bst 2.1 Sst. The strategy used is outlined in 
FIG. 8. 
First, a precursor plasmid was constructed which contained the portion of 
the 3' end of the gene shown as fragment A in FIG. 8A. Purified plasmid 
pGem Bst 2.1 Sst DNA was digested with Hind III plus Sal I and subjected 
to electrophoresis on a 1% agarose gel. A gel slice containing a DNA band 
of approximately 1.6 kb (fragment A) was excised and the DNA was gel 
purified, as described above. Plasmid vector pUC 18 was digested with the 
same two enzymes, and purified at the same time. Approximately 0.25 .mu.g 
of fragment A and 0.15 .mu.g of pUC 18 fragment were ethanol precipitated 
together. The nucleic acids were redissolved and ligated overnight at 
16.degree. C. in a reaction mixture containing 10 units T4 DNA ligase in a 
volume of 15 .mu.l, as described above. The ligation mixture was used to 
transform XL1-Blue MRF' cells and the transformed cells were plated on LB 
agar plates containing ampicillin, IPTG and X-gal. White colonies, 
indicating a DNA insert, were selected and grown in LB broth with 
ampicillin. Plasmid minipreparations were made, as described above, and 
the resulting plasmid DNA was digested with Hind III plus Sal I, subjected 
to electrophoresis on a 1% agarose gel and stained with ethidium bromide. 
A sample was identified which gave rise to DNA bands of the expected sizes 
of 1.6 and 2.7 kb. This plasmid clone was named pUC Bst 3' end. 
pGem Bst 5' end was used for isolating the portion of the 5' end of the 
gene shown as an Aat II/Hind III fragment (fragment B) in FIG. 8B as 
follows. Sequencing and restriction mapping of this clone had revealed the 
Sal I and Aat II restriction sites indicated. Purified pGem Bst 5' end DNA 
was digested with Hind III plus Sph I plus Ssp I and the precursor 2.86 kb 
Hind III/Sph I fragment was gel purified, as described above. The 
precursor fragment was subsequently digested with Aat II and the 2.3 kb 
fragment B was gel purified, as described above. (This fragment was 
prepared in two stages, with the initial Sph I and Ssp I digestions in 
order to eliminate unwanted plasmid fragments that would have co-migrated 
with the desired fragment during electrophoresis.) 
Plasmid pUC Bst 3' end DNA was digested with Hind III plus Aat II and the 
large fragment was gel purified as described above. Approximately 0.6 
.mu.g of the digested pUC Bst 3' end DNA was ethanol precipitated together 
with approximately 0.5 .mu.g fragment B and allowed to ligate overnight at 
16.degree. C. in a reaction mixture containing 10 units of T4 DNA ligase. 
The ligation mixture was used to transform XL1-Blue MRF' cells and the 
transformed cells were plated on LB agar plates containing ampicillin. 
Colonies were selected, grown in LB broth containing ampicillin and 
plasmid minipreparations were made, as described above. The plasmid DNA 
was digested with Sal I digestions and subjected to agarose gel 
electrophoresis, as described above. Three bands having the expected sizes 
of approximately 2.7, 2.6 and 0.7 kb were observed in the majority of the 
plasmid preparations so screened, indicating successful construction of 
the full length DNA polymerase gene, including 5' and 3' genomic flanking 
sequences. One of these clones was selected as a representative clone. 
This plasmid was named pUC Bst I and is shown in FIG. 8B. The Bst DNA 
polymerase gene and its 5' and 3' flanking sequences are shown in SEQ ID 
NO: 19. 
Example 4 
Construction of a Bst DNA Polymerase Clone Lacking the 5'-3' Exonuclease 
Domain 
A plasmid clone was constructed which contained only the 3'-5' exonuclease 
and polymerase domains of the Bst DNA polymerase gene as follows. 
Generally, the plasmid was constructed by first inserting the lac I.sup.q 
repressor gene from plasmid pMAL.TM.-P2 (New England Biolabs) into a 
modified pUC 18 plasmid so that the final clone would be inducible with 
IPTG in a variety of host cells. Previous publications had indicated that 
expression of full length DNA polymerase I is lethal to E. coli host 
cells. See, e.g., Joyce, et al., Proc. Natl. Acad. Sci. USA 80:1830-1834 
(1983). The DNA polymerase gene fragment to be cloned was assembled from 
three components: a 3' gene fragment containing the Hind III to Sal I 
region from pGem Bst 2.1 Sst, a middle gene fragment containing the region 
from a Sty I site to the Hind III site in pGem Bst 5' end, and a fragment 
made using synthetic oligonucleotides to complete the 5' end of the coding 
region for Bst DNA polymerase, and to provide a cloning site. The cloning 
strategy is shown in FIGS. 9A and 9B. 
Step 1: One .mu.g plasmid pMAL.TM.-p2 was digested with restriction 
endonucleases Msc I plus Ssp I, subjected to electrophoresis in an agarose 
gel, and the resulting band of approximately 1.39 kb containing the lac 
I.sup.q repressor gene was gel purified, as described above. This fragment 
was then ligated to 20 pmol of Sph I linkers (New England Biolabs) 
overnight at room temperature in a reaction mixture containing 20 units of 
T4 DNA ligase, as described above. The T4 ligase was heat-inactivated at 
75.degree. C. for 5 minutes and the ligation mixture was ethanol 
precipitated. The DNA fragment was then redissolved and digested with Sph 
I, then subjected to electrophoresis and gel purified, as described above. 
The resulting DNA fragment is shown as fragment "a" in FIG. 9A. Plasmid 
vector pUC 18N had been constructed previously by making a two base 
substitution in pUC 18 which resulted in the creation of a new Nco I 
cloning site. As indicated below, the A nucleotide 11 bases upstream from 
the Eco RI site was substituted with a G, and the T residue 15 bases 
upstream from the EcoR I site was substituted with a C. Nucleotide 
sequences comprising a restriction endonuclease site are indicated by 
underlining. 
pUC 18 5'-.....CTATGACCATGATTACGAATTC.....-3' 
pUC 18N 5'-.....CCATGGCCATGATTACGAATTC.....-3' 
Nco I Eco RI 
Plasmid pUC 18N was digested with Sph I, subjected to electrophoresis in an 
agarose gel and gel purified, as described above. The linearized plasmid 
was then co-ethanol precipitated with the lac I.sup.q fragment described 
above, and the two DNA fragments were ligated overnight at 16.degree. C. 
in a reaction mixture containing 2 units of T4 DNA ligase, as previously 
described. The ligation mixture was used to transform XL1-Blue MRF' cells 
and the transformed cells were plated on LB plates containing ampicillin, 
IPTG and X-gal. White colonies, indicating the presence of DNA inserts, 
were selected and grown in LB broth containing ampicillin and plasmid 
minipreparations were made as previously described. The plasmid DNA preps 
were each digested with Eco RI plus Eco RV and with Hind III plus Eco RV, 
and subjected to electrophoresis in an agarose gel. A plasmid clone was 
selected which displayed DNA bands of the expected size (Eco RI/Eco RV: 
3.15 kb+0.96 kb, Hind III/Eco RV: 3.59 kb+0.52 kb) and was designated pUC 
18N I.sup.q. 
Step 2: The synthetic fragment required to complete the 5' end of the 
cloned gene was constructed using two partially complementary 
single-stranded synthetic oligonucleotides (SEQ ID NOS: 17 and 18). These 
oligonucleotides were designed based on the sequence of the Bst DNA 
polymerase gene obtained by sequencing pGem Bst 5' end DNA. The 
oligonucleotides were structured so that their complementary regions would 
cause the oligonucleotides to overlap each other by 28 bases at their 3' 
ends upon hybridization. The annealed single-stranded oligonucleotides 
were extended with the Klenow fragment from E. coli DNA polymerase I, 
which caused the formation of a double-stranded DNA molecule. The 
resulting duplex DNA molecule contained an Nco I restriction endonuclease 
site near the 5' end, a Sty I restriction endonuclease site near the 3' 
end, and the Bst DNA polymerase gene sequence corresponding to gene 
coordinates 868-1012. This fragment contains the 5' end of the 3'-51 
exonuclease domain of the Bst DNA polymerase gene with a new Nco I cloning 
site added at the 5' end of this domain, and the native Sty I cloning site 
at the 3' end of the fragment. This DNA fragment is represented as 
fragment "b" in FIG. 9A. 
To accomplish step 2, 15 pmol each of oligonucleotides having SED ID NOS: 
17 and 18 were mixed in a total volume of 96 .mu.l of a solution 
containing 50 mM potassium chloride, 2 mM magnesium chloride and 20 mM 
Tris-HCl (pH 8.0). The solution was incubated at 76.degree. C. for 10 
minutes and then allowed to slowly cool to room temperature over a few 
hours in order to anneal the oligonucleotides. The mixture was brought to 
100 .mu.total volume with the addition of 10 units of the Klenow fragment 
of E. coli DNA polymerase I and 0.2 mM each of DATP, dCTP, dGTP and dTTP. 
The resulting reaction mixture was incubated at room temperature for 6 
minutes, 37.degree. C. for 45 minutes and 42.degree. C. for 10 minutes. 
The solution was then sequentially extracted with solutions of 
phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol as 
previously described, then ethanol precipitated. The double-stranded 
fragment was redissolved and digested in a reaction mixture containing 25 
U of Nco I, and the resulting 0.15 kb fragment was gel purified, as 
described above. 
Step 3: Plasmid pUC18N I.sup.q, constructed in Step 1, was digested with 
Nco I, combined with fragment "b" from Step 2, and the plasmid and DNA 
fragment were ligated overnight at 16.degree. C. The ligase was heat 
inactivated at 65.degree. C. for 10 minutes and the ligation products were 
ethanol precipitated. The plasmid was digested with Sty I plus Sal I and 
gel purified, as described above. 
Step 4: Bst DNA polymerase gene fragments were isolated and reassembled as 
follows. Plasmid pGem Bst 5' end was digested with Sty I plus Hind III, 
subjected to electrophoresis, and the resulting 0.68 kb DNA fragment 
(termed fragment "c") was gel purified. Plasmid pGem Bst 2.1 Sst was 
digested with Hind III plus Sal I. This digestion mixture was also 
subjected to electrophoresis, and the 1.57 kb DNA fragment (termed 
fragment "d") was gel purified, as previously described. Purified 
fragments "c" and "d" were combined and co-ethanol precipitated. The 
pelleted DNA was redissolved and allowed to ligate overnight in a 30 .mu.l 
reaction mixture containing 4 U of T4 ligase at 16.degree. C. The ligase 
was heat inactivated at 65.degree. C. for 10 minutes and ligated fragments 
"c" and "d" were ethanol precipitated, then digested with Sal I. Following 
agarose gel electrophoresis, the resulting 2.25 kb ligation fragment "cd" 
was gel purified, as described above. 
Step 5: The gel purified fragment "cd" was ligated with the plasmid 
produced in Step 3 in a 17 .mu.l reaction mixture containing 2 U of T4 
ligase at 16.degree. C. overnight. The ligation reaction mixture was used 
to transform XL1-Blue MRF' cells and the transformants were plated on LB 
agar plates containing ampicillin. Colonies were selected, grown in LB 
broth containing ampicillin and plasmid minipreparations of the selected 
colonies were made. The DNA preparations were analyzed using restriction 
endonuclease digestions with Nco I plus Hind III and with Sph I plus Sty 
I. The restriction digests were subjected to agarose gel electrophoresis, 
and ethidium bromide staining. A clone was identified which gave rise to 
restriction fragments of the expected size (Nco I+Sty I: 2 bands at 2.62 
kb, 0.83 kb, 0.37 kb and Sph I+Sty I: 2.77 kb, 1.41 kb, 1.05 kb, 0.88 kb, 
0.33 kb). This clone was named pUC Bst 2 and is shown in FIG. 9; the Bst 2 
gene insert, without its 5' and 3' untranslated regions (but with the 
untranslated termination codon) has a nucleotide sequence of SEQ ID NO: 
22. 
Example 5 
Construction of Modified Versions of pUC Bst 2 
In order to evaluate the effect of the lac I.sup.q repressor gene on the 
expression of the Bst DNA polymerase gene, modified versions of pUC Bst 2 
were constructed in which the lac I.sup.q repressor gene was either 
deleted or reversed in orientation. To create these clones, pUC Bst 2 DNA 
was digested with Sph I restriction endonuclease to liberate the lac 
I.sup.q insert. The reaction mixture was sequentially extracted with 
solutions of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl 
alcohol as previously described, and then ethanol precipitated. The sample 
was then redissolved and religated in a 20 .mu.l reaction mixture 
containing 1 U of T4 DNA ligase overnight at 16.degree. C. The ligation 
reaction mixture was used to transform E. coli 1200 cells, and the 
transformed cells were plated on LB agar plates containing ampicillin. 
Colonies were selected and grown in LB broth containing ampicillin. 
Plasmid minipreparations were made as described above. The samples were 
then digested with Eco RV plus Hind III, subjected to electrophoresis on a 
1% agarose gel and then stained with ethidium bromide. Plasmids pUC Bst 2 
"AB", "CD" and "EF" were identified based on the expected band sizes 
indicated in the table below and in FIG. 11. 
______________________________________ 
pUC Bst 2 AB 
pUC Bst 2 CD 
pUC Bst 2 EF 
______________________________________ 
Expected Eco 
3445 3445 3445 
RV and Hind 
2477 2094 1573 
III 518 901 
Restriction 
Fragments 
(base pairs) 
______________________________________ 
Example 6 
Construction of a Bst DNA Polymerase Clone with a Deletion in the 5'-3' 
Exonuclease Domain 
A plasmid containing an in-frame deletion in the 5'-3' exonuclease domain 
of the Bst DNA polymerase gene was constructed in order to inactivate or 
diminish the 5'-3' exonuclease activity of the expressed gene product 
without modifying the domains of the gene affecting the 3'-5' exonuclease 
and DNA polymerase activities. 
The experimental strategy is outlined in FIG. 10, and utilized two 
restriction fragments from pUC Bst 1. The first fragment was prepared by 
digesting pUC Bst 1 DNA with Pvu II. The restriction digest was subjected 
to agarose gel electrophoresis. A fragment of 3,321 base pairs was 
identified and gel purified, as described. The purified fragment was then 
partially digested with Hinc II. Conditions suitable for partial digestion 
of this fragment were previously determined; conditions for conducting 
partial restriction digests of a substrate DNA are easily determined and 
well known to those of ordinary skill in the art. Upon agarose gel 
electrophoresis, a 3,126 base pair fragment was identified and gel 
purified. 
To prepare the second restriction fragment, pUC Bst 1 was first digested to 
completion with Aat II in order to eliminate a DNA fragment predicted to 
co-migrate with the desired DNA fragment during agarose gel 
electrophoresis. The DNA was then partially digested with Pvu II under 
conditions previously determined by small scale pilot digestions. 
Following gel electrophoresis, the desired fragment of having a size of 
2754 base pairs was excised from the gel and gel purified. 
The two gel purified fragments so isolated were combined, ethanol 
precipitated, and the pellets were redissolved and allowed to ligate 
overnight in a 10 .mu.l reaction mixture containing 1.5 U of T4 DNA ligase 
at room temperature. The ligation reaction mixture was used to transform 
XL1-Blue MRF' cells and the transformed cells were plated on LB agar 
plates containing ampicillin. Colonies were isolated and grown in LB broth 
containing ampicillin. Plasmid minipreparations were made, as previously 
described. The samples containing plasmid DNA were then digested with Pvu 
II, Sal I, Hind III plus Aat II and Sal I plus Sty I and subjected to 
electrophoresis on 1% agarose gels. A plasmid clone was identified which 
produced restriction fragments of molecular weight predicted from the map 
shown in FIG. 10; this clone was named pUC Bst 3; the DNA sequence of the 
Bst 3 cleavage product, without its 5' and 3' untranslated regions (and 
with the untranslated termination codon) is given in SEQ ID NO: 24. 
Plasmid pUC Bst 3 is 195 base pairs shorter than the full length DNA 
polymerase clone pUC Bst 1 due to the removal of nucleotides from within 
the 5'-3' exonuclease domain of the DNA polymerase gene. This deletion 
results in the absence of 65 amino acid residues from the 5'-3' 
exonuclease domain of the expressed modified enzyme (residues 178-242). 
Among these 65 amino acids are two glycine residues which were thought to 
correspond to amino acids of E. coli DNA polymerase I necessary for 5'-3' 
exonuclease activity (see Joyce, et al., J. Mol. Biol. 186:283-293 
(1985)). 
Example 7 
Insertion of the Tetracycline Resistance Gene into all Bst DNA Polymerase 
Clones 
All of the Bst DNA polymerase containing plasmids described above contained 
a selectable marker gene conferring ampicillin resistance on the 
transformed host cells. This gene encodes .beta.-lactamase. Cultures of 
host cells transformed with plasmids containing this gene and grown in 
media containing ampicillin are often found to have a relatively high rate 
of reversion, with resulting loss of cloned genes. In an attempt to 
stabilize the plasmids within host cells during culture, an additional 
selectable marker gene, conferring tetracycline resistance (tet.sup.r), 
was added to each plasmid. A fragment containing this gene was isolated 
from pBR322 by digesting the plasmid with Eco RI plus Ava I, subjecting 
the digestion mixture to electrophoresis, and gel purifying the 1427 bp 
tet.sup.r fragment, as described above. The purified fragment was 
end-filled using the Klenow fragment of E. coli DNA polymerase I, and the 
resulting blunt-ended tet.sup.r fragment was ligated with Aat II 
oligonucleotide linkers (New England Biolabs); ligation of a gel purified 
DNA fragment with synthetic linkers was previously described above, and is 
well known to those of skill in the art. (S Sambrook, supra, previously 
incorporated by reference herein). The ligation mixture was ethanol 
precipitated and the DNA ligase was heat inactivated. The 
linker-containing fragment was then digested with Aat II, subjected to 
agarose gel electrophoresis and gel purified. Plasmid vector pUC 18 was 
digested with Aat II and following agarose gel electrophoresis, the 
linearized large fragment was gel purified. The Aat II digested vector and 
tet.sup.r fragment containing Aat II linkers were sequentially extracted 
in solutions of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl 
alcohol, as described above. The extracted DNA fragments were combined, 
ethanol precipitated together and allowed to ligate in a reaction mixture 
containing T4 ligase. E. coli JM109 cells were transformed with this 
ligation mixture and plated on LB agar plates containing tetracycline. 
Colonies were isolated, cultured in LB broth containing tetracycline and 
plasmid minipreps were made, as described above. The plasmid preparations 
were digested with Eco RV and with Ssp I plus Hind III and subjected to 
electrophoresis on agarose gels. A clone was identified which gave rise to 
DNA fragments of the sizes expected for a plasmid containing the tet.sup.r 
gene in one of two possible orientations. The expected fragment sizes 
were: Eco RV: 4121, Ssp I+Hind III: 2102, 1868 and 150. This plasmid was 
named pUC Tet(+). Purified pUC Tet(+) DNA was isolated from a cell culture 
of this clone. This DNA was digested with Aat II, the digestion mixture 
subjected to agarose gel electrophoresis, and the 1435 bp tet.sup.r 
fragment was gel purified, as previously described. This fragment was then 
used as a source of the tet.sup.r gene for insertion into each of the Bst 
DNA polymerase clones at their unique Aat II vector site. 
To accomplish this, a preparation of plasmid DNA from each Bst DNA 
polymerase clone was digested with Aat II, subjected to agarose gel 
electrophoresis, and the linearized plasmid gel purified. The purified 
plasmid fragment was sequentially extracted with solutions of 
phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol. The 
purified Aat II linearized plasmid DNA was combined with the 1435 bp 
tet.sup.r fragment, and ethanol precipitated. The DNA pellet was 
dissolved, and the DNA fragments were allowed to ligate in a reaction 
mixture containing T4 DNA ligase, as described above. The ligation 
mixtures were used to transform E. coli 1200 cells, and the transformants 
were plated on LB agar containing tetracycline. Individual colonies were 
cultured in LB broth containing tetracycline, and plasmid minipreps of 
these cultures were made. In order to determine the orientation of the 
tet.sup.r gene in each plasmid, the plasmid DNA from each preparation was 
digested with either Hind III or with Eco RV in combination with another 
restriction endonuclease having a convenient recognition site within the 
cloned Bst DNA polymerase gene. Following gel electrophoresis and ethidium 
bromide staining, clones were selected which contained the tet.sup.r 
insert in each orientation relative to that of the Bst DNA polymerase 
gene. Plus (+) orientation was designated as the same orientation, 
relative to transcription, as the Bst polymerase gene and minus (-) 
orientation was designated as that opposite to the Bst DNA polymerase 
gene. Stock cultures of each of these clones were made, and the clones 
named as indicated below. 
______________________________________ 
Bst DNA Polymerase 
tet.sup.r Gene in (+) 
tet.sup.r Gene in (-) 
Clones without tet.sup.r 
Orientation Orientation 
______________________________________ 
pUC Bst 1 pUC BSt 1 T (+) 
pUC Bst 1 T (-) 
pUC Bst 2 pUC Bst 2 T 
pUC Bst 2 AB pUC Bst 2 A pUC Bst 2 B 
pUC Bst 2 CD pUC Bst 2 C pUC Bst 2 D 
pUC Bst 2 EF pUC Bst 2 E (same 
pUC Bst 2 F 
as PUC Bst 2 T) 
pUC Bst 3 pUC Bst 3 T (+) 
pUC Bst 3 T (-) 
pUC Bst 4 pUC Bst 4 T (+) 
pUC Bst 4 T (-) 
______________________________________ 
Example 8 
Preliminary Evaluation of Enzyme Expression in Bst DNA Polymerase Clones 
As a preliminary determination of the expression of active Bst DNA 
polymerase from the clones constructed as described herein, they were 
grown overnight in cultures of LB broth containing either ampicillin or 
tetracycline. Cultures of pUC Bst 2 containing the lac I.sup.q gene in 
each orientation were also given 1 mM IPTG to induce expression of the 
enzyme. The amino acid sequences of Bst 1, Bst 2, and the cleavage product 
of Bst 3 are shown as SEQ ID NOS: 20, 23, and 25, respectively. Aliquots 
of 0.5 ml of each culture were analysed by SDS gel and by enzyme activity 
assays as follows. 
Each aliquot for enzyme activity assays was centrifuged for 2 minutes in a 
microcentrifuge, and the cell pellets were washed one time with wash 
buffer (50 mM sodium chloride, 5 mM EDTA, 0.25 M sucrose, 50 mM Tris-HCl 
(pH 8.0)). The pellets were frozen at -8.degree. C. and each resuspended 
in 200 .mu.l of lysis buffer (10 mM sodium chloride, 1 mM EDTA, 1% 
glycerol, 25 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 
500 .mu.g/ml lysozyme, 10 mM Tris-HCl (pH 8.0)). After 20 minutes on ice, 
100 .mu.l of 0.75% (v/v) Triton X-100 was added to each sample, and the 
sample was frozen on dry ice and thawed three times. The resulting cell 
lysate was diluted 5,000 fold in enzyme dilution buffer (100 mM sodium 
chloride, 0.1 mM EDTA, 0.01% NP-40 (a nonionic detergent comprising a 
polyglycol ether derivative; Sigma Chemical Co., St. Louis, Mo.), 10% 
glycerol, 20 mM Tris-HCl (pH 7.5)) and 10 .mu.l aliquots were assayed for 
DNA polymerase activity at 60.degree. C., as described above. The results 
of two experiments are shown in the tables below. The assay results are 
expressed in RLU (relative light units). 
The first experiment made use of two E. coli host cell strains, strain 
XL1-Blue MRF' and the E. coli 1200 strain. E. coli XL1-Blue MRF' contains 
an episomal copy of tet.sup.r. Strain XL1-Blue MRF' was transformed with 
plasmids pUC Bst 1, pUC Bst 2 and pUC Bst 3, all lacking the tet.sup.r 
gene. The enzyme activities of lysates from cultures of these clones were 
compared with those of lysates from E. coli 1200 host cells containing 
versions of the same plasmids but with the tet.sup.r gene in each 
orientation. 
______________________________________ 
DNA Polymerase Activity at 60.degree. C. (RLU) 
Host Cell Strain 
E. coli 1200 
XL1-Blue MRF' 
tet.sup.r (+) 
tet.sup.r (-) 
No tet.sup.r Gene 
Orientation 
Orientation 
______________________________________ 
pUC Bst 1 38,834 75,644 70,968 
pUC Bst 2 4,868 9,382 Not Done 
pUC Bst 3 27,737 63,675 45,992 
pUC 18 4,324 
(Negative Control) 
pUC tet.sup.r (+) 4,730 
(Negative Control) 
______________________________________ 
In a second experiment, the versions of pUC Bst 2 (A,B,C,D,E and F), 
constructed as described above, were compared with pUC Bst 1 T(+) and pUC 
Bst 3 T(+) to examine the effect of the lac I.sup.q repressor gene on Bst 
DNA polymerase expression. All clones used for this experiment were in the 
E. coli 1200 host cell line, and all clones which contained the lac Iq 
gene (pUC Bst 2 A, B, C and D) were grown in the presence of 1 mM IPTG to 
induce expression of the Bst DNA polymerase gene, under the control of the 
lac promoter in these plasmids. 
______________________________________ 
DNA Polymerase Activity at 60.degree. C. (RLU) 
______________________________________ 
PUC Bst 1 T (+) 67,747 
pUC Bst 2 A 2,993 
pUC Bst 2 B 2,644 
PUC Bst 2 C 2,729 
pUC Bst 2 D 3,664 
pUC Bst 2 E 3,895 
PUC Bst 2 F 7,876 
pUC Bst 3 T (+) 49,275 
pUC tetr (+) (Negative 
1,094 
Control) 
______________________________________ 
Aliquots of cell lysates generated in both experiments were run on SDS 
polyacrylamide gels, and stained with Coomassie Brilliant Blue as 
described in Sambrook, supra, previously incorporated by reference herein. 
These gels revealed prominent new bands in all cell lysates made from host 
cells containing pUC Bst 1 and pUC Bst 3 as compared to the negative 
controls. By contrast, no new bands were visible in gel lanes 
corresponding to lysates made from host cells containing any of the pUC 
Bst 2 series of plasmids. The newly appearing bands from cells containing 
the pUC Bst 1 series of plasmids ran at approximately the same position as 
a 97 kDa molecular weight marker, while the new bands from host cells 
containing the pUC Bst 3 series of plasmids was several kDa smaller. These 
protein bands migrate at approximately the predicted size of the Bst DNA 
polymerase enzymes encoded by the particular plasmid construct. 
The data obtained from these two experiments indicate several things. The 
Bst DNA polymerase gene is able to be expressed in E. coli host cells 
without the use of a heterologous promoter such as the lac promoter. 
Clones of the pUC Bst 1 series and pUC Bst 3 series contain approximately 
600 base pairs of Bst genomic DNA flanking the 5' end of the polymerase 
gene. Although not wishing to be bound by theory, Applicant believes that 
expression of the DNA polymerase gene product is driven by at least one 
native promoter or promoter-like sequence in this region. Although these 
clones contain a lac promoter in the cloning vector, it is downstream from 
the polymerase gene and directs transcription in the opposite orientation 
than the Bst polymerase gene. Thus, this promoter would not be expected to 
function in expressing the polymerase gene. 
Surprisingly, the recombinant Bst DNA polymerase gene of the present 
invention may be constitutively expressed in E. coli host cells without 
the use of an inducible or repressible promoter, such as the lac promoter 
under the control of the lac I.sup.q gene. By contrast, attempts to 
express full length DNA polymerase genes derived from other organisms 
using E. coli as a host cell have often been unsuccessful. For example, 
Uemori, et al., J. Biochem. 113:401-410 (1983) and Joyce, et al., J. Biol. 
Chem. 257:1958-1964 (1982) report that clones containing full length DNA 
polymerase genes are unstable, and the DNA polymerase gene can only be 
propagated as a Klenow-type fragment where the 5'-3' exonuclease activity 
is greatly diminished or absent. Although not wishing to be limited by 
theory, Applicants believe that the clones of the present invention may 
have improved stability by virtue of the tet.sup.r gene and by the 
relatively low activity level of Bst DNA polymerase at 37.degree. C. as 
compared to the optimal temperature of 60.degree. C. (Kaboev, et al., J. 
Bacteriol. 145:21-26 (1981). 
The experiments demonstrate that the tet.sup.r clones in the E. coli 1200 
host cell line expressed higher levels of enzyme activity than their 
non-tet.sup.r counterparts in the E. coli XL1-Blue MRF' host cell line. 
While not wishing to be bound by theory, the present Applicant believes 
that this is due to a lower frequency of reversion when the tet.sup.r gene 
is used as a selectable marker. 
Clones containing the tet.sup.r gene in the (+) orientation (the same 
orientation as the cloned polymerase gene) also gave rise to higher levels 
of DNA polymerase activity than clones having the tet.sup.r gene in the 
(-) orientation. 
Example 9 
Comparison of pUC BST 1 T (+) Derived Bst DNA Polymerase with a Commercial 
Preparation of Bst DNA Polymerase. 
The full length Bst DNA polymerase was purified from a culture of E. coli 
1200 cells containing plasmid pUC Bst 1 T(+) as described previously, and 
an aliquot was digested with subtilisin. The resulting large "Klenow-type" 
fragment, of approximately 66,000 Daltons, contained the DNA polymerase 
and 3' to 5' exonuclease domains, and was purified as detailed above. A 
commercial preparation of a Bst DNA polymerase subtilisin fragment, 
obtained from Bio-Rad Laboratories was purchased and used for comparison. 
The latter enzyme is reportedly directly purified from a strain of B. 
stearothermophilus prior to subtilisin cleavage; this strain is a 
different strain than the one used as the starting material for the 
compositions of the present invention. This enzyme is described in Ye and 
Hong, Scientia Sinica 30:503-506 (1987), and its use in DNA sequencing 
reactions is reported in Lu et al., BioTechniques 11:465-466 (1991), 
McClary et al., DNA Sequence 1: 173-180 (1991), and Mead et al., 
BioTechniques 11:76-87 (1991). 
An assay of these two enzymes was performed using a nucleic acid having the 
same nucleotide sequence as a portion of the HIV genome as a template for 
DNA synthesis in nucleic acid amplification reactions performed as 
described in Ryder et al., U.S. patent application Ser. No. 08/097262, 
hereby incorporated by reference herein, and which enjoys common ownership 
with the present application. This method makes use of both DNA and RNA 
synthesis to amplify a nucleic acid sequence. Nucleic acid amplification 
was performed using 5 copies of the single-stranded HIV template and the 
same number of units of each DNA polymerase enzyme. The results of the 
comparison experiments are shown below and are expressed in relative light 
units (RLU). 
______________________________________ 
Gen-Probe 
Bio-Rad Bst 
Bst 1 Gen-Probe 
No added 
Subtilisin Subtilisin 
Bst 1 Full 
Bst Fragment Fragment Length 
______________________________________ 
No 1,008 1,123 1,007 1,186 
Template 
Template 897 499,250 431,779 
398,745 
938 478,090 412,632 
414,696 
966 511,314 421,338 
317,848 
993 499,573 392,560 
441,114 
959 464,196 399,665 
326,355 
(Geometric 
950 490,188 411,349 
376,510 
Mean) 
______________________________________ 
The results indicate that both the full length and the subtilisin fragments 
of the recombinant enzymes of the present invention are able to promote 
the amplification of HIV DNA. 
Example 10 
Nucleic Acid Amplification in the Presence of a Cell Lysate from Normal 
White Blood Cells. 
Another set of amplification reactions was performed as above, except the 
reactions were performed in the presence of of normal human white blood 
cell lysate purified from 0.5 ml of whole blood, as described in Ryder, 
supra. In this experiment, 10 copies of the HIV template DNA were used 
rather than 5 as in the previous experiment. The results were as indicated 
in the table below. 
______________________________________ 
Gen-Probe 
Bio-Rad Bst 1 Gen-Probe 
No added 
Subtilisin Subtilisin 
Bst 1 Full 
Bst Fragment Fragment Length 
______________________________________ 
No 1,099 1,144 1,129 1,245 
Template 
Template 1,123 1,002,133 1,088,906 
1,009,661 
1,095 1,041,312 1,035,826 
1,007,071 
1,058 1,030,350 1,000,339 
1,020,751 
(Geometric 
1,091 1,024,464 1,014,911 
1,012,476 
Mean) 
______________________________________ 
These data indicate that both the full-length Bst DNA polymerase and the 
subtilisin-generated large fragment recombinant enzymes of the present 
invention supported amplification reactions in the presence of a cell 
lysate. 
Example 11 
Sensitivity Assay of Recombinant Bst DNA Polymerase Enzymes 
Another set of nucleic acid amplification experiments was performed as in 
Example 9, except that the number of template molecules was lowered to 
either 2.5 or 0.5 copies per reaction, and both the pol and gag regions of 
the HIV genome were used as target sequences for primer binding and 
amplification. Detection of the resulting amplicons was performed as 
described in Ryder et al., supra, previously incorporated by reference. In 
place of the subtilisin large fragment of pUC Bst 1 T, a Bst DNA 
polymerase fragment of similar size, from E. coli 1200/pUC Bst 3 T (+) was 
used. This fragment is spontaneously produced by an endogenous protease 
activity during the purification of the pUC Bst 3 T enzyme. 
__________________________________________________________________________ 
2.5 Copies Template per Reaction 
0.5 Copies Template per Reaction 
Commercial Full Commercial Full 
Subtilisin 
pUC Bst 3 T 
Length 
Subtilisin 
pUC Bst 3 T 
Length 
Fragment 
Fragment 
Enzyme 
Fragment 
Fragment 
Enzyme 
__________________________________________________________________________ 
75,351 
1,414,081 
1,514,059 
685,121 
4,937 1,778 
880,648 
1,137,354 
2,101,167 
973 5,909 2,248 
125,304 
1,515,839 
1,565,670 
481,529 
52,426 
1,355,728 
384,228 
2,173,285 
1,585,148 
906 647,230 
1,465 
430,392 
356,737 
1,879,384 
290,032 
18,428 
1,796 
3,019 968,199 
942,562 
780,481 
20,518 
1,666 
492,167 
1,351,785 
944,147 
1,122 878,148 
1,632 
433,327 
2,468,726 
423,967 
1,100 352,646 
1,215 
729,439 
1,374,685 
684,638 
2,241 1,251,116 
4,698 
232,912 
2,414,018 
642,848 
8,149 4,384 21,432 
207,839 
1,351,069 
1,094,169 
16,484 
60,301 
4,651 
(mean) 
(mean) 
(mean) 
(mean) 
(mean) 
(mean) 
__________________________________________________________________________ 
These data indicate that, especially at the lower template levels, both the 
full length and "Klenow" forms of the preferred enzymes of the present 
invention support nucleic acid amplification reactions. 
Example 12 
N-terminal Sequencing of Selected DNA Polymerase Enzymes 
In order to better understand the structure/function relationships between 
the different truncated Bst DNA polymerase enzymes, samples of the active 
subtilisin fragment ("Klenow" fragment) of Bst 1, a naturally-occuring 
breakdown product of the E. coli-expressed cloned Bst 3 DNA polymerase, 
and a biologically active subtilisin fragment from a preparation of an 
uncloned Bst DNA polymerase (obtained from Bio-Rad Laboratories, Inc.) 
were purified as described above, and subjected to N-terminal amino acid 
sequencing. Methods for amino acid sequence determination are well-known 
to those of skill in the art; such methods are described in Hewick et al., 
J. Biol. Chem. 256:7990-7997 (1981), the disclosure of which is hereby 
incorporated by reference herein. Automated methods of N-terminal amino 
acid sequence determination are also well known in the art; the amino acid 
sequencing described herein was performed using an Applied Biosystems-470A 
Gas-Phase sequencer with an in-line HPLC (Applied Biosystems, Foster City, 
Calif.) according to the manufacturer's instructions. 
The polypeptides described above were subjected to amino acid sequence 
determination and the resulting sequences aligned and compared in the 
region corresponding to amino acid residue 285 of the full length Bst DNA 
polymerase (as encoded by the pUC Bst 1 clone). The resulting alignment is 
shown in FIG. 12; the amino acid sequences of Bst 1, Bst 2 and Bst 4 (see 
Example 13) are those predicted by the nucleic acid sequences. In the 
cases of Bst 2 and Bst 4, the translational start codon ATG (which encodes 
methionine) was the first codon of the coding region. Thus, these enzymes 
may have a Met residue at the N- terminus before the indicated residue. 
Alternatively, this residue may be removed by E. coli in the expressed 
protein. As can be seen, the subtilisin fragment of the full length Bst 
polymerase of the present invention is a polypeptide fragment beginning 
with a threonine residue corresponding to amino acid position 289 of the 
full length DNA polymerase. This peptide has DNA polymerase activity. 
The Bst 2 protein, encoded by pUC Bst 2 in which a restriction fragment 
corresponding to the 5'-3' exonuclease domain of the full length protein 
had been engineered out of the Bst DNA polymerase gene, begins with 
aspartic acid. This amino acid occupies a position corresponding to amino 
acid 290 of the full length DNA polymerase, and is the second residue of 
the subtilisin fragment of Bst 1. This enzyme, as expressed in E. coli, is 
active in DNA polymerase assays, but at a lower level of activity than Bst 
1 or its subtilisin fragment. 
The protein expressed by cells including pUC Bst 3 is found in two forms. 
In the first of these forms, the uncleaved protein contains a deletion in 
the 5'-3' exonuclease domain of the full length Bst 1 protein. However, 
both proteins have the same N-terminus, and the region corresponding to 
amino acid residue 285 of the Bst 1 protein is similar in both proteins. 
The second form of the Bst 3 enzyme appears to be a cleavage product of 
the Bst 3 protein by an E. coli protease. This fragment begins with a 
valine as the first amino acid residue; this residue corresponds to amino 
acid 287 of the full length Bst polymerase clone of the present invention. 
The third residue of this proteolytic fragment is the threonine residue 
that begins the Bst 1 subtilisin fragment's amino acid sequence; the 
fourth residue is the aspartic acid residue which begins the amino acid 
sequence of the Bst 2 protein. 
Surprisingly, the sequence information derived from the commercial Bst DNA 
polymerase preparation ("Klenow" fragment) revealed that the N-terminal 
residue of this subtlisin fragment began with an alanine residue at a 
position corresponding to amino acid 290 of the full length Bst 1 protein 
sequence. As disclosed above, the Bst 2 protein begins with an aspartic 
acid residue at this position. All the other enzymes of the present 
invention that were sequenced in this region also showed an aspartic acid 
residue at this position. Moreover, the sequence of the N-terminal first 
21 amino acids of this fragment revealed that 7 residues (or 33%) of the 
amino acids were different between the commercial, uncloned Bst DNA 
polymerase preparation and the enzymes of the present invention in this 
region. See FIG. 12. 
Additionally, a comparison of the amino acid sequences of the proteins of 
the present invention with the published Bca DNA polymerase sequence shows 
that 12 out of 25 residues, or almost 50% of the amino acids, are 
different between the published Bca DNA polymerase sequence, previously 
incorporated by reference, and the Bst DNA polymerase of the present 
invention in this region (see FIG. 12). Overall, 105 out of 876 (almost 
12%) of the amino acids of the Bst DNA polymerase amino acid sequence are 
not found in the corresponding position of the published Bca DNA 
polymerase sequence. 
Example 13 
Construction of a Bst DNA Polymerase (Bst 4) Having the Same N-Terminus as 
the Active Proteolytic Fragment of Bst 3 
A plasmid clone was constructed similarly to the method used in the 
construction of plasmid pUC Bst 2 in order to encode a protein beginning 
with a valine residue and having the amino acid sequence of the 
naturally-occurring degradation product of Bst 3, as described in Example 
12 above. The coding region of the DNA gene insert had a nucleotide 
sequence of SEQ ID NO: 26. The plasmid was used to transform strain 1200. 
A lysate from a culture of this transformant was electrophoresed by 
SDS-PAGE, and a protein band of the expected mobility was observed as 
shown in FIG. 14. This protein was termed Bst 4. The N-terminal amino 
acids predicted for the clone are indicated in FIG. 12, and the entire 
deduced amino acid sequence of Bst 4 is shown as SEQ ID NO: 27. 
FIG. 13 shows a schematic diagram of the Bst DNA polymerase gene inserts 
and their relation to the genomic Bst gene and its 3 domains. 
Example 14 
Construction of Bst DNA Polymerase Point Mutants Lacking 5'-3' Exonuclease 
Activity. 
Two different additional plasmid clones were constructed, each of which 
encoded Bst DNA polymerase enzymes having a single amino acid substitution 
in the 5'-3' exonuclease domain. Because a single substitution in this 
domain was unlikely to significantly affect the polymerase activity or 
expression of the enzyme, it was thought that such a substitution 
presented a strategy for constructing mutant enzymes with DNA polymerase 
activity but being defective in the 5'-3' exonuclease activity. 
The first strategy was to cause the change of the tyrosine at position 73 
of the wild-type Bst 1 enzyme (SEQ ID NO: 20) to a phenylalanine residue. 
This substitution was chosen because the hydroxyl group of the tyrosine 
residue would no longer be available for reaction at or near the active 
site of the 5'-3' exonuclease domain, but the overall conformation of the 
enzyme should be otherwise little affected, since the space-filling phenyl 
ring is common to both tyrosine and phenylalanine. The Phe.sub.73 mutant 
is termed Bst 5 (SEQ. ID NO:32). 
The other mutant enzyme, termed Bst 6 (SEQ. ID NO:34), results from the 
substitution of an alanine residue for the tyrosine at position 73 of the 
Bst 1 amino acid sequence (SEQ. ID NO:34). Since this residue not only 
replaces a polar group with a non-polar group, but replaces a sterically 
large amino acid side group with a much smaller side group, this 
substitution would be expected to change the conformation of the 
polymerase enzyme to a greater degree than was seen in Bst 5. 
A diagramatic representation of the pUC Bst 5 and pUC Bst 6 DNA inserts 
(SEQ ID NO:31 and SEQ ID NO:33, respectively) in relation to the other Bst 
inserts and to the three domains of the Bst DNA polymerase gene is shown 
in FIG. 13. 
Construction of Bst 5 
Plasmid pUC Bst 1 was partially digested with Acc I and Xmn I restriction 
enzymes and electrophoresed on an agarose gel. An Acc I/Xmn I DNA band 
corresponding to the full length plasmid minus a 153 bp region from an Acc 
I site at Bst 1 (SEQ ID NO: 21) coordinate 103 to an Xmn I site at Bst 1 
coordinate 256 was excised from the gel and gel purified using standard 
methods. 
Synthetic oligonucleotides of SEQ ID NOs: 28 and 29 were synthesized using 
a method similar to that described in Example 4 above. Fifteen picomoles 
of each oligonucleotide were combined in duplicate reactions and incubated 
at 72.degree. C. for 5 minutes in a solution of 20 mM Tris-HCl (pH 8.0), 2 
mM MgCl2 and 50 mM KCl. The solutions were then cooled slowly to 
40.degree. C. to anneal the oligonucleotides, which had complementary 
nucleotide sequences at their 3' ends. The solutions were then given 0.2 
mM each DNTP and 10 units of the Klenow fragment of E. coli DNA polymerase 
I to create a blunt-ended double-stranded DNA fragment which contained the 
native Bst DNA polymerase nucleotide sequence with the desired changes at 
the codon corresponding to amino acid 72 of the Bst DNA polymerase enzyme, 
as well as an Acc I site near the 5' end of the coding strand and an Xmn I 
site near the 3' end of the coding strand. A single degenerate mutation 
was also introduced by the synthetic oligonucleotides into the nucleotide 
sequence in order to create a new diagnostically useful restriction site; 
this mutation did not result in additional amino acid substitutions in the 
Bst enzyme. The reaction mixtures were incubated at 37.degree. C. for 50 
minutes. The duplicate reactions were pooled and extracted, first with 
phenol/chloroform, then with chloroform, and finally the double-standed 
oligonucleotide fragment was precipitated with ethanol. The resulting 
fragment was redissolved and phosphroylated using 30 units T4 
polynucleotide kinase and 0.5 mM ATP at 37.degree. C. for one hour. 
Plasmid pGem-3Z (1.22 .mu.g) was digested with 10 units of Sma I at room 
temperature for 65 minutes, then extracted with phenol/chloroform and 
chloroform alone. Approximately 11 picomoles of the phosphorylated 
synthetic double-stranded fragments were combined with 0.24 .mu.g of the 
Sma I-digested plasmid pGem-3Z and the nucleic acids co-ethanol 
precipitated. The pellet was reconstituted and ligated using 15 units of 
T4 DNA ligase at room temperature overnight. The resulting ligation 
mixture was used to transform E. coli strain 1200, and the transformants 
plated onto LB agar plus ampicillin. Following incubation overnight at 
37.degree. C., ampicillin-resistant colonies were picked, grown in LB plus 
ampicillin, and the plasmids purified and screened using restriction 
endonuclease digestion (Xmn I). Clones were identified which had the 
expected synthetic DNA fragment insert; plasmid preparations were made of 
these clones and the plasmids were digested with Acc I and Xmn I. The 
restriction digests were then electrophoresed and the 153 bp fragment was 
gel isolated and ligated with the pUC Bst I fragment previously gel 
isolated as described above. 
The ligation mixture was used to transform XL1-Blue MRF' cells, and the 
transformants were plated onto LB agar containing ampicillin. 
Ampicillin-resistant colonies were chosen, grown in LB plus ampicillin, 
and the plasmids purified and screened using restriction endonuclease 
digestion. The plasmids containing the expected Bst 5 insert were digested 
with Aat II and ligated with the 1435 bp tetracycline resistance gene 
fragment, as described in Example 7 above. The ligation mixture was used 
to transform E. coli strain 1200, and the transformants were plated onto 
LB agar containing tetracycline. Tetracycline resistant colonies were 
grown in LB plus tetracycline and the plasmids purified and screened using 
restriction endonuclease digestion. Clones containing the tetracycline 
resistance gene in both orientations were identified and named pUC Bst 5 T 
[+] and pUC Bst 5 T [-]. An SDS-PAGE analysis of the protein expressed by 
these transformants showed protein bands migrating at the position 
expected for Bst DNA polymerase. Lysates of these transformants displayed 
DNA polymerase activity. The plasmid DNA from these transformants was also 
sequenced in the region of the mutations and confirmed to have the 
expected DNA sequence within the Bst polymerase gene. The sequencing 
reactions were as described above. 
Construction of Bst 6 
Bst 6 was constructed exactly as was Bst 5, except the synthetic 
oligonucleotide pair used for this construction were oligonucleotides of 
SEQ ID NOS: 28 AND 30. 
The tetracycline-resistant clones of Bst 6 having the tetracycline 
resistance gene in both orientations were named pUC Bst 6 T [+] and pUC 
Bst 6 T [-]. These also expressed a protein migrating on SDS-PAGE gels at 
the position correlating with Bst DNA polymerase and lysates from cultures 
of these transformants expressed a DNA polymerase activity. Sequencing of 
the plasmid DNA revealed the expected nucleotide sequence within the Bst 6 
gene. 
DNA Polymerase Activity Assays for Bst 5 and Bst 6 
Cultures of each of the four Bst 5 and Bst 6 clones were grown overnight in 
LB plus tetracycline and analyzed for the expression of DNA polymerase 
activity as described in Example 8. Results of the assay are shown below. 
______________________________________ 
DNA Polymerase Activity at 60.degree. C. (in RLU) 
______________________________________ 
pUC Bst 1 T [+] 58,837 
pUC Bst 5 T [+] 58,729 
PUC Bst 5 T [-] 53,118 
pUC Bst 6 T [+] 63,206 
pUC Bst 6 T [-] 66,582 
pUC Tet [+] (negative 
704 
control) 
______________________________________ 
Analysis of lysates from the Bst 5 and 6 clones by SDS-PAGE showed 
approximately equal amounts of a prominent band at around 97 KDa; this 
band was absent from a lysate from E. coli 1200/pUC Tet [+]. 
5'-3' Exonuclease Activity Assays of the Bst 5 and Bst 6 Clones 
The Bst 5 and Bst 6 enzymes were purified in substantially the same manner 
as described above. The purified Bst 1, Bst 5 and Bst 6 enzymes, and the 
purified subtilisn DNA polymerase fragment from Bst 1 were assayed for 
5'-3' exonuclease activity. Vent DNA polymerase from New England Biolabs, 
which is known to be deficient in 5'-3' exonuclease activity, was used as 
a negative control. rTth DNA polymerase, obtained from Perkin Elmer, is 
known to contain a 5'-3' exonuclease activity; this was used as a positive 
control. 
The assay was performed as follows. Plasmid pGem 3Z DNA was linearized 
using Hind III restriction endonuclease, then treated with alkaline 
phosphatase to dephosphorylate the 5' ends. The DNA was then labeled at 
the 5' ends with .sup.32 P using T4 polynucleotide kinase, as described 
above. Approximately 0.015 pmoles (130,000 cpm) of this labeled substrate 
was used in each assay reaction. 
For each assay of Bst 1, Bst 5, and Bst 6 enzymes, different amounts of 
each enzyme were added to the substrate nucleic acid in a reaction mixture 
containing 0.5 mM of each dNTP, 1.5 mM MgCl.sub.2, 90 mM KCl and 10 mM 
Tris-HCl(pH 8.3); the total volume of each reaction was 50 .mu.l. The 
reaction mixtures were incubated at 60.degree. C. for 3 hours, then 
chilled on ice. Ten microliters of 10 mg/ml BSA was then added to each 
tube as a carrier, then each reaction tube was given 20 .mu.l of cold 50% 
trichloroacetic acid. The tubes were incubated for 20 minutes on ice, then 
centrifuged for 5 minutes in a microcentrifuge. The supernatants and 
pellets were separated and each was counted in a scintillation counter for 
the presence of radioactivity. The percentage of total cpm released in the 
supernatant was used as a measure of 5'-3' exonuclease activity. 
The Vent.TM. and rTth enzymes were assayed in a similar manner with the 
following changes, made according to the manufacturer's instructions. For 
the Vent.RTM. enzyme, the enzyme was added to the substrate in a reaction 
mixture containing 0.5 mM each dNTP, 10 mM KCl, 10 mM (NH.sub.4).sub.2 
SO.sub.4, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO.sub.4, and 0.1% (v/v) 
Triton.RTM. X-100 in a total volume of 50 .mu.l. The reaction mixtures 
were incubated at 70.degree. C. 
For the rTth enzyme, the enzyme was added to the same reaction mixture as 
for the Bst enzymes with the further addition of 0.6 mM MnCl2, 100 mM KCl, 
0.75 mM EGTA, 0.05% (v/v) Tween.RTM. 20, and 5% (v/v) glycerol in a total 
volume of 50 .mu.l. The reaction mixtures were incubated at 70.degree. C. 
Because the manufacturers' units of enzyme activity are not the same as 
Gen-Probe's units of enzyme activity, the concentrations of enzyme added 
to the Vent.RTM. and rTth reactions was based on the amount of enzyme 
determined to be active in DNA polymerase assays. 
The following table presents data which are the averages of duplicate 
assays. 
______________________________________ 
5'-3' Exonuclease Assay 
Gen-Probe Units or 
Manufacturer's Units 
% cpm in supernatant 
______________________________________ 
Bst 1 96,000 95 
19,100 69 
2,000 26 
200 12 
Bst 5 136,100 16 
68,000 15 
27,200 15 
2,700 13 
Bst 6 136,100 17 
68,000 18 
27,200 18 
2,700 14 
Bst 1 78,500 17 
subtilisin no enzyme 13 
fragment 
rTth (+) 25 (Mfr's units) 
42 
control no enzyme 13 
Vent .RTM. (-) 
5 (Mfr's units) 
16 
control no enzyme 14 
______________________________________ 
These data shown that the Bst 5 and Bst 6 enzymes do not contain detectable 
5'-3' exonuclease activities, even at high enzyme concentrations. The data 
also confirm that the purified subtilisin polymerase fragment of Bst 1 
also contains no detectable 5'-3' exonuclease activity. 
Example 15 
Ability of Bst 5 and Bst 6 to Support Nucleic Acid Amplification 
The purified Bst enzymes were tested for their ability to support nucleic 
acid amplification. Nucleic acid amplification was performed substantially 
as described in Example 9, except the commercial source of the 
non-recombinant Bst DNA polymerase subtilisin fragment was Molecular 
Biological Resources (Milwaukee Wis.). An equal number of units of each 
enzyme was used for each assay. 
______________________________________ 
Copies 
Commercial 
of Subtilisin 
HIV Fragment of Subtilisin 
Temp- Native Fragment of 
late Enzyme Bst-1 Bst-1 Bst-5 Bst-6 
______________________________________ 
5 2,538,405 2,190,958 
2,680,877 
2,560,438 
2,600,262 
2,503,380 2,520,161 
2,645,578 
2,571,370 
2,651,576 
2,654,329 2,651,948 
2,703,753 
2,433,015 
2,630,750 
2,714,339 2,486,977 
2,581,356 
2,495,086 
2,658,697 
2,572,544 2,521,970 
2,622,247 
2,492,034 
2,686,534 
2,700,737 2,624,428 
2,601,453 
2,401,619 
2,655,092 
2,719,892 2,574,901 
2,638,163 
2,639,461 
2,667,916 
2,712,654 2,572,914 
2,638,399 
2,294,487 
2,672,463 
2,603,278 2,633,240 
2,535,452 
2,675,323 
2,663,664 
0 7,016 5,114 6,698 6,845 6,449 
______________________________________ 
Example 16 
Use of Purified Bst 1 Subtilisin Fragment and Bst 5 and 6 Enzymes in 
Sequencing Reactions 
Bst 1, Bst 5 and Bst 6 enzymes and the subtilisin fragment from the Bst 1 
clone were purified as described above and tested for their ability to 
support sequencing reactions. Sequencing reactions were done using the 
Bio-Rad (Hercules, Calif.) Bst sequencing reagents according to the 
manufacturers protocol and were compared with reactions done using the 
Bio-Rad Bst DNA polymerase, which is the subtilisin fragment of the 
non-recombinant (native) enzyme. The primer and template used were the T7 
promoter-primer and pGem 3Z plasmid obtained from Promega Corp. 
Both the Bst 1 and native enzyme subtilisin fragments, as well as both of 
the Bst 5 and 6 enzymes, produced clear sequencing ladders, whereas the 
use of the Bst 1 holoenzyme resulted in no signal at all. Because the full 
length Bst 1 enzyme has a 5'-3' exonuclease activity, the rate of 
degradation of newly synthesized stands is in equilibrium with the rate of 
synthesis of these strands, and sequencing is not effective. Thus, the 
results indicate the single amino acid substitutions of the Bst 5 and 6 
enzymes have eliminated the undesired 5'-3' exonuclease activity to the 
extent that the Bst 5 and Bst 6 enzymes are comparable to the subtilisin 
fragment of Bst DNA polymerase in these sequencing reactions, with the 
added advantage of obviating the need for subtilisin digestion and 
repurification. 
The foregoing examples exemplify various embodiments of the present 
invention and are not intended to limit the invention, the scope of the 
invention and its equivalents being determined solely by the claims which 
follow. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
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#pairs (A) LENGTH: 38 base 
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# 38 AGCT CGCCGGCCAA GAATTCAA 
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# 42 ATGT CAGCGGCGCT CCCTTGAATC GG 
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# 36 CATC AGCGGATGTG CTGGAA 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 45 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
#45 GCGT GGCGTACCGC GCCTTTTTCG CCTTG 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 45 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
#45 CGGT ACGCCACGCT GCTGCCGTCG ATTAA 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 19 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
# 19 TAG 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
# 20 AGGG 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 94 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- AGCATGCCAT GGATGAAGGC GAAAAGCCGC TCGCCGGGAT GGATTTTGCG AT - #CGCCGACA 
60 
# 94 GCTC GCCGACAAAG CGGC 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 96 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- CAGACACCAA GGCGATCCCG ACAATCGGGG CATGGTGATA GTTGTCGCCC AC - #CACCTCCA 
60 
# 96 GTCG GCGAGCATTT CGTCCG 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2761 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 103...2730 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- TTTACGATTC ATTTCCCGAA GCCGGAGCGG TAGCCGGCTT CTTTTTATGG CC - #GCCCGCCG 
60 
- GCGTGGTACA ATAGAACAAG GAACGTCCGA GGAGGGATGA TG TTG AAA - # AAC AAG 
114 
# Leu Lys Asn Lys 
# 1 
- CTC GTC TTA ATT GAC GGC AAC AGC GTG GCG TA - #C CGC GCC TTT TTC GCG 
162 
Leu Val Leu Ile Asp Gly Asn Ser Val Ala Ty - #r Arg Ala Phe Phe Ala 
# 20 
- TTG CCG CTT TTG CAT AAC GAT AAA GGG ATT CA - #T ACG AAC GCA GTC TAC 
210 
Leu Pro Leu Leu His Asn Asp Lys Gly Ile Hi - #s Thr Asn Ala Val Tyr 
# 35 
- GGG TTT ACG ATG ATG TTA AAC AAA ATT TTG GC - #G GAA GAG CAG CCG ACC 
258 
Gly Phe Thr Met Met Leu Asn Lys Ile Leu Al - #a Glu Glu Gln Pro Thr 
# 50 
- CAC ATT CTC GTG GCG TTT GAC GCC GGG AAA AC - #G ACG TTC CGC CAT GAA 
306 
His Ile Leu Val Ala Phe Asp Ala Gly Lys Th - #r Thr Phe Arg His Glu 
# 65 
- ACG TTC CAA GAC TAT AAA GGC GGG CGG CAG CA - #G ACG CCG CCG GAA CTG 
354 
Thr Phe Gln Asp Tyr Lys Gly Gly Arg Gln Gl - #n Thr Pro Pro Glu Leu 
# 80 
- TCG GAA CAG TTT CCG CTG CTG CGC GAA TTG CT - #C AAG GCG TAC CGC ATC 
402 
Ser Glu Gln Phe Pro Leu Leu Arg Glu Leu Le - #u Lys Ala Tyr Arg Ile 
#100 
- CCC GCC TAT GAG CTC GAC CAT TAC GAA GCG GA - #C GAT ATT ATC GGA ACG 
450 
Pro Ala Tyr Glu Leu Asp His Tyr Glu Ala As - #p Asp Ile Ile Gly Thr 
# 115 
- ATG GCG GCG CGG GCT GAG CGA GAA GGG TTT GC - #A GTG AAA GTC ATT TCC 
498 
Met Ala Ala Arg Ala Glu Arg Glu Gly Phe Al - #a Val Lys Val Ile Ser 
# 130 
- GGC GAC CGC GAT TTA ACC CAG CTT GCT TCC CC - #G CAA GTG ACG GTG GAG 
546 
Gly Asp Arg Asp Leu Thr Gln Leu Ala Ser Pr - #o Gln Val Thr Val Glu 
# 145 
- ATT ACG AAA AAA GGG ATT ACC GAC ATC GAG TC - #G TAC ACG CCG GAG ACG 
594 
Ile Thr Lys Lys Gly Ile Thr Asp Ile Glu Se - #r Tyr Thr Pro Glu Thr 
# 160 
- GTC GTG GAA AAA TAC GGC CTC ACC CCG GAG CA - #A ATT GTC GAC TTG AAA 
642 
Val Val Glu Lys Tyr Gly Leu Thr Pro Glu Gl - #n Ile Val Asp Leu Lys 
165 1 - #70 1 - #75 1 - 
#80 
- GGA TTG ATG GGC GAC AAA TCC GAC AAC ATC CC - #T GGC GTG CCC GGC ATC 
690 
Gly Leu Met Gly Asp Lys Ser Asp Asn Ile Pr - #o Gly Val Pro Gly Ile 
# 195 
- GGG GAA AAA ACA GCC GTC AAG CTG CTC AAG CA - #A TTC GGC ACG GTC GAA 
738 
Gly Glu Lys Thr Ala Val Lys Leu Leu Lys Gl - #n Phe Gly Thr Val Glu 
# 210 
- AAC GTA CTG GCA TCG ATC GAT GAG ATC AAA GG - #G GAG AAG CTG AAA GAA 
786 
Asn Val Leu Ala Ser Ile Asp Glu Ile Lys Gl - #y Glu Lys Leu Lys Glu 
# 225 
- AAT TTG CGC CAA TAC CGG GAT TTG GCG CTT TT - #A AGC AAA CAG CTG GCC 
834 
Asn Leu Arg Gln Tyr Arg Asp Leu Ala Leu Le - #u Ser Lys Gln Leu Ala 
# 240 
- GCT ATT TGC CGC GAC GCC CCG GTT GAG CTG AC - #G CTC GAT GAC ATT GTC 
882 
Ala Ile Cys Arg Asp Ala Pro Val Glu Leu Th - #r Leu Asp Asp Ile Val 
245 2 - #50 2 - #55 2 - 
#60 
- TAC AAA GGA GAA GAC CGG GAA AAA GTG GTC GC - #C TTG TTT CAG GAG CTC 
930 
Tyr Lys Gly Glu Asp Arg Glu Lys Val Val Al - #a Leu Phe Gln Glu Leu 
# 275 
- GGA TTC CAG TCG TTT CTC GAC AAG ATG GCC GT - #C CAA ACG GAT GAA GGC 
978 
Gly Phe Gln Ser Phe Leu Asp Lys Met Ala Va - #l Gln Thr Asp Glu Gly 
# 290 
- GAA AAG CCG CTC GCC GGG ATG GAT TTT GCG AT - #C GCC GAC AGC GTC ACG 
1026 
Glu Lys Pro Leu Ala Gly Met Asp Phe Ala Il - #e Ala Asp Ser Val Thr 
# 305 
- GAC GAA ATG CTC GCC GAC AAA GCG GCC CTC GT - #C GTG GAG GTG GTG GGC 
1074 
Asp Glu Met Leu Ala Asp Lys Ala Ala Leu Va - #l Val Glu Val Val Gly 
# 320 
- GAC AAC TAT CAC CAT GCC CCG ATT GTC GGG AT - #C GCC TTG GCC AAC GAA 
1122 
Asp Asn Tyr His His Ala Pro Ile Val Gly Il - #e Ala Leu Ala Asn Glu 
325 3 - #30 3 - #35 3 - 
#40 
- CGC GGG CGG TTT TTC CTG CGC CCG GAG ACG GC - #G CTC GCC GAT CCG AAA 
1170 
Arg Gly Arg Phe Phe Leu Arg Pro Glu Thr Al - #a Leu Ala Asp Pro Lys 
# 355 
- TTT CTC GCT TGG CTT GGC GAT GAG ACG AAG AA - #A AAA ACG ATG TTT GAT 
1218 
Phe Leu Ala Trp Leu Gly Asp Glu Thr Lys Ly - #s Lys Thr Met Phe Asp 
# 370 
- TCA AAG CGG GCG GCC GTC GCG CTA AAA TGG AA - #A GGA ATC GAA CTG CGC 
1266 
Ser Lys Arg Ala Ala Val Ala Leu Lys Trp Ly - #s Gly Ile Glu Leu Arg 
# 385 
- GGC GTC GTG TTC GAT CTG TTG CTG GCC GCT TA - #C TTG CTC GAT CCG GCG 
1314 
Gly Val Val Phe Asp Leu Leu Leu Ala Ala Ty - #r Leu Leu Asp Pro Ala 
# 400 
- CAG GCG GCG GGC GAC GTT GCC GCG GTG GCG AA - #A ATG CAT CAG TAC GAG 
1362 
Gln Ala Ala Gly Asp Val Ala Ala Val Ala Ly - #s Met His Gln Tyr Glu 
405 4 - #10 4 - #15 4 - 
#20 
- GCG GTG CGA TCG GAT GAG GCG GTC TAT GGA AA - #A GGA GCG AAG CGG ACG 
1410 
Ala Val Arg Ser Asp Glu Ala Val Tyr Gly Ly - #s Gly Ala Lys Arg Thr 
# 435 
- GTT CCT GAT GAA CCG ACG CTT GCC GAG CAT CT - #C GCC CGC AAG GCG GCG 
1458 
Val Pro Asp Glu Pro Thr Leu Ala Glu His Le - #u Ala Arg Lys Ala Ala 
# 450 
- GCC ATT TGG GCG CTT GAA GAG CCG TTG ATG GA - #C GAA CTG CGC CGC AAC 
1506 
Ala Ile Trp Ala Leu Glu Glu Pro Leu Met As - #p Glu Leu Arg Arg Asn 
# 465 
- GAA CAA GAT CGG CTG CTG ACC GAG CTC GAA CA - #G CCG CTG GCT GGC ATT 
1554 
Glu Gln Asp Arg Leu Leu Thr Glu Leu Glu Gl - #n Pro Leu Ala Gly Ile 
# 480 
- TTG GCC AAT ATG GAA TTT ACT GGA GTG AAA GT - #G GAC ACG AAG CGG CTT 
1602 
Leu Ala Asn Met Glu Phe Thr Gly Val Lys Va - #l Asp Thr Lys Arg Leu 
485 4 - #90 4 - #95 5 - 
#00 
- GAA CAG ATG GGG GCG GAG CTC ACC GAG CAG CT - #G CAG GCG GTC GAG CGG 
1650 
Glu Gln Met Gly Ala Glu Leu Thr Glu Gln Le - #u Gln Ala Val Glu Arg 
# 515 
- CGC ATT TAC GAA CTC GCC GGC CAA GAG TTC AA - #C ATT AAC TCG CCG AAA 
1698 
Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe As - #n Ile Asn Ser Pro Lys 
# 530 
- CAG CTC GGG ACG GTT TTA TTT GAC AAG CTG CA - #G CTC CCG GTG TTG AAA 
1746 
Gln Leu Gly Thr Val Leu Phe Asp Lys Leu Gl - #n Leu Pro Val Leu Lys 
# 545 
- AAG ACA AAA ACC GGC TAT TCG ACT TCA GCC GA - #T GTG CTT GAG AAG CTT 
1794 
Lys Thr Lys Thr Gly Tyr Ser Thr Ser Ala As - #p Val Leu Glu Lys Leu 
# 560 
- GCA CCG CAC CAT GAA ATC GTC GAA CAT ATT TT - #G CAT TAC CGC CAA CTC 
1842 
Ala Pro His His Glu Ile Val Glu His Ile Le - #u His Tyr Arg Gln Leu 
565 5 - #70 5 - #75 5 - 
#80 
- GGC AAG CTG CAG TCA ACG TAT ATT GAA GGG CT - #G CTG AAA GTG GTG CAC 
1890 
Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Le - #u Leu Lys Val Val His 
# 595 
- CCC GTG ACG GGC AAA GTG CAC ACG ATG TTC AA - #T CAG GCG TTG ACG CAA 
1938 
Pro Val Thr Gly Lys Val His Thr Met Phe As - #n Gln Ala Leu Thr Gln 
# 610 
- ACC GGG CGC CTC AGC TCC GTC GAA CCG AAT TT - #G CAA AAC ATT CCG ATT 
1986 
Thr Gly Arg Leu Ser Ser Val Glu Pro Asn Le - #u Gln Asn Ile Pro Ile 
# 625 
- CGG CTT GAG GAA GGG CGG AAA ATC CGC CAG GC - #G TTC GTG CCG TCG GAG 
2034 
Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Al - #a Phe Val Pro Ser Glu 
# 640 
- CCG GAC TGG CTC ATC TTT GCG GCC GAC TAT TC - #G CAA ATC GAG CTG CGC 
2082 
Pro Asp Trp Leu Ile Phe Ala Ala Asp Tyr Se - #r Gln Ile Glu Leu Arg 
645 6 - #50 6 - #55 6 - 
#60 
- GTC CTC GCC CAT ATC GCG GAA GAT GAC AAT TT - #G ATT GAA GCG TTC CGG 
2130 
Val Leu Ala His Ile Ala Glu Asp Asp Asn Le - #u Ile Glu Ala Phe Arg 
# 675 
- CGC GGG TTG GAC ATC CAT ACG AAA ACA GCC AT - #G GAC ATT TTC CAT GTG 
2178 
Arg Gly Leu Asp Ile His Thr Lys Thr Ala Me - #t Asp Ile Phe His Val 
# 690 
- AGC GAA GAA GAC GTG ACA GCC AAC ATG CGC CG - #C CAA GCG AAG GCC GTC 
2226 
Ser Glu Glu Asp Val Thr Ala Asn Met Arg Ar - #g Gln Ala Lys Ala Val 
# 705 
- AAT TTT GGC ATC GTG TAC GGC ATT AGT GAT TA - #C GGT CTG GCG CAA AAC 
2274 
Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Ty - #r Gly Leu Ala Gln Asn 
# 720 
- TTG AAC ATT ACG CGC AAA GAA GCG GCT GAA TT - #T ATT GAG CGA TAT TTT 
2322 
Leu Asn Ile Thr Arg Lys Glu Ala Ala Glu Ph - #e Ile Glu Arg Tyr Phe 
725 7 - #30 7 - #35 7 - 
#40 
- GCC AGT TTT CCA GGT GTA AAG CAA TAT ATG GA - #C AAC ATT GTG CAA GAA 
2370 
Ala Ser Phe Pro Gly Val Lys Gln Tyr Met As - #p Asn Ile Val Gln Glu 
# 755 
- GCG AAA CAA AAA GGG TAT GTG ACG ACG CTG CT - #G CAT CGG CGC CGC TAT 
2418 
Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Le - #u His Arg Arg Arg Tyr 
# 770 
- TTG CCC GAT ATT ACA AGC CGC AAC TTC AAC GT - #C CGC AGC TTC GCC GAG 
2466 
Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Va - #l Arg Ser Phe Ala Glu 
# 785 
- CGG ACG GCG ATG AAC ACA CCG ATC CAA GGG AG - #T GCC GCT GAT ATT ATT 
2514 
Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Se - #r Ala Ala Asp Ile Ile 
# 800 
- AAA AAA GCG ATG ATC GAT CTA AGC GTG AGG CT - #G CGC GAA GAA CGG CTG 
2562 
Lys Lys Ala Met Ile Asp Leu Ser Val Arg Le - #u Arg Glu Glu Arg Leu 
805 8 - #10 8 - #15 8 - 
#20 
- CAG GCG CGC CTG TTG CTG CAA GTG CAT GAC GA - #A CTC ATT TTG GAG GCG 
2610 
Gln Ala Arg Leu Leu Leu Gln Val His Asp Gl - #u Leu Ile Leu Glu Ala 
# 835 
- CCG AAA GAG GAA ATC GAG CGG CTG TGC CGC CT - #C GTT CCA GAG GTG ATG 
2658 
Pro Lys Glu Glu Ile Glu Arg Leu Cys Arg Le - #u Val Pro Glu Val Met 
# 850 
- GAG CAA GCC GTC GCA CTC CGC GTG CCG CTG AA - #A GTC GAT TAC CAT TAC 
2706 
Glu Gln Ala Val Ala Leu Arg Val Pro Leu Ly - #s Val Asp Tyr His Tyr 
# 865 
- GGT CCG ACG TGG TAC GAC GCC AAA TAAAAGCGGC CT - #GCCCGCCA GCTGCTCGGT T 
2761 
Gly Pro Thr Trp Tyr Asp Ala Lys 
# 875 
- (2) INFORMATION FOR SEQ ID NO:20: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 876 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- Leu Lys Asn Lys Leu Val Leu Ile Asp Gly As - #n Ser Val Ala Tyr Arg 
# 15 
- Ala Phe Phe Ala Leu Pro Leu Leu His Asn As - #p Lys Gly Ile His Thr 
# 30 
- Asn Ala Val Tyr Gly Phe Thr Met Met Leu As - #n Lys Ile Leu Ala Glu 
# 45 
- Glu Gln Pro Thr His Ile Leu Val Ala Phe As - #p Ala Gly Lys Thr Thr 
# 60 
- Phe Arg His Glu Thr Phe Gln Asp Tyr Lys Gl - #y Gly Arg Gln Gln Thr 
#80 
- Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Le - #u Arg Glu Leu Leu Lys 
# 95 
- Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp Hi - #s Tyr Glu Ala Asp Asp 
# 110 
- Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Ar - #g Glu Gly Phe Ala Val 
# 125 
- Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gl - #n Leu Ala Ser Pro Gln 
# 140 
- Val Thr Val Glu Ile Thr Lys Lys Gly Ile Th - #r Asp Ile Glu Ser Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Le - #u Thr Pro Glu Gln Ile 
# 175 
- Val Asp Leu Lys Gly Leu Met Gly Asp Lys Se - #r Asp Asn Ile Pro Gly 
# 190 
- Val Pro Gly Ile Gly Glu Lys Thr Ala Val Ly - #s Leu Leu Lys Gln Phe 
# 205 
- Gly Thr Val Glu Asn Val Leu Ala Ser Ile As - #p Glu Ile Lys Gly Glu 
# 220 
- Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg As - #p Leu Ala Leu Leu Ser 
225 2 - #30 2 - #35 2 - 
#40 
- Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pr - #o Val Glu Leu Thr Leu 
# 255 
- Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Gl - #u Lys Val Val Ala Leu 
# 270 
- Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu As - #p Lys Met Ala Val Gln 
# 285 
- Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 300 
- Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 335 
- Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 350 
- Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
# 365 
- Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 380 
- Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
385 3 - #90 3 - #95 4 - 
#00 
- Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 415 
- His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 430 
- Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
# 445 
- Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 460 
- Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
465 4 - #70 4 - #75 4 - 
#80 
- Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 495 
- Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 510 
- Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
# 525 
- Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 540 
- Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
545 5 - #50 5 - #55 5 - 
#60 
- Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 575 
- Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 590 
- Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
# 605 
- Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 620 
- Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
625 6 - #30 6 - #35 6 - 
#40 
- Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 655 
- Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 670 
- Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
# 685 
- Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 700 
- Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
705 7 - #10 7 - #15 7 - 
#20 
- Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 735 
- Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 750 
- Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
# 765 
- Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 780 
- Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
785 7 - #90 7 - #95 8 - 
#00 
- Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 815 
- Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 830 
- Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
# 845 
- Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 860 
- Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
865 8 - #70 8 - #75 
- (2) INFORMATION FOR SEQ ID NO:21: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2631 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...2628 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- TTG AAA AAC AAG CTC GTC TTA ATT GAC GGC AA - #C AGC GTG GCG TAC CGC 
48 
Leu Lys Asn Lys Leu Val Leu Ile Asp Gly As - #n Ser Val Ala Tyr Arg 
# 15 
- GCC TTT TTC GCG TTG CCG CTT TTG CAT AAC GA - #T AAA GGG ATT CAT ACG 
96 
Ala Phe Phe Ala Leu Pro Leu Leu His Asn As - #p Lys Gly Ile His Thr 
# 30 
- AAC GCA GTC TAC GGG TTT ACG ATG ATG TTA AA - #C AAA ATT TTG GCG GAA 
144 
Asn Ala Val Tyr Gly Phe Thr Met Met Leu As - #n Lys Ile Leu Ala Glu 
# 45 
- GAG CAG CCG ACC CAC ATT CTC GTG GCG TTT GA - #C GCC GGG AAA ACG ACG 
192 
Glu Gln Pro Thr His Ile Leu Val Ala Phe As - #p Ala Gly Lys Thr Thr 
# 60 
- TTC CGC CAT GAA ACG TTC CAA GAC TAT AAA GG - #C GGG CGG CAG CAG ACG 
240 
Phe Arg His Glu Thr Phe Gln Asp Tyr Lys Gl - #y Gly Arg Gln Gln Thr 
#80 
- CCG CCG GAA CTG TCG GAA CAG TTT CCG CTG CT - #G CGC GAA TTG CTC AAG 
288 
Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Le - #u Arg Glu Leu Leu Lys 
# 95 
- GCG TAC CGC ATC CCC GCC TAT GAG CTC GAC CA - #T TAC GAA GCG GAC GAT 
336 
Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp Hi - #s Tyr Glu Ala Asp Asp 
# 110 
- ATT ATC GGA ACG ATG GCG GCG CGG GCT GAG CG - #A GAA GGG TTT GCA GTG 
384 
Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Ar - #g Glu Gly Phe Ala Val 
# 125 
- AAA GTC ATT TCC GGC GAC CGC GAT TTA ACC CA - #G CTT GCT TCC CCG CAA 
432 
Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gl - #n Leu Ala Ser Pro Gln 
# 140 
- GTG ACG GTG GAG ATT ACG AAA AAA GGG ATT AC - #C GAC ATC GAG TCG TAC 
480 
Val Thr Val Glu Ile Thr Lys Lys Gly Ile Th - #r Asp Ile Glu Ser Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- ACG CCG GAG ACG GTC GTG GAA AAA TAC GGC CT - #C ACC CCG GAG CAA ATT 
528 
Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Le - #u Thr Pro Glu Gln Ile 
# 175 
- GTC GAC TTG AAA GGA TTG ATG GGC GAC AAA TC - #C GAC AAC ATC CCT GGC 
576 
Val Asp Leu Lys Gly Leu Met Gly Asp Lys Se - #r Asp Asn Ile Pro Gly 
# 190 
- GTG CCC GGC ATC GGG GAA AAA ACA GCC GTC AA - #G CTG CTC AAG CAA TTC 
624 
Val Pro Gly Ile Gly Glu Lys Thr Ala Val Ly - #s Leu Leu Lys Gln Phe 
# 205 
- GGC ACG GTC GAA AAC GTA CTG GCA TCG ATC GA - #T GAG ATC AAA GGG GAG 
672 
Gly Thr Val Glu Asn Val Leu Ala Ser Ile As - #p Glu Ile Lys Gly Glu 
# 220 
- AAG CTG AAA GAA AAT TTG CGC CAA TAC CGG GA - #T TTG GCG CTT TTA AGC 
720 
Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg As - #p Leu Ala Leu Leu Ser 
225 2 - #30 2 - #35 2 - 
#40 
- AAA CAG CTG GCC GCT ATT TGC CGC GAC GCC CC - #G GTT GAG CTG ACG CTC 
768 
Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pr - #o Val Glu Leu Thr Leu 
# 255 
- GAT GAC ATT GTC TAC AAA GGA GAA GAC CGG GA - #A AAA GTG GTC GCC TTG 
816 
Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Gl - #u Lys Val Val Ala Leu 
# 270 
- TTT CAG GAG CTC GGA TTC CAG TCG TTT CTC GA - #C AAG ATG GCC GTC CAA 
864 
Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu As - #p Lys Met Ala Val Gln 
# 285 
- ACG GAT GAA GGC GAA AAG CCG CTC GCC GGG AT - #G GAT TTT GCG ATC GCC 
912 
Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 300 
- GAC AGC GTC ACG GAC GAA ATG CTC GCC GAC AA - #A GCG GCC CTC GTC GTG 
960 
Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- GAG GTG GTG GGC GAC AAC TAT CAC CAT GCC CC - #G ATT GTC GGG ATC GCC 
1008 
Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 335 
- TTG GCC AAC GAA CGC GGG CGG TTT TTC CTG CG - #C CCG GAG ACG GCG CTC 
1056 
Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 350 
- GCC GAT CCG AAA TTT CTC GCT TGG CTT GGC GA - #T GAG ACG AAG AAA AAA 
1104 
Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
# 365 
- ACG ATG TTT GAT TCA AAG CGG GCG GCC GTC GC - #G CTA AAA TGG AAA GGA 
1152 
Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 380 
- ATC GAA CTG CGC GGC GTC GTG TTC GAT CTG TT - #G CTG GCC GCT TAC TTG 
1200 
Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
385 3 - #90 3 - #95 4 - 
#00 
- CTC GAT CCG GCG CAG GCG GCG GGC GAC GTT GC - #C GCG GTG GCG AAA ATG 
1248 
Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 415 
- CAT CAG TAC GAG GCG GTG CGA TCG GAT GAG GC - #G GTC TAT GGA AAA GGA 
1296 
His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 430 
- GCG AAG CGG ACG GTT CCT GAT GAA CCG ACG CT - #T GCC GAG CAT CTC GCC 
1344 
Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
# 445 
- CGC AAG GCG GCG GCC ATT TGG GCG CTT GAA GA - #G CCG TTG ATG GAC GAA 
1392 
Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 460 
- CTG CGC CGC AAC GAA CAA GAT CGG CTG CTG AC - #C GAG CTC GAA CAG CCG 
1440 
Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
465 4 - #70 4 - #75 4 - 
#80 
- CTG GCT GGC ATT TTG GCC AAT ATG GAA TTT AC - #T GGA GTG AAA GTG GAC 
1488 
Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 495 
- ACG AAG CGG CTT GAA CAG ATG GGG GCG GAG CT - #C ACC GAG CAG CTG CAG 
1536 
Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 510 
- GCG GTC GAG CGG CGC ATT TAC GAA CTC GCC GG - #C CAA GAG TTC AAC ATT 
1584 
Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
# 525 
- AAC TCG CCG AAA CAG CTC GGG ACG GTT TTA TT - #T GAC AAG CTG CAG CTC 
1632 
Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 540 
- CCG GTG TTG AAA AAG ACA AAA ACC GGC TAT TC - #G ACT TCA GCC GAT GTG 
1680 
Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
545 5 - #50 5 - #55 5 - 
#60 
- CTT GAG AAG CTT GCA CCG CAC CAT GAA ATC GT - #C GAA CAT ATT TTG CAT 
1728 
Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 575 
- TAC CGC CAA CTC GGC AAG CTG CAG TCA ACG TA - #T ATT GAA GGG CTG CTG 
1776 
Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 590 
- AAA GTG GTG CAC CCC GTG ACG GGC AAA GTG CA - #C ACG ATG TTC AAT CAG 
1824 
Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
# 605 
- GCG TTG ACG CAA ACC GGG CGC CTC AGC TCC GT - #C GAA CCG AAT TTG CAA 
1872 
Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 620 
- AAC ATT CCG ATT CGG CTT GAG GAA GGG CGG AA - #A ATC CGC CAG GCG TTC 
1920 
Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
625 6 - #30 6 - #35 6 - 
#40 
- GTG CCG TCG GAG CCG GAC TGG CTC ATC TTT GC - #G GCC GAC TAT TCG CAA 
1968 
Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 655 
- ATC GAG CTG CGC GTC CTC GCC CAT ATC GCG GA - #A GAT GAC AAT TTG ATT 
2016 
Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 670 
- GAA GCG TTC CGG CGC GGG TTG GAC ATC CAT AC - #G AAA ACA GCC ATG GAC 
2064 
Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
# 685 
- ATT TTC CAT GTG AGC GAA GAA GAC GTG ACA GC - #C AAC ATG CGC CGC CAA 
2112 
Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 700 
- GCG AAG GCC GTC AAT TTT GGC ATC GTG TAC GG - #C ATT AGT GAT TAC GGT 
2160 
Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
705 7 - #10 7 - #15 7 - 
#20 
- CTG GCG CAA AAC TTG AAC ATT ACG CGC AAA GA - #A GCG GCT GAA TTT ATT 
2208 
Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 735 
- GAG CGA TAT TTT GCC AGT TTT CCA GGT GTA AA - #G CAA TAT ATG GAC AAC 
2256 
Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 750 
- ATT GTG CAA GAA GCG AAA CAA AAA GGG TAT GT - #G ACG ACG CTG CTG CAT 
2304 
Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
# 765 
- CGG CGC CGC TAT TTG CCC GAT ATT ACA AGC CG - #C AAC TTC AAC GTC CGC 
2352 
Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 780 
- AGC TTC GCC GAG CGG ACG GCG ATG AAC ACA CC - #G ATC CAA GGG AGT GCC 
2400 
Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
785 7 - #90 7 - #95 8 - 
#00 
- GCT GAT ATT ATT AAA AAA GCG ATG ATC GAT CT - #A AGC GTG AGG CTG CGC 
2448 
Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 815 
- GAA GAA CGG CTG CAG GCG CGC CTG TTG CTG CA - #A GTG CAT GAC GAA CTC 
2496 
Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 830 
- ATT TTG GAG GCG CCG AAA GAG GAA ATC GAG CG - #G CTG TGC CGC CTC GTT 
2544 
Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
# 845 
- CCA GAG GTG ATG GAG CAA GCC GTC GCA CTC CG - #C GTG CCG CTG AAA GTC 
2592 
Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 860 
# 2631C CAT TAC GGT CCG ACG TGG TAC GAC GC - #C AAA TAA 
Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
865 8 - #70 8 - #75 
- (2) INFORMATION FOR SEQ ID NO:22: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1764 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...1761 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- GAT GAA GGC GAA AAG CCG CTC GCC GGG ATG GA - #T TTT GCG ATC GCC GAC 
48 
Asp Glu Gly Glu Lys Pro Leu Ala Gly Met As - #p Phe Ala Ile Ala Asp 
# 15 
- AGC GTC ACG GAC GAA ATG CTC GCC GAC AAA GC - #G GCC CTC GTC GTG GAG 
96 
Ser Val Thr Asp Glu Met Leu Ala Asp Lys Al - #a Ala Leu Val Val Glu 
# 30 
- GTG GTG GGC GAC AAC TAT CAC CAT GCC CCG AT - #T GTC GGG ATC GCC TTG 
144 
Val Val Gly Asp Asn Tyr His His Ala Pro Il - #e Val Gly Ile Ala Leu 
# 45 
- GCC AAC GAA CGC GGG CGG TTT TTC CTG CGC CC - #G GAG ACG GCG CTC GCC 
192 
Ala Asn Glu Arg Gly Arg Phe Phe Leu Arg Pr - #o Glu Thr Ala Leu Ala 
# 60 
- GAT CCG AAA TTT CTC GCT TGG CTT GGC GAT GA - #G ACG AAG AAA AAA ACG 
240 
Asp Pro Lys Phe Leu Ala Trp Leu Gly Asp Gl - #u Thr Lys Lys Lys Thr 
#80 
- ATG TTT GAT TCA AAG CGG GCG GCC GTC GCG CT - #A AAA TGG AAA GGA ATC 
288 
Met Phe Asp Ser Lys Arg Ala Ala Val Ala Le - #u Lys Trp Lys Gly Ile 
# 95 
- GAA CTG CGC GGC GTC GTG TTC GAT CTG TTG CT - #G GCC GCT TAC TTG CTC 
336 
Glu Leu Arg Gly Val Val Phe Asp Leu Leu Le - #u Ala Ala Tyr Leu Leu 
# 110 
- GAT CCG GCG CAG GCG GCG GGC GAC GTT GCC GC - #G GTG GCG AAA ATG CAT 
384 
Asp Pro Ala Gln Ala Ala Gly Asp Val Ala Al - #a Val Ala Lys Met His 
# 125 
- CAG TAC GAG GCG GTG CGA TCG GAT GAG GCG GT - #C TAT GGA AAA GGA GCG 
432 
Gln Tyr Glu Ala Val Arg Ser Asp Glu Ala Va - #l Tyr Gly Lys Gly Ala 
# 140 
- AAG CGG ACG GTT CCT GAT GAA CCG ACG CTT GC - #C GAG CAT CTC GCC CGC 
480 
Lys Arg Thr Val Pro Asp Glu Pro Thr Leu Al - #a Glu His Leu Ala Arg 
145 1 - #50 1 - #55 1 - 
#60 
- AAG GCG GCG GCC ATT TGG GCG CTT GAA GAG CC - #G TTG ATG GAC GAA CTG 
528 
Lys Ala Ala Ala Ile Trp Ala Leu Glu Glu Pr - #o Leu Met Asp Glu Leu 
# 175 
- CGC CGC AAC GAA CAA GAT CGG CTG CTG ACC GA - #G CTC GAA CAG CCG CTG 
576 
Arg Arg Asn Glu Gln Asp Arg Leu Leu Thr Gl - #u Leu Glu Gln Pro Leu 
# 190 
- GCT GGC ATT TTG GCC AAT ATG GAA TTT ACT GG - #A GTG AAA GTG GAC ACG 
624 
Ala Gly Ile Leu Ala Asn Met Glu Phe Thr Gl - #y Val Lys Val Asp Thr 
# 205 
- AAG CGG CTT GAA CAG ATG GGG GCG GAG CTC AC - #C GAG CAG CTG CAG GCG 
672 
Lys Arg Leu Glu Gln Met Gly Ala Glu Leu Th - #r Glu Gln Leu Gln Ala 
# 220 
- GTC GAG CGG CGC ATT TAC GAA CTC GCC GGC CA - #A GAG TTC AAC ATT AAC 
720 
Val Glu Arg Arg Ile Tyr Glu Leu Ala Gly Gl - #n Glu Phe Asn Ile Asn 
225 2 - #30 2 - #35 2 - 
#40 
- TCG CCG AAA CAG CTC GGG ACG GTT TTA TTT GA - #C AAG CTG CAG CTC CCG 
768 
Ser Pro Lys Gln Leu Gly Thr Val Leu Phe As - #p Lys Leu Gln Leu Pro 
# 255 
- GTG TTG AAA AAG ACA AAA ACC GGC TAT TCG AC - #T TCA GCC GAT GTG CTT 
816 
Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Th - #r Ser Ala Asp Val Leu 
# 270 
- GAG AAG CTT GCA CCG CAC CAT GAA ATC GTC GA - #A CAT ATT TTG CAT TAC 
864 
Glu Lys Leu Ala Pro His His Glu Ile Val Gl - #u His Ile Leu His Tyr 
# 285 
- CGC CAA CTC GGC AAG CTG CAG TCA ACG TAT AT - #T GAA GGG CTG CTG AAA 
912 
Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Il - #e Glu Gly Leu Leu Lys 
# 300 
- GTG GTG CAC CCC GTG ACG GGC AAA GTG CAC AC - #G ATG TTC AAT CAG GCG 
960 
Val Val His Pro Val Thr Gly Lys Val His Th - #r Met Phe Asn Gln Ala 
305 3 - #10 3 - #15 3 - 
#20 
- TTG ACG CAA ACC GGG CGC CTC AGC TCC GTC GA - #A CCG AAT TTG CAA AAC 
1008 
Leu Thr Gln Thr Gly Arg Leu Ser Ser Val Gl - #u Pro Asn Leu Gln Asn 
# 335 
- ATT CCG ATT CGG CTT GAG GAA GGG CGG AAA AT - #C CGC CAG GCG TTC GTG 
1056 
Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Il - #e Arg Gln Ala Phe Val 
# 350 
- CCG TCG GAG CCG GAC TGG CTC ATC TTT GCG GC - #C GAC TAT TCG CAA ATC 
1104 
Pro Ser Glu Pro Asp Trp Leu Ile Phe Ala Al - #a Asp Tyr Ser Gln Ile 
# 365 
- GAG CTG CGC GTC CTC GCC CAT ATC GCG GAA GA - #T GAC AAT TTG ATT GAA 
1152 
Glu Leu Arg Val Leu Ala His Ile Ala Glu As - #p Asp Asn Leu Ile Glu 
# 380 
- GCG TTC CGG CGC GGG TTG GAC ATC CAT ACG AA - #A ACA GCC ATG GAC ATT 
1200 
Ala Phe Arg Arg Gly Leu Asp Ile His Thr Ly - #s Thr Ala Met Asp Ile 
385 3 - #90 3 - #95 4 - 
#00 
- TTC CAT GTG AGC GAA GAA GAC GTG ACA GCC AA - #C ATG CGC CGC CAA GCG 
1248 
Phe His Val Ser Glu Glu Asp Val Thr Ala As - #n Met Arg Arg Gln Ala 
# 415 
- AAG GCC GTC AAT TTT GGC ATC GTG TAC GGC AT - #T AGT GAT TAC GGT CTG 
1296 
Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Il - #e Ser Asp Tyr Gly Leu 
# 430 
- GCG CAA AAC TTG AAC ATT ACG CGC AAA GAA GC - #G GCT GAA TTT ATT GAG 
1344 
Ala Gln Asn Leu Asn Ile Thr Arg Lys Glu Al - #a Ala Glu Phe Ile Glu 
# 445 
- CGA TAT TTT GCC AGT TTT CCA GGT GTA AAG CA - #A TAT ATG GAC AAC ATT 
1392 
Arg Tyr Phe Ala Ser Phe Pro Gly Val Lys Gl - #n Tyr Met Asp Asn Ile 
# 460 
- GTG CAA GAA GCG AAA CAA AAA GGG TAT GTG AC - #G ACG CTG CTG CAT CGG 
1440 
Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Th - #r Thr Leu Leu His Arg 
465 4 - #70 4 - #75 4 - 
#80 
- CGC CGC TAT TTG CCC GAT ATT ACA AGC CGC AA - #C TTC AAC GTC CGC AGC 
1488 
Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg As - #n Phe Asn Val Arg Ser 
# 495 
- TTC GCC GAG CGG ACG GCG ATG AAC ACA CCG AT - #C CAA GGG AGT GCC GCT 
1536 
Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Il - #e Gln Gly Ser Ala Ala 
# 510 
- GAT ATT ATT AAA AAA GCG ATG ATC GAT CTA AG - #C GTG AGG CTG CGC GAA 
1584 
Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Se - #r Val Arg Leu Arg Glu 
# 525 
- GAA CGG CTG CAG GCG CGC CTG TTG CTG CAA GT - #G CAT GAC GAA CTC ATT 
1632 
Glu Arg Leu Gln Ala Arg Leu Leu Leu Gln Va - #l His Asp Glu Leu Ile 
# 540 
- TTG GAG GCG CCG AAA GAG GAA ATC GAG CGG CT - #G TGC CGC CTC GTT CCA 
1680 
Leu Glu Ala Pro Lys Glu Glu Ile Glu Arg Le - #u Cys Arg Leu Val Pro 
545 5 - #50 5 - #55 5 - 
#60 
- GAG GTG ATG GAG CAA GCC GTC GCA CTC CGC GT - #G CCG CTG AAA GTC GAT 
1728 
Glu Val Met Glu Gln Ala Val Ala Leu Arg Va - #l Pro Leu Lys Val Asp 
# 575 
# 1764TAC GGT CCG ACG TGG TAC GAC GCC AA - #A TAA 
Tyr His Tyr Gly Pro Thr Trp Tyr Asp Ala Ly - #s 
# 585 
- (2) INFORMATION FOR SEQ ID NO:23: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 587 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- Asp Glu Gly Glu Lys Pro Leu Ala Gly Met As - #p Phe Ala Ile Ala Asp 
# 15 
- Ser Val Thr Asp Glu Met Leu Ala Asp Lys Al - #a Ala Leu Val Val Glu 
# 30 
- Val Val Gly Asp Asn Tyr His His Ala Pro Il - #e Val Gly Ile Ala Leu 
# 45 
- Ala Asn Glu Arg Gly Arg Phe Phe Leu Arg Pr - #o Glu Thr Ala Leu Ala 
# 60 
- Asp Pro Lys Phe Leu Ala Trp Leu Gly Asp Gl - #u Thr Lys Lys Lys Thr 
#80 
- Met Phe Asp Ser Lys Arg Ala Ala Val Ala Le - #u Lys Trp Lys Gly Ile 
# 95 
- Glu Leu Arg Gly Val Val Phe Asp Leu Leu Le - #u Ala Ala Tyr Leu Leu 
# 110 
- Asp Pro Ala Gln Ala Ala Gly Asp Val Ala Al - #a Val Ala Lys Met His 
# 125 
- Gln Tyr Glu Ala Val Arg Ser Asp Glu Ala Va - #l Tyr Gly Lys Gly Ala 
# 140 
- Lys Arg Thr Val Pro Asp Glu Pro Thr Leu Al - #a Glu His Leu Ala Arg 
145 1 - #50 1 - #55 1 - 
#60 
- Lys Ala Ala Ala Ile Trp Ala Leu Glu Glu Pr - #o Leu Met Asp Glu Leu 
# 175 
- Arg Arg Asn Glu Gln Asp Arg Leu Leu Thr Gl - #u Leu Glu Gln Pro Leu 
# 190 
- Ala Gly Ile Leu Ala Asn Met Glu Phe Thr Gl - #y Val Lys Val Asp Thr 
# 205 
- Lys Arg Leu Glu Gln Met Gly Ala Glu Leu Th - #r Glu Gln Leu Gln Ala 
# 220 
- Val Glu Arg Arg Ile Tyr Glu Leu Ala Gly Gl - #n Glu Phe Asn Ile Asn 
225 2 - #30 2 - #35 2 - 
#40 
- Ser Pro Lys Gln Leu Gly Thr Val Leu Phe As - #p Lys Leu Gln Leu Pro 
# 255 
- Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Th - #r Ser Ala Asp Val Leu 
# 270 
- Glu Lys Leu Ala Pro His His Glu Ile Val Gl - #u His Ile Leu His Tyr 
# 285 
- Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Il - #e Glu Gly Leu Leu Lys 
# 300 
- Val Val His Pro Val Thr Gly Lys Val His Th - #r Met Phe Asn Gln Ala 
305 3 - #10 3 - #15 3 - 
#20 
- Leu Thr Gln Thr Gly Arg Leu Ser Ser Val Gl - #u Pro Asn Leu Gln Asn 
# 335 
- Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Il - #e Arg Gln Ala Phe Val 
# 350 
- Pro Ser Glu Pro Asp Trp Leu Ile Phe Ala Al - #a Asp Tyr Ser Gln Ile 
# 365 
- Glu Leu Arg Val Leu Ala His Ile Ala Glu As - #p Asp Asn Leu Ile Glu 
# 380 
- Ala Phe Arg Arg Gly Leu Asp Ile His Thr Ly - #s Thr Ala Met Asp Ile 
385 3 - #90 3 - #95 4 - 
#00 
- Phe His Val Ser Glu Glu Asp Val Thr Ala As - #n Met Arg Arg Gln Ala 
# 415 
- Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Il - #e Ser Asp Tyr Gly Leu 
# 430 
- Ala Gln Asn Leu Asn Ile Thr Arg Lys Glu Al - #a Ala Glu Phe Ile Glu 
# 445 
- Arg Tyr Phe Ala Ser Phe Pro Gly Val Lys Gl - #n Tyr Met Asp Asn Ile 
# 460 
- Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Th - #r Thr Leu Leu His Arg 
465 4 - #70 4 - #75 4 - 
#80 
- Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg As - #n Phe Asn Val Arg Ser 
# 495 
- Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Il - #e Gln Gly Ser Ala Ala 
# 510 
- Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Se - #r Val Arg Leu Arg Glu 
# 525 
- Glu Arg Leu Gln Ala Arg Leu Leu Leu Gln Va - #l His Asp Glu Leu Ile 
# 540 
- Leu Glu Ala Pro Lys Glu Glu Ile Glu Arg Le - #u Cys Arg Leu Val Pro 
545 5 - #50 5 - #55 5 - 
#60 
- Glu Val Met Glu Gln Ala Val Ala Leu Arg Va - #l Pro Leu Lys Val Asp 
# 575 
- Tyr His Tyr Gly Pro Thr Trp Tyr Asp Ala Ly - #s 
# 585 
- (2) INFORMATION FOR SEQ ID NO:24: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1767 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...1764 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- ACG GAT GAA GGC GAA AAG CCG CTC GCC GGG AT - #G GAT TTT GCG ATC GCC 
48 
Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 15 
- GAC AGC GTC ACG GAC GAA ATG CTC GCC GAC AA - #A GCG GCC CTC GTC GTG 
96 
Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
# 30 
- GAG GTG GTG GGC GAC AAC TAT CAC CAT GCC CC - #G ATT GTC GGG ATC GCC 
144 
Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 45 
- TTG GCC AAC GAA CGC GGG CGG TTT TTC CTG CG - #C CCG GAG ACG GCG CTC 
192 
Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 60 
- GCC GAT CCG AAA TTT CTC GCT TGG CTT GGC GA - #T GAG ACG AAG AAA AAA 
240 
Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
#80 
- ACG ATG TTT GAT TCA AAG CGG GCG GCC GTC GC - #G CTA AAA TGG AAA GGA 
288 
Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 95 
- ATC GAA CTG CGC GGC GTC GTG TTC GAT CTG TT - #G CTG GCC GCT TAC TTG 
336 
Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
# 110 
- CTC GAT CCG GCG CAG GCG GCG GGC GAC GTT GC - #C GCG GTG GCG AAA ATG 
384 
Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 125 
- CAT CAG TAC GAG GCG GTG CGA TCG GAT GAG GC - #G GTC TAT GGA AAA GGA 
432 
His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 140 
- GCG AAG CGG ACG GTT CCT GAT GAA CCG ACG CT - #T GCC GAG CAT CTC GCC 
480 
Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
145 1 - #50 1 - #55 1 - 
#60 
- CGC AAG GCG GCG GCC ATT TGG GCG CTT GAA GA - #G CCG TTG ATG GAC GAA 
528 
Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 175 
- CTG CGC CGC AAC GAA CAA GAT CGG CTG CTG AC - #C GAG CTC GAA CAG CCG 
576 
Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
# 190 
- CTG GCT GGC ATT TTG GCC AAT ATG GAA TTT AC - #T GGA GTG AAA GTG GAC 
624 
Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 205 
- ACG AAG CGG CTT GAA CAG ATG GGG GCG GAG CT - #C ACC GAG CAG CTG CAG 
672 
Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 220 
- GCG GTC GAG CGG CGC ATT TAC GAA CTC GCC GG - #C CAA GAG TTC AAC ATT 
720 
Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
225 2 - #30 2 - #35 2 - 
#40 
- AAC TCG CCG AAA CAG CTC GGG ACG GTT TTA TT - #T GAC AAG CTG CAG CTC 
768 
Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 255 
- CCG GTG TTG AAA AAG ACA AAA ACC GGC TAT TC - #G ACT TCA GCC GAT GTG 
816 
Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
# 270 
- CTT GAG AAG CTT GCA CCG CAC CAT GAA ATC GT - #C GAA CAT ATT TTG CAT 
864 
Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 285 
- TAC CGC CAA CTC GGC AAG CTG CAG TCA ACG TA - #T ATT GAA GGG CTG CTG 
912 
Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 300 
- AAA GTG GTG CAC CCC GTG ACG GGC AAA GTG CA - #C ACG ATG TTC AAT CAG 
960 
Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
305 3 - #10 3 - #15 3 - 
#20 
- GCG TTG ACG CAA ACC GGG CGC CTC AGC TCC GT - #C GAA CCG AAT TTG CAA 
1008 
Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 335 
- AAC ATT CCG ATT CGG CTT GAG GAA GGG CGG AA - #A ATC CGC CAG GCG TTC 
1056 
Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
# 350 
- GTG CCG TCG GAG CCG GAC TGG CTC ATC TTT GC - #G GCC GAC TAT TCG CAA 
1104 
Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 365 
- ATC GAG CTG CGC GTC CTC GCC CAT ATC GCG GA - #A GAT GAC AAT TTG ATT 
1152 
Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 380 
- GAA GCG TTC CGG CGC GGG TTG GAC ATC CAT AC - #G AAA ACA GCC ATG GAC 
1200 
Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
385 3 - #90 3 - #95 4 - 
#00 
- ATT TTC CAT GTG AGC GAA GAA GAC GTG ACA GC - #C AAC ATG CGC CGC CAA 
1248 
Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 415 
- GCG AAG GCC GTC AAT TTT GGC ATC GTG TAC GG - #C ATT AGT GAT TAC GGT 
1296 
Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
# 430 
- CTG GCG CAA AAC TTG AAC ATT ACG CGC AAA GA - #A GCG GCT GAA TTT ATT 
1344 
Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 445 
- GAG CGA TAT TTT GCC AGT TTT CCA GGT GTA AA - #G CAA TAT ATG GAC AAC 
1392 
Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 460 
- ATT GTG CAA GAA GCG AAA CAA AAA GGG TAT GT - #G ACG ACG CTG CTG CAT 
1440 
Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
465 4 - #70 4 - #75 4 - 
#80 
- CGG CGC CGC TAT TTG CCC GAT ATT ACA AGC CG - #C AAC TTC AAC GTC CGC 
1488 
Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 495 
- AGC TTC GCC GAG CGG ACG GCG ATG AAC ACA CC - #G ATC CAA GGG AGT GCC 
1536 
Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
# 510 
- GCT GAT ATT ATT AAA AAA GCG ATG ATC GAT CT - #A AGC GTG AGG CTG CGC 
1584 
Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 525 
- GAA GAA CGG CTG CAG GCG CGC CTG TTG CTG CA - #A GTG CAT GAC GAA CTC 
1632 
Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 540 
- ATT TTG GAG GCG CCG AAA GAG GAA ATC GAG CG - #G CTG TGC CGC CTC GTT 
1680 
Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
545 5 - #50 5 - #55 5 - 
#60 
- CCA GAG GTG ATG GAG CAA GCC GTC GCA CTC CG - #C GTG CCG CTG AAA GTC 
1728 
Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 575 
# 1767C CAT TAC GGT CCG ACG TGG TAC GAC GC - #C AAA TAA 
Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
# 585 
- (2) INFORMATION FOR SEQ ID NO:25: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 588 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 15 
- Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
# 30 
- Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 45 
- Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 60 
- Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
#80 
- Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 95 
- Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
# 110 
- Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 125 
- His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 140 
- Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
145 1 - #50 1 - #55 1 - 
#60 
- Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 175 
- Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
# 190 
- Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 205 
- Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 220 
- Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
225 2 - #30 2 - #35 2 - 
#40 
- Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 255 
- Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
# 270 
- Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 285 
- Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 300 
- Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
305 3 - #10 3 - #15 3 - 
#20 
- Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 335 
- Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
# 350 
- Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 365 
- Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 380 
- Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
385 3 - #90 3 - #95 4 - 
#00 
- Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 415 
- Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
# 430 
- Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 445 
- Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 460 
- Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
465 4 - #70 4 - #75 4 - 
#80 
- Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 495 
- Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
# 510 
- Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 525 
- Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 540 
- Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
545 5 - #50 5 - #55 5 - 
#60 
- Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 575 
- Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
# 585 
- (2) INFORMATION FOR SEQ ID NO:26: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1773 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...1770 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- GTC CAA ACG GAT GAA GGC GAA AAG CCG CTC GC - #C GGG ATG GAT TTT GCG 
48 
Val Gln Thr Asp Glu Gly Glu Lys Pro Leu Al - #a Gly Met Asp Phe Ala 
# 15 
- ATC GCC GAC AGC GTC ACG GAC GAA ATG CTC GC - #C GAC AAA GCG GCC CTC 
96 
Ile Ala Asp Ser Val Thr Asp Glu Met Leu Al - #a Asp Lys Ala Ala Leu 
# 30 
- GTC GTG GAG GTG GTG GGC GAC AAC TAT CAC CA - #T GCC CCG ATT GTC GGG 
144 
Val Val Glu Val Val Gly Asp Asn Tyr His Hi - #s Ala Pro Ile Val Gly 
# 45 
- ATC GCC TTG GCC AAC GAA CGC GGG CGG TTT TT - #C CTG CGC CCG GAG ACG 
192 
Ile Ala Leu Ala Asn Glu Arg Gly Arg Phe Ph - #e Leu Arg Pro Glu Thr 
# 60 
- GCG CTC GCC GAT CCG AAA TTT CTC GCT TGG CT - #T GGC GAT GAG ACG AAG 
240 
Ala Leu Ala Asp Pro Lys Phe Leu Ala Trp Le - #u Gly Asp Glu Thr Lys 
#80 
- AAA AAA ACG ATG TTT GAT TCA AAG CGG GCG GC - #C GTC GCG CTA AAA TGG 
288 
Lys Lys Thr Met Phe Asp Ser Lys Arg Ala Al - #a Val Ala Leu Lys Trp 
# 95 
- AAA GGA ATC GAA CTG CGC GGC GTC GTG TTC GA - #T CTG TTG CTG GCC GCT 
336 
Lys Gly Ile Glu Leu Arg Gly Val Val Phe As - #p Leu Leu Leu Ala Ala 
# 110 
- TAC TTG CTC GAT CCG GCG CAG GCG GCG GGC GA - #C GTT GCC GCG GTG GCG 
384 
Tyr Leu Leu Asp Pro Ala Gln Ala Ala Gly As - #p Val Ala Ala Val Ala 
# 125 
- AAA ATG CAT CAG TAC GAG GCG GTG CGA TCG GA - #T GAG GCG GTC TAT GGA 
432 
Lys Met His Gln Tyr Glu Ala Val Arg Ser As - #p Glu Ala Val Tyr Gly 
# 140 
- AAA GGA GCG AAG CGG ACG GTT CCT GAT GAA CC - #G ACG CTT GCC GAG CAT 
480 
Lys Gly Ala Lys Arg Thr Val Pro Asp Glu Pr - #o Thr Leu Ala Glu His 
145 1 - #50 1 - #55 1 - 
#60 
- CTC GCC CGC AAG GCG GCG GCC ATT TGG GCG CT - #T GAA GAG CCG TTG ATG 
528 
Leu Ala Arg Lys Ala Ala Ala Ile Trp Ala Le - #u Glu Glu Pro Leu Met 
# 175 
- GAC GAA CTG CGC CGC AAC GAA CAA GAT CGG CT - #G CTG ACC GAG CTC GAA 
576 
Asp Glu Leu Arg Arg Asn Glu Gln Asp Arg Le - #u Leu Thr Glu Leu Glu 
# 190 
- CAG CCG CTG GCT GGC ATT TTG GCC AAT ATG GA - #A TTT ACT GGA GTG AAA 
624 
Gln Pro Leu Ala Gly Ile Leu Ala Asn Met Gl - #u Phe Thr Gly Val Lys 
# 205 
- GTG GAC ACG AAG CGG CTT GAA CAG ATG GGG GC - #G GAG CTC ACC GAG CAG 
672 
Val Asp Thr Lys Arg Leu Glu Gln Met Gly Al - #a Glu Leu Thr Glu Gln 
# 220 
- CTG CAG GCG GTC GAG CGG CGC ATT TAC GAA CT - #C GCC GGC CAA GAG TTC 
720 
Leu Gln Ala Val Glu Arg Arg Ile Tyr Glu Le - #u Ala Gly Gln Glu Phe 
225 2 - #30 2 - #35 2 - 
#40 
- AAC ATT AAC TCG CCG AAA CAG CTC GGG ACG GT - #T TTA TTT GAC AAG CTG 
768 
Asn Ile Asn Ser Pro Lys Gln Leu Gly Thr Va - #l Leu Phe Asp Lys Leu 
# 255 
- CAG CTC CCG GTG TTG AAA AAG ACA AAA ACC GG - #C TAT TCG ACT TCA GCC 
816 
Gln Leu Pro Val Leu Lys Lys Thr Lys Thr Gl - #y Tyr Ser Thr Ser Ala 
# 270 
- GAT GTG CTT GAG AAG CTT GCA CCG CAC CAT GA - #A ATC GTC GAA CAT ATT 
864 
Asp Val Leu Glu Lys Leu Ala Pro His His Gl - #u Ile Val Glu His Ile 
# 285 
- TTG CAT TAC CGC CAA CTC GGC AAG CTG CAG TC - #A ACG TAT ATT GAA GGG 
912 
Leu His Tyr Arg Gln Leu Gly Lys Leu Gln Se - #r Thr Tyr Ile Glu Gly 
# 300 
- CTG CTG AAA GTG GTG CAC CCC GTG ACG GGC AA - #A GTG CAC ACG ATG TTC 
960 
Leu Leu Lys Val Val His Pro Val Thr Gly Ly - #s Val His Thr Met Phe 
305 3 - #10 3 - #15 3 - 
#20 
- AAT CAG GCG TTG ACG CAA ACC GGG CGC CTC AG - #C TCC GTC GAA CCG AAT 
1008 
Asn Gln Ala Leu Thr Gln Thr Gly Arg Leu Se - #r Ser Val Glu Pro Asn 
# 335 
- TTG CAA AAC ATT CCG ATT CGG CTT GAG GAA GG - #G CGG AAA ATC CGC CAG 
1056 
Leu Gln Asn Ile Pro Ile Arg Leu Glu Glu Gl - #y Arg Lys Ile Arg Gln 
# 350 
- GCG TTC GTG CCG TCG GAG CCG GAC TGG CTC AT - #C TTT GCG GCC GAC TAT 
1104 
Ala Phe Val Pro Ser Glu Pro Asp Trp Leu Il - #e Phe Ala Ala Asp Tyr 
# 365 
- TCG CAA ATC GAG CTG CGC GTC CTC GCC CAT AT - #C GCG GAA GAT GAC AAT 
1152 
Ser Gln Ile Glu Leu Arg Val Leu Ala His Il - #e Ala Glu Asp Asp Asn 
# 380 
- TTG ATT GAA GCG TTC CGG CGC GGG TTG GAC AT - #C CAT ACG AAA ACA GCC 
1200 
Leu Ile Glu Ala Phe Arg Arg Gly Leu Asp Il - #e His Thr Lys Thr Ala 
385 3 - #90 3 - #95 4 - 
#00 
- ATG GAC ATT TTC CAT GTG AGC GAA GAA GAC GT - #G ACA GCC AAC ATG CGC 
1248 
Met Asp Ile Phe His Val Ser Glu Glu Asp Va - #l Thr Ala Asn Met Arg 
# 415 
- CGC CAA GCG AAG GCC GTC AAT TTT GGC ATC GT - #G TAC GGC ATT AGT GAT 
1296 
Arg Gln Ala Lys Ala Val Asn Phe Gly Ile Va - #l Tyr Gly Ile Ser Asp 
# 430 
- TAC GGT CTG GCG CAA AAC TTG AAC ATT ACG CG - #C AAA GAA GCG GCT GAA 
1344 
Tyr Gly Leu Ala Gln Asn Leu Asn Ile Thr Ar - #g Lys Glu Ala Ala Glu 
# 445 
- TTT ATT GAG CGA TAT TTT GCC AGT TTT CCA GG - #T GTA AAG CAA TAT ATG 
1392 
Phe Ile Glu Arg Tyr Phe Ala Ser Phe Pro Gl - #y Val Lys Gln Tyr Met 
# 460 
- GAC AAC ATT GTG CAA GAA GCG AAA CAA AAA GG - #G TAT GTG ACG ACG CTG 
1440 
Asp Asn Ile Val Gln Glu Ala Lys Gln Lys Gl - #y Tyr Val Thr Thr Leu 
465 4 - #70 4 - #75 4 - 
#80 
- CTG CAT CGG CGC CGC TAT TTG CCC GAT ATT AC - #A AGC CGC AAC TTC AAC 
1488 
Leu His Arg Arg Arg Tyr Leu Pro Asp Ile Th - #r Ser Arg Asn Phe Asn 
# 495 
- GTC CGC AGC TTC GCC GAG CGG ACG GCG ATG AA - #C ACA CCG ATC CAA GGG 
1536 
Val Arg Ser Phe Ala Glu Arg Thr Ala Met As - #n Thr Pro Ile Gln Gly 
# 510 
- AGT GCC GCT GAT ATT ATT AAA AAA GCG ATG AT - #C GAT CTA AGC GTG AGG 
1584 
Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Il - #e Asp Leu Ser Val Arg 
# 525 
- CTG CGC GAA GAA CGG CTG CAG GCG CGC CTG TT - #G CTG CAA GTG CAT GAC 
1632 
Leu Arg Glu Glu Arg Leu Gln Ala Arg Leu Le - #u Leu Gln Val His Asp 
# 540 
- GAA CTC ATT TTG GAG GCG CCG AAA GAG GAA AT - #C GAG CGG CTG TGC CGC 
1680 
Glu Leu Ile Leu Glu Ala Pro Lys Glu Glu Il - #e Glu Arg Leu Cys Arg 
545 5 - #50 5 - #55 5 - 
#60 
- CTC GTT CCA GAG GTG ATG GAG CAA GCC GTC GC - #A CTC CGC GTG CCG CTG 
1728 
Leu Val Pro Glu Val Met Glu Gln Ala Val Al - #a Leu Arg Val Pro Leu 
# 575 
- AAA GTC GAT TAC CAT TAC GGT CCG ACG TGG TA - #C GAC GCC AAA TAA 
1773 
Lys Val Asp Tyr His Tyr Gly Pro Thr Trp Ty - #r Asp Ala Lys 
# 590 
- (2) INFORMATION FOR SEQ ID NO:27: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 590 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- Val Gln Thr Asp Glu Gly Glu Lys Pro Leu Al - #a Gly Met Asp Phe Ala 
# 15 
- Ile Ala Asp Ser Val Thr Asp Glu Met Leu Al - #a Asp Lys Ala Ala Leu 
# 30 
- Val Val Glu Val Val Gly Asp Asn Tyr His Hi - #s Ala Pro Ile Val Gly 
# 45 
- Ile Ala Leu Ala Asn Glu Arg Gly Arg Phe Ph - #e Leu Arg Pro Glu Thr 
# 60 
- Ala Leu Ala Asp Pro Lys Phe Leu Ala Trp Le - #u Gly Asp Glu Thr Lys 
#80 
- Lys Lys Thr Met Phe Asp Ser Lys Arg Ala Al - #a Val Ala Leu Lys Trp 
# 95 
- Lys Gly Ile Glu Leu Arg Gly Val Val Phe As - #p Leu Leu Leu Ala Ala 
# 110 
- Tyr Leu Leu Asp Pro Ala Gln Ala Ala Gly As - #p Val Ala Ala Val Ala 
# 125 
- Lys Met His Gln Tyr Glu Ala Val Arg Ser As - #p Glu Ala Val Tyr Gly 
# 140 
- Lys Gly Ala Lys Arg Thr Val Pro Asp Glu Pr - #o Thr Leu Ala Glu His 
145 1 - #50 1 - #55 1 - 
#60 
- Leu Ala Arg Lys Ala Ala Ala Ile Trp Ala Le - #u Glu Glu Pro Leu Met 
# 175 
- Asp Glu Leu Arg Arg Asn Glu Gln Asp Arg Le - #u Leu Thr Glu Leu Glu 
# 190 
- Gln Pro Leu Ala Gly Ile Leu Ala Asn Met Gl - #u Phe Thr Gly Val Lys 
# 205 
- Val Asp Thr Lys Arg Leu Glu Gln Met Gly Al - #a Glu Leu Thr Glu Gln 
# 220 
- Leu Gln Ala Val Glu Arg Arg Ile Tyr Glu Le - #u Ala Gly Gln Glu Phe 
225 2 - #30 2 - #35 2 - 
#40 
- Asn Ile Asn Ser Pro Lys Gln Leu Gly Thr Va - #l Leu Phe Asp Lys Leu 
# 255 
- Gln Leu Pro Val Leu Lys Lys Thr Lys Thr Gl - #y Tyr Ser Thr Ser Ala 
# 270 
- Asp Val Leu Glu Lys Leu Ala Pro His His Gl - #u Ile Val Glu His Ile 
# 285 
- Leu His Tyr Arg Gln Leu Gly Lys Leu Gln Se - #r Thr Tyr Ile Glu Gly 
# 300 
- Leu Leu Lys Val Val His Pro Val Thr Gly Ly - #s Val His Thr Met Phe 
305 3 - #10 3 - #15 3 - 
#20 
- Asn Gln Ala Leu Thr Gln Thr Gly Arg Leu Se - #r Ser Val Glu Pro Asn 
# 335 
- Leu Gln Asn Ile Pro Ile Arg Leu Glu Glu Gl - #y Arg Lys Ile Arg Gln 
# 350 
- Ala Phe Val Pro Ser Glu Pro Asp Trp Leu Il - #e Phe Ala Ala Asp Tyr 
# 365 
- Ser Gln Ile Glu Leu Arg Val Leu Ala His Il - #e Ala Glu Asp Asp Asn 
# 380 
- Leu Ile Glu Ala Phe Arg Arg Gly Leu Asp Il - #e His Thr Lys Thr Ala 
385 3 - #90 3 - #95 4 - 
#00 
- Met Asp Ile Phe His Val Ser Glu Glu Asp Va - #l Thr Ala Asn Met Arg 
# 415 
- Arg Gln Ala Lys Ala Val Asn Phe Gly Ile Va - #l Tyr Gly Ile Ser Asp 
# 430 
- Tyr Gly Leu Ala Gln Asn Leu Asn Ile Thr Ar - #g Lys Glu Ala Ala Glu 
# 445 
- Phe Ile Glu Arg Tyr Phe Ala Ser Phe Pro Gl - #y Val Lys Gln Tyr Met 
# 460 
- Asp Asn Ile Val Gln Glu Ala Lys Gln Lys Gl - #y Tyr Val Thr Thr Leu 
465 4 - #70 4 - #75 4 - 
#80 
- Leu His Arg Arg Arg Tyr Leu Pro Asp Ile Th - #r Ser Arg Asn Phe Asn 
# 495 
- Val Arg Ser Phe Ala Glu Arg Thr Ala Met As - #n Thr Pro Ile Gln Gly 
# 510 
- Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Il - #e Asp Leu Ser Val Arg 
# 525 
- Leu Arg Glu Glu Arg Leu Gln Ala Arg Leu Le - #u Leu Gln Val His Asp 
# 540 
- Glu Leu Ile Leu Glu Ala Pro Lys Glu Glu Il - #e Glu Arg Leu Cys Arg 
545 5 - #50 5 - #55 5 - 
#60 
- Leu Val Pro Glu Val Met Glu Gln Ala Val Al - #a Leu Arg Val Pro Leu 
# 575 
- Lys Val Asp Tyr His Tyr Gly Pro Thr Trp Ty - #r Asp Ala Lys 
# 590 
- (2) INFORMATION FOR SEQ ID NO:28: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 99 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- AACGCAGTCT ACGGGTTTAC GATGATGTTA AACAAAATTT TGGCGGAAGA GC - #AGCCGACC 
60 
# 99 TTGA CGCCGGGAAA ACGACGTTC 
- (2) INFORMATION FOR SEQ ID NO:29: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 97 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- GCAGCGGAAA CTGTTCCGAC AGTTCCGGCG GCGTCTGCTG CCGCCCGCCT TT - #AAAGTCTT 
60 
# 97 GAAC GTCGTTTTCC CGGCGTC 
- (2) INFORMATION FOR SEQ ID NO:30: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 97 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- GCAGCGGAAA CTGTTCCGAC AGTTCCGGCG GCGTCTGCTG CCGGCCGCCT TT - #CGCGTCTT 
60 
# 97 GAAC GTCGTTTTCC CGGCGTC 
- (2) INFORMATION FOR SEQ ID NO:31: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2631 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...2631 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- TTG AAA AAC AAG CTC GTC TTA ATT GAC GGC AA - #C AGC GTG GCG TAC CGC 
48 
Leu Lys Asn Lys Leu Val Leu Ile Asp Gly As - #n Ser Val Ala Tyr Arg 
# 15 
- GCC TTT TTC GCG TTG CCG CTT TTG CAT AAC GA - #T AAA GGG ATT CAT ACG 
96 
Ala Phe Phe Ala Leu Pro Leu Leu His Asn As - #p Lys Gly Ile His Thr 
# 30 
- AAC GCA GTC TAC GGG TTT ACG ATG ATG TTA AA - #C AAA ATT TTG GCG GAA 
144 
Asn Ala Val Tyr Gly Phe Thr Met Met Leu As - #n Lys Ile Leu Ala Glu 
# 45 
- GAG CAG CCG ACC CAC ATT CTC GTG GCG TTT GA - #C GCC GGG AAA ACG ACG 
192 
Glu Gln Pro Thr His Ile Leu Val Ala Phe As - #p Ala Gly Lys Thr Thr 
# 60 
- TTC CGC CAT GAA ACG TTC CAA GAC TTT AAA GG - #C GGG CGG CAG CAG ACG 
240 
Phe Arg His Glu Thr Phe Gln Asp Phe Lys Gl - #y Gly Arg Gln Gln Thr 
#80 
- CCG CCG GAA CTG TCG GAA CAG TTT CCG CTG CT - #G CGC GAA TTG CTC AAG 
288 
Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Le - #u Arg Glu Leu Leu Lys 
# 95 
- GCG TAC CGC ATC CCC GCC TAT GAG CTC GAC CA - #T TAC GAA GCG GAC GAT 
336 
Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp Hi - #s Tyr Glu Ala Asp Asp 
# 110 
- ATT ATC GGA ACG ATG GCG GCG CGG GCT GAG CG - #A GAA GGG TTT GCA GTG 
384 
Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Ar - #g Glu Gly Phe Ala Val 
# 125 
- AAA GTC ATT TCC GGC GAC CGC GAT TTA ACC CA - #G CTT GCT TCC CCG CAA 
432 
Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gl - #n Leu Ala Ser Pro Gln 
# 140 
- GTG ACG GTG GAG ATT ACG AAA AAA GGG ATT AC - #C GAC ATC GAG TCG TAC 
480 
Val Thr Val Glu Ile Thr Lys Lys Gly Ile Th - #r Asp Ile Glu Ser Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- ACG CCG GAG ACG GTC GTG GAA AAA TAC GGC CT - #C ACC CCG GAG CAA ATT 
528 
Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Le - #u Thr Pro Glu Gln Ile 
# 175 
- GTC GAC TTG AAA GGA TTG ATG GGC GAC AAA TC - #C GAC AAC ATC CCT GGC 
576 
Val Asp Leu Lys Gly Leu Met Gly Asp Lys Se - #r Asp Asn Ile Pro Gly 
# 190 
- GTG CCC GGC ATC GGG GAA AAA ACA GCC GTC AA - #G CTG CTC AAG CAA TTC 
624 
Val Pro Gly Ile Gly Glu Lys Thr Ala Val Ly - #s Leu Leu Lys Gln Phe 
# 205 
- GGC ACG GTC GAA AAC GTA CTG GCA TCG ATC GA - #T GAG ATC AAA GGG GAG 
672 
Gly Thr Val Glu Asn Val Leu Ala Ser Ile As - #p Glu Ile Lys Gly Glu 
# 220 
- AAG CTG AAA GAA AAT TTG CGC CAA TAC CGG GA - #T TTG GCG CTT TTA AGC 
720 
Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg As - #p Leu Ala Leu Leu Ser 
225 2 - #30 2 - #35 2 - 
#40 
- AAA CAG CTG GCC GCT ATT TGC CGC GAC GCC CC - #G GTT GAG CTG ACG CTC 
768 
Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pr - #o Val Glu Leu Thr Leu 
# 255 
- GAT GAC ATT GTC TAC AAA GGA GAA GAC CGG GA - #A AAA GTG GTC GCC TTG 
816 
Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Gl - #u Lys Val Val Ala Leu 
# 270 
- TTT CAG GAG CTC GGA TTC CAG TCG TTT CTC GA - #C AAG ATG GCC GTC CAA 
864 
Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu As - #p Lys Met Ala Val Gln 
# 285 
- ACG GAT GAA GGC GAA AAG CCG CTC GCC GGG AT - #G GAT TTT GCG ATC GCC 
912 
Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 300 
- GAC AGC GTC ACG GAC GAA ATG CTC GCC GAC AA - #A GCG GCC CTC GTC GTG 
960 
Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- GAG GTG GTG GGC GAC AAC TAT CAC CAT GCC CC - #G ATT GTC GGG ATC GCC 
1008 
Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 335 
- TTG GCC AAC GAA CGC GGG CGG TTT TTC CTG CG - #C CCG GAG ACG GCG CTC 
1056 
Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 350 
- GCC GAT CCG AAA TTT CTC GCT TGG CTT GGC GA - #T GAG ACG AAG AAA AAA 
1104 
Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
# 365 
- ACG ATG TTT GAT TCA AAG CGG GCG GCC GTC GC - #G CTA AAA TGG AAA GGA 
1152 
Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 380 
- ATC GAA CTG CGC GGC GTC GTG TTC GAT CTG TT - #G CTG GCC GCT TAC TTG 
1200 
Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
385 3 - #90 3 - #95 4 - 
#00 
- CTC GAT CCG GCG CAG GCG GCG GGC GAC GTT GC - #C GCG GTG GCG AAA ATG 
1248 
Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 415 
- CAT CAG TAC GAG GCG GTG CGA TCG GAT GAG GC - #G GTC TAT GGA AAA GGA 
1296 
His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 430 
- GCG AAG CGG ACG GTT CCT GAT GAA CCG ACG CT - #T GCC GAG CAT CTC GCC 
1344 
Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
# 445 
- CGC AAG GCG GCG GCC ATT TGG GCG CTT GAA GA - #G CCG TTG ATG GAC GAA 
1392 
Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 460 
- CTG CGC CGC AAC GAA CAA GAT CGG CTG CTG AC - #C GAG CTC GAA CAG CCG 
1440 
Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
465 4 - #70 4 - #75 4 - 
#80 
- CTG GCT GGC ATT TTG GCC AAT ATG GAA TTT AC - #T GGA GTG AAA GTG GAC 
1488 
Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 495 
- ACG AAG CGG CTT GAA CAG ATG GGG GCG GAG CT - #C ACC GAG CAG CTG CAG 
1536 
Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 510 
- GCG GTC GAG CGG CGC ATT TAC GAA CTC GCC GG - #C CAA GAG TTC AAC ATT 
1584 
Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
# 525 
- AAC TCG CCG AAA CAG CTC GGG ACG GTT TTA TT - #T GAC AAG CTG CAG CTC 
1632 
Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 540 
- CCG GTG TTG AAA AAG ACA AAA ACC GGC TAT TC - #G ACT TCA GCC GAT GTG 
1680 
Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
545 5 - #50 5 - #55 5 - 
#60 
- CTT GAG AAG CTT GCA CCG CAC CAT GAA ATC GT - #C GAA CAT ATT TTG CAT 
1728 
Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 575 
- TAC CGC CAA CTC GGC AAG CTG CAG TCA ACG TA - #T ATT GAA GGG CTG CTG 
1776 
Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 590 
- AAA GTG GTG CAC CCC GTG ACG GGC AAA GTG CA - #C ACG ATG TTC AAT CAG 
1824 
Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
# 605 
- GCG TTG ACG CAA ACC GGG CGC CTC AGC TCC GT - #C GAA CCG AAT TTG CAA 
1872 
Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 620 
- AAC ATT CCG ATT CGG CTT GAG GAA GGG CGG AA - #A ATC CGC CAG GCG TTC 
1920 
Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
625 6 - #30 6 - #35 6 - 
#40 
- GTG CCG TCG GAG CCG GAC TGG CTC ATC TTT GC - #G GCC GAC TAT TCG CAA 
1968 
Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 655 
- ATC GAG CTG CGC GTC CTC GCC CAT ATC GCG GA - #A GAT GAC AAT TTG ATT 
2016 
Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 670 
- GAA GCG TTC CGG CGC GGG TTG GAC ATC CAT AC - #G AAA ACA GCC ATG GAC 
2064 
Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
# 685 
- ATT TTC CAT GTG AGC GAA GAA GAC GTG ACA GC - #C AAC ATG CGC CGC CAA 
2112 
Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 700 
- GCG AAG GCC GTC AAT TTT GGC ATC GTG TAC GG - #C ATT AGT GAT TAC GGT 
2160 
Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
705 7 - #10 7 - #15 7 - 
#20 
- CTG GCG CAA AAC TTG AAC ATT ACG CGC AAA GA - #A GCG GCT GAA TTT ATT 
2208 
Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 735 
- GAG CGA TAT TTT GCC AGT TTT CCA GGT GTA AA - #G CAA TAT ATG GAC AAC 
2256 
Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 750 
- ATT GTG CAA GAA GCG AAA CAA AAA GGG TAT GT - #G ACG ACG CTG CTG CAT 
2304 
Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
# 765 
- CGG CGC CGC TAT TTG CCC GAT ATT ACA AGC CG - #C AAC TTC AAC GTC CGC 
2352 
Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 780 
- AGC TTC GCC GAG CGG ACG GCG ATG AAC ACA CC - #G ATC CAA GGG AGT GCC 
2400 
Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
785 7 - #90 7 - #95 8 - 
#00 
- GCT GAT ATT ATT AAA AAA GCG ATG ATC GAT CT - #A AGC GTG AGG CTG CGC 
2448 
Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 815 
- GAA GAA CGG CTG CAG GCG CGC CTG TTG CTG CA - #A GTG CAT GAC GAA CTC 
2496 
Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 830 
- ATT TTG GAG GCG CCG AAA GAG GAA ATC GAG CG - #G CTG TGC CGC CTC GTT 
2544 
Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
# 845 
- CCA GAG GTG ATG GAG CAA GCC GTC GCA CTC CG - #C GTG CCG CTG AAA GTC 
2592 
Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 860 
# 2631C CAT TAC GGT CCG ACG TGG TAC GAC GC - #C AAA TAA 
Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys * 
865 8 - #70 8 - #75 
- (2) INFORMATION FOR SEQ ID NO:32: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 876 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- Leu Lys Asn Lys Leu Val Leu Ile Asp Gly As - #n Ser Val Ala Tyr Arg 
# 15 
- Ala Phe Phe Ala Leu Pro Leu Leu His Asn As - #p Lys Gly Ile His Thr 
# 30 
- Asn Ala Val Tyr Gly Phe Thr Met Met Leu As - #n Lys Ile Leu Ala Glu 
# 45 
- Glu Gln Pro Thr His Ile Leu Val Ala Phe As - #p Ala Gly Lys Thr Thr 
# 60 
- Phe Arg His Glu Thr Phe Gln Asp Phe Lys Gl - #y Gly Arg Gln Gln Thr 
#80 
- Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Le - #u Arg Glu Leu Leu Lys 
# 95 
- Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp Hi - #s Tyr Glu Ala Asp Asp 
# 110 
- Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Ar - #g Glu Gly Phe Ala Val 
# 125 
- Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gl - #n Leu Ala Ser Pro Gln 
# 140 
- Val Thr Val Glu Ile Thr Lys Lys Gly Ile Th - #r Asp Ile Glu Ser Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Le - #u Thr Pro Glu Gln Ile 
# 175 
- Val Asp Leu Lys Gly Leu Met Gly Asp Lys Se - #r Asp Asn Ile Pro Gly 
# 190 
- Val Pro Gly Ile Gly Glu Lys Thr Ala Val Ly - #s Leu Leu Lys Gln Phe 
# 205 
- Gly Thr Val Glu Asn Val Leu Ala Ser Ile As - #p Glu Ile Lys Gly Glu 
# 220 
- Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg As - #p Leu Ala Leu Leu Ser 
225 2 - #30 2 - #35 2 - 
#40 
- Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pr - #o Val Glu Leu Thr Leu 
# 255 
- Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Gl - #u Lys Val Val Ala Leu 
# 270 
- Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu As - #p Lys Met Ala Val Gln 
# 285 
- Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 300 
- Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 335 
- Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 350 
- Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
# 365 
- Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 380 
- Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
385 3 - #90 3 - #95 4 - 
#00 
- Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 415 
- His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 430 
- Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
# 445 
- Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 460 
- Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
465 4 - #70 4 - #75 4 - 
#80 
- Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 495 
- Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 510 
- Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
# 525 
- Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 540 
- Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
545 5 - #50 5 - #55 5 - 
#60 
- Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 575 
- Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 590 
- Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
# 605 
- Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 620 
- Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
625 6 - #30 6 - #35 6 - 
#40 
- Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 655 
- Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 670 
- Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
# 685 
- Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 700 
- Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
705 7 - #10 7 - #15 7 - 
#20 
- Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 735 
- Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 750 
- Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
# 765 
- Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 780 
- Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
785 7 - #90 7 - #95 8 - 
#00 
- Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 815 
- Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 830 
- Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
# 845 
- Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 860 
- Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
865 8 - #70 8 - #75 
- (2) INFORMATION FOR SEQ ID NO:33: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2631 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...2631 
(D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
- TTG AAA AAC AAG CTC GTC TTA ATT GAC GGC AA - #C AGC GTG GCG TAC CGC 
48 
Leu Lys Asn Lys Leu Val Leu Ile Asp Gly As - #n Ser Val Ala Tyr Arg 
# 15 
- GCC TTT TTC GCG TTG CCG CTT TTG CAT AAC GA - #T AAA GGG ATT CAT ACG 
96 
Ala Phe Phe Ala Leu Pro Leu Leu His Asn As - #p Lys Gly Ile His Thr 
# 30 
- AAC GCA GTC TAC GGG TTT ACG ATG ATG TTA AA - #C AAA ATT TTG GCG GAA 
144 
Asn Ala Val Tyr Gly Phe Thr Met Met Leu As - #n Lys Ile Leu Ala Glu 
# 45 
- GAG CAG CCG ACC CAC ATT CTC GTG GCG TTT GA - #C GCC GGG AAA ACG ACG 
192 
Glu Gln Pro Thr His Ile Leu Val Ala Phe As - #p Ala Gly Lys Thr Thr 
# 60 
- TTC CGC CAT GAA ACG TTC CAA GAC GCG AAA GG - #C GGC CGG CAG CAG ACG 
240 
Phe Arg His Glu Thr Phe Gln Asp Ala Lys Gl - #y Gly Arg Gln Gln Thr 
#80 
- CCG CCG GAA CTG TCG GAA CAG TTT CCG CTG CT - #G CGC GAA TTG CTC AAG 
288 
Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Le - #u Arg Glu Leu Leu Lys 
# 95 
- GCG TAC CGC ATC CCC GCC TAT GAG CTC GAC CA - #T TAC GAA GCG GAC GAT 
336 
Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp Hi - #s Tyr Glu Ala Asp Asp 
# 110 
- ATT ATC GGA ACG ATG GCG GCG CGG GCT GAG CG - #A GAA GGG TTT GCA GTG 
384 
Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Ar - #g Glu Gly Phe Ala Val 
# 125 
- AAA GTC ATT TCC GGC GAC CGC GAT TTA ACC CA - #G CTT GCT TCC CCG CAA 
432 
Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gl - #n Leu Ala Ser Pro Gln 
# 140 
- GTG ACG GTG GAG ATT ACG AAA AAA GGG ATT AC - #C GAC ATC GAG TCG TAC 
480 
Val Thr Val Glu Ile Thr Lys Lys Gly Ile Th - #r Asp Ile Glu Ser Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- ACG CCG GAG ACG GTC GTG GAA AAA TAC GGC CT - #C ACC CCG GAG CAA ATT 
528 
Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Le - #u Thr Pro Glu Gln Ile 
# 175 
- GTC GAC TTG AAA GGA TTG ATG GGC GAC AAA TC - #C GAC AAC ATC CCT GGC 
576 
Val Asp Leu Lys Gly Leu Met Gly Asp Lys Se - #r Asp Asn Ile Pro Gly 
# 190 
- GTG CCC GGC ATC GGG GAA AAA ACA GCC GTC AA - #G CTG CTC AAG CAA TTC 
624 
Val Pro Gly Ile Gly Glu Lys Thr Ala Val Ly - #s Leu Leu Lys Gln Phe 
# 205 
- GGC ACG GTC GAA AAC GTA CTG GCA TCG ATC GA - #T GAG ATC AAA GGG GAG 
672 
Gly Thr Val Glu Asn Val Leu Ala Ser Ile As - #p Glu Ile Lys Gly Glu 
# 220 
- AAG CTG AAA GAA AAT TTG CGC CAA TAC CGG GA - #T TTG GCG CTT TTA AGC 
720 
Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg As - #p Leu Ala Leu Leu Ser 
225 2 - #30 2 - #35 2 - 
#40 
- AAA CAG CTG GCC GCT ATT TGC CGC GAC GCC CC - #G GTT GAG CTG ACG CTC 
768 
Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pr - #o Val Glu Leu Thr Leu 
# 255 
- GAT GAC ATT GTC TAC AAA GGA GAA GAC CGG GA - #A AAA GTG GTC GCC TTG 
816 
Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Gl - #u Lys Val Val Ala Leu 
# 270 
- TTT CAG GAG CTC GGA TTC CAG TCG TTT CTC GA - #C AAG ATG GCC GTC CAA 
864 
Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu As - #p Lys Met Ala Val Gln 
# 285 
- ACG GAT GAA GGC GAA AAG CCG CTC GCC GGG AT - #G GAT TTT GCG ATC GCC 
912 
Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 300 
- GAC AGC GTC ACG GAC GAA ATG CTC GCC GAC AA - #A GCG GCC CTC GTC GTG 
960 
Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- GAG GTG GTG GGC GAC AAC TAT CAC CAT GCC CC - #G ATT GTC GGG ATC GCC 
1008 
Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 335 
- TTG GCC AAC GAA CGC GGG CGG TTT TTC CTG CG - #C CCG GAG ACG GCG CTC 
1056 
Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 350 
- GCC GAT CCG AAA TTT CTC GCT TGG CTT GGC GA - #T GAG ACG AAG AAA AAA 
1104 
Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
# 365 
- ACG ATG TTT GAT TCA AAG CGG GCG GCC GTC GC - #G CTA AAA TGG AAA GGA 
1152 
Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 380 
- ATC GAA CTG CGC GGC GTC GTG TTC GAT CTG TT - #G CTG GCC GCT TAC TTG 
1200 
Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
385 3 - #90 3 - #95 4 - 
#00 
- CTC GAT CCG GCG CAG GCG GCG GGC GAC GTT GC - #C GCG GTG GCG AAA ATG 
1248 
Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 415 
- CAT CAG TAC GAG GCG GTG CGA TCG GAT GAG GC - #G GTC TAT GGA AAA GGA 
1296 
His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 430 
- GCG AAG CGG ACG GTT CCT GAT GAA CCG ACG CT - #T GCC GAG CAT CTC GCC 
1344 
Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
# 445 
- CGC AAG GCG GCG GCC ATT TGG GCG CTT GAA GA - #G CCG TTG ATG GAC GAA 
1392 
Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 460 
- CTG CGC CGC AAC GAA CAA GAT CGG CTG CTG AC - #C GAG CTC GAA CAG CCG 
1440 
Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
465 4 - #70 4 - #75 4 - 
#80 
- CTG GCT GGC ATT TTG GCC AAT ATG GAA TTT AC - #T GGA GTG AAA GTG GAC 
1488 
Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 495 
- ACG AAG CGG CTT GAA CAG ATG GGG GCG GAG CT - #C ACC GAG CAG CTG CAG 
1536 
Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 510 
- GCG GTC GAG CGG CGC ATT TAC GAA CTC GCC GG - #C CAA GAG TTC AAC ATT 
1584 
Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
# 525 
- AAC TCG CCG AAA CAG CTC GGG ACG GTT TTA TT - #T GAC AAG CTG CAG CTC 
1632 
Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 540 
- CCG GTG TTG AAA AAG ACA AAA ACC GGC TAT TC - #G ACT TCA GCC GAT GTG 
1680 
Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
545 5 - #50 5 - #55 5 - 
#60 
- CTT GAG AAG CTT GCA CCG CAC CAT GAA ATC GT - #C GAA CAT ATT TTG CAT 
1728 
Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 575 
- TAC CGC CAA CTC GGC AAG CTG CAG TCA ACG TA - #T ATT GAA GGG CTG CTG 
1776 
Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 590 
- AAA GTG GTG CAC CCC GTG ACG GGC AAA GTG CA - #C ACG ATG TTC AAT CAG 
1824 
Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
# 605 
- GCG TTG ACG CAA ACC GGG CGC CTC AGC TCC GT - #C GAA CCG AAT TTG CAA 
1872 
Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 620 
- AAC ATT CCG ATT CGG CTT GAG GAA GGG CGG AA - #A ATC CGC CAG GCG TTC 
1920 
Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
625 6 - #30 6 - #35 6 - 
#40 
- GTG CCG TCG GAG CCG GAC TGG CTC ATC TTT GC - #G GCC GAC TAT TCG CAA 
1968 
Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 655 
- ATC GAG CTG CGC GTC CTC GCC CAT ATC GCG GA - #A GAT GAC AAT TTG ATT 
2016 
Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 670 
- GAA GCG TTC CGG CGC GGG TTG GAC ATC CAT AC - #G AAA ACA GCC ATG GAC 
2064 
Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
# 685 
- ATT TTC CAT GTG AGC GAA GAA GAC GTG ACA GC - #C AAC ATG CGC CGC CAA 
2112 
Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 700 
- GCG AAG GCC GTC AAT TTT GGC ATC GTG TAC GG - #C ATT AGT GAT TAC GGT 
2160 
Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
705 7 - #10 7 - #15 7 - 
#20 
- CTG GCG CAA AAC TTG AAC ATT ACG CGC AAA GA - #A GCG GCT GAA TTT ATT 
2208 
Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 735 
- GAG CGA TAT TTT GCC AGT TTT CCA GGT GTA AA - #G CAA TAT ATG GAC AAC 
2256 
Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 750 
- ATT GTG CAA GAA GCG AAA CAA AAA GGG TAT GT - #G ACG ACG CTG CTG CAT 
2304 
Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
# 765 
- CGG CGC CGC TAT TTG CCC GAT ATT ACA AGC CG - #C AAC TTC AAC GTC CGC 
2352 
Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 780 
- AGC TTC GCC GAG CGG ACG GCG ATG AAC ACA CC - #G ATC CAA GGG AGT GCC 
2400 
Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
785 7 - #90 7 - #95 8 - 
#00 
- GCT GAT ATT ATT AAA AAA GCG ATG ATC GAT CT - #A AGC GTG AGG CTG CGC 
2448 
Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 815 
- GAA GAA CGG CTG CAG GCG CGC CTG TTG CTG CA - #A GTG CAT GAC GAA CTC 
2496 
Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 830 
- ATT TTG GAG GCG CCG AAA GAG GAA ATC GAG CG - #G CTG TGC CGC CTC GTT 
2544 
Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
# 845 
- CCA GAG GTG ATG GAG CAA GCC GTC GCA CTC CG - #C GTG CCG CTG AAA GTC 
2592 
Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 860 
# 2631C CAT TAC GGT CCG ACG TGG TAC GAC GC - #C AAA TAA 
Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
865 8 - #70 8 - #75 
- (2) INFORMATION FOR SEQ ID NO:34: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 876 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
- Leu Lys Asn Lys Leu Val Leu Ile Asp Gly As - #n Ser Val Ala Tyr Arg 
# 15 
- Ala Phe Phe Ala Leu Pro Leu Leu His Asn As - #p Lys Gly Ile His Thr 
# 30 
- Asn Ala Val Tyr Gly Phe Thr Met Met Leu As - #n Lys Ile Leu Ala Glu 
# 45 
- Glu Gln Pro Thr His Ile Leu Val Ala Phe As - #p Ala Gly Lys Thr Thr 
# 60 
- Phe Arg His Glu Thr Phe Gln Asp Ala Lys Gl - #y Gly Arg Gln Gln Thr 
#80 
- Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Le - #u Arg Glu Leu Leu Lys 
# 95 
- Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp Hi - #s Tyr Glu Ala Asp Asp 
# 110 
- Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Ar - #g Glu Gly Phe Ala Val 
# 125 
- Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gl - #n Leu Ala Ser Pro Gln 
# 140 
- Val Thr Val Glu Ile Thr Lys Lys Gly Ile Th - #r Asp Ile Glu Ser Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Le - #u Thr Pro Glu Gln Ile 
# 175 
- Val Asp Leu Lys Gly Leu Met Gly Asp Lys Se - #r Asp Asn Ile Pro Gly 
# 190 
- Val Pro Gly Ile Gly Glu Lys Thr Ala Val Ly - #s Leu Leu Lys Gln Phe 
# 205 
- Gly Thr Val Glu Asn Val Leu Ala Ser Ile As - #p Glu Ile Lys Gly Glu 
# 220 
- Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg As - #p Leu Ala Leu Leu Ser 
225 2 - #30 2 - #35 2 - 
#40 
- Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pr - #o Val Glu Leu Thr Leu 
# 255 
- Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Gl - #u Lys Val Val Ala Leu 
# 270 
- Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu As - #p Lys Met Ala Val Gln 
# 285 
- Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Me - #t Asp Phe Ala Ile Ala 
# 300 
- Asp Ser Val Thr Asp Glu Met Leu Ala Asp Ly - #s Ala Ala Leu Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- Glu Val Val Gly Asp Asn Tyr His His Ala Pr - #o Ile Val Gly Ile Ala 
# 335 
- Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Ar - #g Pro Glu Thr Ala Leu 
# 350 
- Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly As - #p Glu Thr Lys Lys Lys 
# 365 
- Thr Met Phe Asp Ser Lys Arg Ala Ala Val Al - #a Leu Lys Trp Lys Gly 
# 380 
- Ile Glu Leu Arg Gly Val Val Phe Asp Leu Le - #u Leu Ala Ala Tyr Leu 
385 3 - #90 3 - #95 4 - 
#00 
- Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Al - #a Ala Val Ala Lys Met 
# 415 
- His Gln Tyr Glu Ala Val Arg Ser Asp Glu Al - #a Val Tyr Gly Lys Gly 
# 430 
- Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Le - #u Ala Glu His Leu Ala 
# 445 
- Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gl - #u Pro Leu Met Asp Glu 
# 460 
- Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Th - #r Glu Leu Glu Gln Pro 
465 4 - #70 4 - #75 4 - 
#80 
- Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Th - #r Gly Val Lys Val Asp 
# 495 
- Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Le - #u Thr Glu Gln Leu Gln 
# 510 
- Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gl - #y Gln Glu Phe Asn Ile 
# 525 
- Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Ph - #e Asp Lys Leu Gln Leu 
# 540 
- Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Se - #r Thr Ser Ala Asp Val 
545 5 - #50 5 - #55 5 - 
#60 
- Leu Glu Lys Leu Ala Pro His His Glu Ile Va - #l Glu His Ile Leu His 
# 575 
- Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Ty - #r Ile Glu Gly Leu Leu 
# 590 
- Lys Val Val His Pro Val Thr Gly Lys Val Hi - #s Thr Met Phe Asn Gln 
# 605 
- Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Va - #l Glu Pro Asn Leu Gln 
# 620 
- Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Ly - #s Ile Arg Gln Ala Phe 
625 6 - #30 6 - #35 6 - 
#40 
- Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Al - #a Ala Asp Tyr Ser Gln 
# 655 
- Ile Glu Leu Arg Val Leu Ala His Ile Ala Gl - #u Asp Asp Asn Leu Ile 
# 670 
- Glu Ala Phe Arg Arg Gly Leu Asp Ile His Th - #r Lys Thr Ala Met Asp 
# 685 
- Ile Phe His Val Ser Glu Glu Asp Val Thr Al - #a Asn Met Arg Arg Gln 
# 700 
- Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gl - #y Ile Ser Asp Tyr Gly 
705 7 - #10 7 - #15 7 - 
#20 
- Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Gl - #u Ala Ala Glu Phe Ile 
# 735 
- Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Ly - #s Gln Tyr Met Asp Asn 
# 750 
- Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Va - #l Thr Thr Leu Leu His 
# 765 
- Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Ar - #g Asn Phe Asn Val Arg 
# 780 
- Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pr - #o Ile Gln Gly Ser Ala 
785 7 - #90 7 - #95 8 - 
#00 
- Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Le - #u Ser Val Arg Leu Arg 
# 815 
- Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gl - #n Val His Asp Glu Leu 
# 830 
- Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Ar - #g Leu Cys Arg Leu Val 
# 845 
- Pro Glu Val Met Glu Gln Ala Val Ala Leu Ar - #g Val Pro Leu Lys Val 
# 860 
- Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Al - #a Lys 
865 8 - #70 8 - #75 
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