Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL POLYNUCLEOTIDES AND METHOD OF USE THEREOF
FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of
novel nucleic acid molecules
which constitute at least a portion of full-length cDNA molecules that encode
human polypeptides.
BACKGROUND OF THE INVENTION
Extracellular proteins play important roles in, among other things, the
formation, differentiation and
maintenance of multicellular organisms. The fate of many individual cells,
e.g., proliferation, migration,
differentiation, or interaction with other cells, is typically governed by
information received from other cells
and/or the immediate environment. This information is often transmitted by
secreted polypeptides (for instance,
mitogenic factors, survival factors, cytotoxic factors, differentiation
factors, neuropeptides, and hormones) which
are, in turn, received and interpreted by diverse cell receptors or membrane-
bound proteins. These secreted
polypeptides or signaling molecules normally pass through the cellular
secretory pathway to reach their site of
action in the extracellular environment.
Secreted proteins have various industrial applications, including as
pharmaceuticals, diagnostics,
biosensors and bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons,
interleukins, erythropoietins, colony stimulating factors, and various other
cytokines, are secretory proteins.
Their receptors, which are membrane proteins, also have potential as
therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new, native
secreted proteins. Many efforts are
focused on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel
secreted proteins. Examples of screening methods and techniques are described
in the literature [see, for
example, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S.
Patent No. 5,536,637)].
Membrane-bound proteins and receptors can play important roles in, among other
things, the formation,
differentiation and maintenance of multicellular organisms. The fate of many
individual cells, e. g. , proliferation,
migration, differentiation, or interaction with other cells, is typically
governed by information received from
other cells and/or the immediate environment. This information is often
transmitted by secreted polypeptides
(for instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and
hormones) which are, in turn, received and interpreted by diverse cell
receptors or membrane-bound proteins.
Such membrane-bound proteins and cell receptors include, but are not limited
to, cytokine receptors, receptor
kinases, receptor phosphatases, receptors involved in cell-cell interactions,
and cellular adhesin molecules like
selectins and integrins. For instance, transduction of signals that regulate
cell growth and differentiation is
regulated in part by phosphorylation of various cellular proteins. Protein
tyrosine kinases, enzymes that catalyze
that process, can also act as growth factor receptors. Examples include
fibroblast growth factor receptor and
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nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial
applications, including as
pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance,
can be employed as therapeutic
agents to block receptor-ligand interactions. The membrane-bound proteins can
also be employed for screening
of potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. Efforts are being
undertaken by both industry and academia to identify new, native receptor or
membrane-bound proteins. Many
efforts are focused on the screening of mammalian recombinant DNA libraries to
identify the coding sequences
for novel receptor or membrane-bound proteins.
Recently, significant progress has been made in identifying and isolating
unique nucleic acid moelcules
which encode all or a portion of many mammalian proteins. We herein describe
the identification and
characterization of novel polynucleotides which constitute at least partial
cDNA molecules that encode various
human polypeptides.
SUMMARY OF THE INVENTION
Novel polynucleotides have been identified and isolated which constitute at
least partial cDNA molecules
that encode human polypeptides.
In one embodiment, the invention provides an isolated nucleic acid molecule
comprising any one of the
nucleic acid sequences shown in the accompanying figures, or the complement
thereof, or polynucleotide variants
of those nucleic acid sequences as defined below.
In another embodiment, the invention provides an isolated nucleic acid
molecule consisting essentially
of any one of the nucleic acid sequences shown in the accompanying figures, or
the complement thereof, or
polynucleotide variants of those nucleic acid sequences as defined below.
In another embodiment, the invention provides an isolated nucleic acid
molecule consisting of any one
of the nucleic acid sequences shown in the accompanying figures, or the
complement thereof, or polynucleotide
variants of those nucleic acid sequences as defined below.
In yet another embodiment, the invention provides an isolated nucleic acid
molecule that comprises a
nucleotide sequence having at least about 80 % sequence identity, preferably
at least about 81 % sequence identity,
more preferably at least about 82 % sequence identity, yet more preferably at
least about 83 % sequence identity,
yet more preferably at least about 84 % sequence identity, yet more preferably
at least about 85 % sequence
identity, yet more preferably at least about 86 % sequence identity, yet more
preferably at least about 87
sequence identity, yet more preferably at least about 88 % sequence identity,
yet more preferably at least about
89% sequence identity, yet more preferably at least about 90% sequence
identity, yet more preferably at least
about 91 % sequence identity, yet more preferably at least about 92 % sequence
identity, yet more preferably at
least about 93 % sequence identity, yet more preferably at least about 94 %
sequence identity, yet more preferably
at least about 95 % sequence identity, yet more preferably at least about 96 %
sequence identity, yet more
preferably at least about 97 % sequence identity, yet more preferably at least
about 98 % sequence identity and
yet more preferably at least about 99 % sequence identity to (a) the DNA
molecule of any one of Figure 1 to 562,
or (b) the complement of the DNA molecule of (a).
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In another aspect, the isolated nucleic acid molecule consists essentially of
a nucleotide sequence having
at least about 80 % sequence identity, preferably at least about 81 % sequence
identity, more preferably at least
about 82% sequence identity, yet more preferably at least about 83 % sequence
identity, yet more preferably at
least about 84 % sequence identity, yet more preferably at least about 85 %
sequence identity, yet more preferably
at least about 86% sequence identity, yet more preferably at least about 87%
sequence identity, yet more
preferably at least about 88% sequence identity, yet more preferably at least
about 89% sequence identity, yet
more preferably at least about 90 % sequence identity, yet more preferably at
least about 91 % sequence identity,
yet more preferably at least about 92 % sequence identity, yet more preferably
at least about 93 % sequence
identity, yet more preferably at least about 94% sequence identity, yet more
preferably at least about 95%
sequence identity, yet more preferably at least about 96 % sequence identity,
yet more preferably at least about
97 % sequence identity, yet more preferably at least about 98 % sequence
identity and yet more preferably at least
about 99% sequence identity to (a) the DNA molecule of any one of Figure 1 to
562, or (b) the complement of
the DNA molecule of (a).
In yet another aspect, the isolated nucleic acid molecule consists of a
nucleotide sequence having at least
about 80 % sequence identity, preferably at least about 81 % sequence
identity, more preferably at least about
82% sequence identity, yet more preferably at least about 83% sequence
identity, yet more preferably at least
about 84% sequence identity, yet more preferably at least about 85 % sequence
identity, yet more preferably at
least about 86 % sequence identity, yet more preferably at least about 87 %
sequence identity, yet more preferably
at least about 88 % sequence identity, yet more preferably at least about 89 %
sequence identity, yet more
preferably at least about 90% sequence identity, yet more preferably at least
about 91 % sequence identity, yet
more preferably at least about 92 % sequence identity, yet more preferably at
least about 93 % sequence identity,
yet more preferably at least about 94 % sequence identity, yet more preferably
at least about 95 % sequence
identity, yet more preferably at least about 96% sequence identity, yet more
preferably at least about 97%
sequence identity, yet more preferably at least about 98% sequence identity
and yet more preferably at least
about 99 % sequence identity to (a) the DNA molecule of any one of Figure 1 to
562, or (b) the complement of
the DNA molecule of (a).
In another embodiment, the invention concerns an isolated nucleic acid
molecule which comprises a
nucleotide sequence that hybridizes to (a) the DNA molecule of any one of
Figure 1 to 562, or (b) the
complement of the DNA molecule of (a). Preferably, hybridization occurs under
stringent hybridization and
wash conditions. Also, it is preferred that the isolated nucleic acid molecule
is fully complementary to (a) the
DNA molecule of any one of Figure 1 to 562, or (b) the complement of the DNA
molecule of (a).
In yet another embodiment, the present invention provides an isolated nucleic
acid molecule which
comprises at least about 10 consecutive nucleotides contained within (a) the
DNA molecule of any one of Figure
1 to 562, or (b) the complement of the DNA molecule of (a) which may fmd use
as, for example, hybridizing
oligonucleotide probes or for encoding polypeptide fragments that may
optionally comprise a binding site for
an antibody. In particular aspects, the isolated nucleic acid molecule is from
about 10 to about 1000, about 10
to about 900, about 10 to about 800, about 10 to about 700, about 10 to about
600, about 10 to about 500, about
10 to about 400, about 10 to about 300, about 10 to about 200, about 10 to
about 100, about 10 to about 90,
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about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to
about 50, about 10 to about 40,
about 10 to about 30 or about 10 to about 20 nucleotides in length, where the
term "about" means the referenced
nucleotide sequence length plus or minus 10 % of that referenced length. In
yet other aspects, the isolated nucleic
acid molecule comprises at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95 or 100
consecutive nucleotides contained within (a) the DNA molecule of any one of
Figure 1 to 562, or (b) the
complement of the DNA molecule of (a).
The present invention is also directed to a method of using an oligonucleotide
probe having a nucleotide
sequence derived from a nucleic acid molecule described herein for detecting
the presence of and/or obtaining
a full-length mammalian cDNA molecule from a mammalian cDNA library which
encodes a mammalian
polypeptide. Preferably, the mammal is human. The methods comprise the step of
screening a mammalian
cDNA library with one or more of the herein described oligonucleotides to
detect the presence of a full-length
cDNA and, optionally, obtaining the full-length cDNA from that library.
In another embodiment, the invention provides a vector comprising any of the
isolated nucleic acid
molecules described herein or their variants.
A host cell comprising such a vector is also provided. By way of example, the
host cells may be CHO
cells, E. coli, or yeast. A process for producing polypeptides is further
provided and comprises culturing the
host cells under conditions suitable for expression of a polypeptide and
recovering the polypeptide from the cell
culture.
In another embodiment, the invention provides isolated polypeptides encoded by
any of the isolated
nucleic acids described herein, wherein thise polypeptides are herein
designated as SRT polypeptides.
In yet another embodiment, the invention provides antibodies which
specifically bind to a polypeptide
encoded by a nucleic acid molecule described herein. Preferably, the
antibodies are monoclonal antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO:1) designated herein as
DNA8284.
Figure 2 shows a nucleotide sequence (SEQ ID N0:2) designated herein as
DNA8328.
Figure 3 shows a nucleotide sequence (SEQ ID N0:3) designated herein as
DNA8350.
Figure 4 shows a nucleotide sequence (SEQ ID N0:4) designated herein as
DNA8369.
Figure 5 shows a nucleotide sequence (SEQ ID NO:S) designated herein as
DNA8377.
Figure 6 shows a nucleotide sequence (SEQ ID N0:6) designated herein as
DNA8456.
Figure 7 shows a nucleotide sequence (SEQ ID N0:7) designated herein as
DNA8555.
Figure 8 shows a nucleotide sequence (SEQ ID N0:8) designated herein as
DNA8576.
Figure 9 shows a nucleotide sequence (SEQ ID N0:9) designated herein as
DNA9383.
Figure 10 shows a nucleotide sequence (SEQ ID NO:10) designated herein as
DNA9840.
Figure 11 shows a nucleotide sequence (SEQ ID NO:11) designated herein as
DNA10028.
Figure 12 shows a nucleotide sequence (SEQ ID N0:12) designated herein as
DNA10072.
Figure 13 shows a nucleotide sequence (SEQ ID N0:13) designated herein as
DNA10242.
Figure 14 shows a nucleotide sequence (SEQ ID N0:14) designated herein as
DNA10281.
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Figure 15 shows a nucleotide sequence (SEQ ID NO:15) designated herein as
DNA12628.
Figure 16 shows a nucleotide sequence (SEQ ID N0:16) designated herein as
DNA12646.
Figure 17 shows a nucleotide sequence (SEQ ID N0:17) designated herein as
DNA12655.
Figure 18 shows a nucleotide sequence (SEQ ID N0:18) designated herein as
DNA12660.
Figure 19 shows a nucleotide sequence (SEQ ID N0:19) designated herein as
DNA12668.
Figure 20 shows a nucleotide sequence (SEQ ID N0:20) designated herein as
DNA12726.
Figure 21 shows a nucleotide sequence (SEQ ID N0:21) designated herein as
DNA12728.
Figure 22 shows a nucleotide sequence (SEQ ID N0:22) designated herein as
DNA12729.
Figure 23 shows a nucleotide sequence (SEQ ID N0:23) designated herein as
DNA12732.
Figure 24 shows a nucleotide sequence (SEQ ID N0:24) designated herein as
DNA12733.
Figure 25 shows a nucleotide sequence (SEQ ID N0:25) designated herein as
DNA12741.
Figure 26 shows a nucleotide sequence (SEQ ID N0:26) designated herein as
DNA12742.
Figure 27 shows a nucleotide sequence (SEQ ID N0:27) designated herein as DNA
12747.
Figure 28 shows a nucleotide sequence (SEQ ID N0:28) designated herein as
DNA12752.
Figure 29 shows a nucleotide sequence (SEQ ID N0:29) designated herein as
DNA12797.
Figure 30 shows a nucleotide sequence (SEQ ID N0:30) designated herein as
DNA12801.
Figure 31 shows a nucleotide sequence (SEQ ID N0:31) designated herein as
DNA12802.
Figure 32 shows a nucleotide sequence (SEQ ID N0:32) designated herein as
DNA12817.
Figure 33 shows a nucleotide sequence (SEQ ID N0:33) designated herein as
DNA12819.
Figure 34 shows a nucleotide sequence (SEQ ID N0:34) designated herein as
DNA12829.
Figure 35 shows a nucleotide sequence (SEQ ID N0:35) designated herein as
DNA12830.
Figure 36 shows a nucleotide sequence (SEQ ID N0:36) designated herein as
DNA12834.
Figure 37 shows a nucleotide sequence (SEQ ID N0:37) designated herein as
DNA12837.
Figure 38 shows a nucleotide sequence (SEQ ID N0:38) designated herein as
DNA12840.
Figure 39 shows a nucleotide sequence (SEQ ID N0:39) designated herein as
DNA12841.
Figure 40 shows a nucleotide sequence (SEQ ID N0:40) designated herein as
DNA12844.
Figure 41 shows a nucleotide sequence (SEQ ID N0:41) designated herein as
DNA12846.
Figure 42 shows a nucleotide sequence (SEQ ID N0:42) designated herein as DNA
12850.
Figure 43 shows a nucleotide sequence (SEQ ID N0:43) designated herein as
DNA12865.
Figure 44 shows a nucleotide sequence (SEQ ID N0:44) designated herein as
DNA12867.
Figure 45 shows a nucleotide sequence (SEQ ID N0:45) designated herein as
DNA12884.
Figure 46 shows a nucleotide sequence (SEQ ID N0:46) designated herein as
DNA12889.
Figure 47 shows a nucleotide sequence (SEQ ID N0:47) designated herein as
DNA12891.
Figure 48 shows a nucleotide sequence (SEQ ID N0:48) designated herein as
DNA12900.
Figure 49 shows a nucleotide sequence (SEQ ID N0:49) designated herein as
DNA12922.
Figure 50 shows a nucleotide sequence (SEQ ID NO:50) designated herein as
DNA12946.
Figure 51 shows a nucleotide sequence (SEQ ID NO:51) designated herein as
DNA12967.
Figure 52 shows a nucleotide sequence (SEQ ID N0:52) designated herein as
DNA12974.
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Figure 53 shows a nucleotide sequence (SEQ ID N0:53) designated herein as
DNA12982.
Figure 54 shows a nucleotide sequence (SEQ ID N0:54) designated herein as
DNA12983.
Figure 55 shows a nucleotide sequence (SEQ ID NO:55) designated herein as
DNA12991.
Figure 56 shows a nucleotide sequence (SEQ ID N0:56) designated herein as
DNA12998.
Figure 57 shows a nucleotide sequence (SEQ ID N0:57) designated herein as
DNA12999.
Figure 58 shows a nucleotide sequence (SEQ ID N0:58) designated herein as
DNA13101.
Figure 59 shows a nucleotide sequence (SEQ ID N0:59) designated herein as DNA
13104.
Figure 60 shows a nucleotide sequence (SEQ ID N0:60) designated herein as DNA
13110.
Figure 61 shows a nucleotide sequence (SEQ ID N0:61) designated herein as
DNA13114.
Figure 62 shows a nucleotide sequence (SEQ ID N0:62) designated herein as
DNA13115.
Figure 63 shows a nucleotide sequence (SEQ ID N0:63) designated herein as
DNA13116.
Figure 64 shows a nucleotide sequence (SEQ ID N0:64) designated herein as
DNA13118.
Figure 65 shows a nucleotide sequence (SEQ ID N0:65) designated herein as
DNA13124.
Figure 66 shows a nucleotide sequence (SEQ ID N0:66) designated herein as
DNA13132.
Figure 67 shows a nucleotide sequence (SEQ ID N0:67) designated herein as DNA
13133.
Figure 68 shows a nucleotide sequence (SEQ ID N0:68) designated herein as
DNA13146.
Figure 69 shows a nucleotide sequence (SEQ ID N0:69) designated herein as
DNA13152.
Figure 70 shows a nucleotide sequence (SEQ ID N0:70) designated herein as
DNA13156.
Figure 71 shows a nucleotide sequence (SEQ ID N0:71) designated herein as
DNA13163.
Figure 72 shows a nucleotide sequence (SEQ ID N0:72) designated herein as
DNA13185.
Figure 73 shows a nucleotide sequence (SEQ ID N0:73) designated herein as
DNA13992.
Figure 74 shows a nucleotide sequence (SEQ ID N0:74) designated herein as
DNA14523.
Figure 75 shows a nucleotide sequence (SEQ ID N0:75) designated herein as
DNA14656.
Figure 76 shows a nucleotide sequence (SEQ ID N0:76) designated herein as
DNA14938.
Figure 77 shows a nucleotide sequence (SEQ ID N0:77) designated herein as
DNA15172.
Figure 78 shows a nucleotide sequence (SEQ ID N0:78) designated herein as
DNA15618.
Figure 79 shows a nucleotide sequence (SEQ ID N0:79) designated herein as
DNA16546.
Figure 80 shows a nucleotide sequence (SEQ ID N0:80) designated herein as
DNA16669.
Figure 81 shows a nucleotide sequence (SEQ ID N0:81) designated herein as
DNA17244.
Figure 82 shows a nucleotide sequence (SEQ ID N0:82) designated herein as
DNA18382.
Figure 83 shows a nucleotide sequence (SEQ ID N0:83) designated herein as
DNA18444.
Figure 84 shows a nucleotide sequence (SEQ ID N0:84) designated herein as
DNA18649.
Figure 85 shows a nucleotide sequence (SEQ ID N0:85) designated herein as
DNA19597.
Figure 86 shows a nucleotide sequence (SEQ ID N0:86) designated herein as
DNA19601.
Figure 87 shows a nucleotide sequence (SEQ ID N0:87) designated herein as
DNA21386.
Figure 88 shows a nucleotide sequence (SEQ ID N0:88) designated herein as
DNA22868.
Figure 89 shows a nucleotide sequence (SEQ ID N0:89) designated herein as
DNA23694.
Figure 90 shows a nucleotide sequence (SEQ ID N0:90) designated herein as
DNA24050.
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Figure 91 shows a nucleotide sequence (SEQ ID N0:91) designated herein as
DNA24074.
Figure 92 shows a nucleotide sequence (SEQ ID N0:92) designated herein as
DNA24787.
Figure 93 shows a nucleotide sequence (SEQ ID N0:93) designated herein as
DNA28242.
Figure 94 shows a nucleotide sequence (SEQ ID N0:94) designated herein as
DNA28254.
Figure 95 shows a nucleotide sequence (SEQ ID N0:95) designated herein as
DNA31751.
Figure 96 shows a nucleotide sequence (SEQ ID N0:96) designated herein as
DNA32922.
Figure 97 shows a nucleotide sequence (SEQ ID N0:97) designated herein as
DNA33439.
Figure 98 shows a nucleotide sequence (SEQ ID N0:98) designated herein as
DNA34508.
Figure 99 shows a nucleotide sequence (SEQ ID N0:99) designated herein as
DNA34807.
Figure 100 shows a nucleotide sequence (SEQ ID NO:100) designated herein as
DNA34832.
Figure 101 shows a nucleotide sequence (SEQ ID NO:101) designated herein as
DNA36223.
Figure 102 shows a nucleotide sequence (SEQ ID N0:102) designated herein as
DNA36240.
Figure 103 shows a nucleotide sequence (SEQ ID N0:103) designated herein as
DNA36490.
Figure 104 shows a nucleotide sequence (SEQ ID N0:104) designated herein as
DNA36516.
Figure 105 shows a nucleotide sequence (SEQ ID NO:105) designated herein as
DNA36533.
Figure 106 shows a nucleotide sequence (SEQ ID N0:106) designated herein as
DNA36538.
Figure 107 shows a nucleotide sequence (SEQ ID N0:107) designated herein as
DNA36788.
Figure 108 shows a nucleotide sequence (SEQ ID N0:108) designated herein as
DNA36818.
Figure 109 shows a nucleotide sequence (SEQ ID N0:109) designated herein as
DNA36868.
Figure 110 shows a nucleotide sequence (SEQ ID NO:110) designated herein as
DNA37393.
Figure 111 shows a nucleotide sequence (SEQ ID NO:111) designated herein as
DNA27588.
Figure 112 shows a nucleotide sequence (SEQ ID N0:112) designated herein as
DNA37602.
Figure 113 shows a nucleotide sequence (SEQ ID N0:113) designated herein as
DNA37642.
Figure 114 shows a nucleotide sequence (SEQ ID N0:114) designated herein as
DNA37676.
Figure 115 shows a nucleotide sequence (SEQ ID NO:115) designated herein as
DNA37721.
Figure 116 shows a nucleotide sequence (SEQ ID N0:116) designated herein as
DNA37759.
Figure 117 shows a nucleotide sequence (SEQ ID N0:117) designated herein as
DNA37857.
Figure 118 shows a nucleotide sequence (SEQ ID NO:118) designated herein as
DNA37937.
Figure 119 shows a nucleotide sequence (SEQ ID N0:119) designated herein as
DNA38037.
Figure 120 shows a nucleotide sequence (SEQ ID N0:120) designated herein as
DNA38050.
Figure 121 shows a nucleotide sequence (SEQ ID N0:121) designated herein as
DNA38053.
Figure 122 shows a nucleotide sequence (SEQ ID N0:122) designated herein as
DNA38312.
Figure 123 shows a nucleotide sequence (SEQ ID N0:123) designated herein as
DNA38360.
Figure 124 shows a nucleotide sequence (SEQ ID N0:124) designated herein as
DNA38600.
Figure 125 shows a nucleotide sequence (SEQ ID N0:125) designated herein as
DNA38720.
Figure 126 shows a nucleotide sequence (SEQ ID N0:126) designated herein as
DNA38727.
Figure 127 shows a nucleotide sequence (SEQ ID N0:127) designated herein as
DNA38731.
Figure 128 shows a nucleotide sequence (SEQ ID N0:128) designated herein as
DNA38810.
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Figure 129 shows a nucleotide sequence (SEQ ID N0:129) designated herein as
DNA38814.
Figure 130 shows a nucleotide sequence (SEQ ID N0:130) designated herein as
DNA39378.
Figure 131 shows a nucleotide sequence (SEQ ID N0:131) designated herein as
DNA40050.
Figure 132 shows a nucleotide sequence (SEQ ID N0:132) designated herein as
DNA40375.
Figure 133 shows a nucleotide sequence (SEQ ID N0:133) designated herein as
DNA40382.
Figure 134 shows a nucleotide sequence (SEQ ID N0:134) designated herein as
DNA40394.
Figure 135 shows a nucleotide sequence (SEQ ID N0:135) designated herein as
DNA40461.
Figure 136 shows a nucleotide sequence (SEQ ID N0:136) designated herein as
DNA40735.
Figure 137 shows a nucleotide sequence (SEQ ID N0:137) designated herein as
DNA40736.
Figure 138 shows a nucleotide sequence (SEQ ID N0:138) designated herein as
DNA40738.
Figure 139 shows a nucleotide sequence (SEQ ID N0:139) designated herein as
DNA40739.
Figure 140 shows a nucleotide sequence (SEQ ID N0:140) designated herein as
DNA41144.
Figure 141 shows a nucleotide sequence (SEQ ID N0:141) designated herein as
DNA41161.
Figure 142 shows a nucleotide sequence (SEQ ID N0:142) designated herein as
DNA41186.
Figure 143 shows a nucleotide sequence (SEQ ID N0:143) designated herein as
DNA41250.
Figure 144 shows a nucleotide sequence (SEQ ID N0:144) designated herein as
DNA41284.
Figure 145 shows a nucleotide sequence (SEQ ID N0:145) designated herein as
DNA41303.
Figure 146 shows a nucleotide sequence (SEQ ID N0:146) designated herein as
DNA41326.
Figure 147 shows a nucleotide sequence (SEQ ID N0:147) designated herein as
DNA41444.
Figure 148 shows a nucleotide sequence (SEQ ID N0:148) designated herein as
DNA41445.
Figure 149 shows a nucleotide sequence (SEQ ID N0:149) designated herein as
DNA41452.
Figure 150 shows a nucleotide sequence (SEQ ID NO:150) designated herein as
DNA41456.
Figure 151 shows a nucleotide sequence (SEQ ID NO:151) designated herein as
DNA41458.
Figure 152 shows a nucleotide sequence (SEQ ID N0:152) designated herein as
DNA41462.
Figure 153 shows a nucleotide sequence (SEQ ID N0:153) designated herein as
DNA41465.
Figure 154 shows a nucleotide sequence (SEQ ID N0:154) designated herein as
DNA41475.
Figure 155 shows a nucleotide sequence (SEQ ID NO:155) designated herein as
DNA41514.
Figure 156 shows a nucleotide sequence (SEQ ID N0:156) designated herein as
DNA41565.
Figure 157 shows a nucleotide sequence (SEQ ID N0:157) designated herein as
DNA41566.
Figure 158 shows a nucleotide sequence (SEQ ID N0:158) designated herein as
DNA41626.
Figure 159 shows a nucleotide sequence (SEQ ID N0:159) designated herein as
DNA41709.
Figure 160 shows a nucleotide sequence (SEQ ID N0:160) designated herein as
DNA41775.
Figure 161 shows a nucleotide sequence (SEQ ID N0:161) designated herein as
DNA41784.
Figure 162 shows a nucleotide sequence (SEQ ID N0:162) designated herein as
DNA42194.
Figure 163 shows a nucleotide sequence (SEQ ID N0:163) designated herein as
DNA42279.
Figure 164 shows a nucleotide sequence (SEQ ID N0:164) designated herein as
DNA42314.
Figure 165 shows a nucleotide sequence (SEQ ID N0:165) designated herein as
DNA42331.
Figure 166 shows a nucleotide sequence (SEQ ID N0:166) designated herein as
DNA42358.
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Figure 167 shows a nucleotide sequence (SEQ ID N0:167) designated herein as
DNA42858.
Figure 168 shows a nucleotide sequence (SEQ ID N0:168) designated herein as
DNA42870.
Figure 169 shows a nucleotide sequence (SEQ ID N0:169) designated herein as
DNA42875.
Figure 170 shows a nucleotide sequence (SEQ ID N0:170) designated herein as
DNA43197.
Figure 171 shows a nucleotide sequence (SEQ ID N0:171) designated herein as
DNA43203.
S Figure 172 shows a nucleotide sequence (SEQ ID N0:172) designated herein as
DNA43295.
Figure 173 shows a nucleotide sequence (SEQ ID N0:173) designated herein as
DNA43301.
Figure 174 shows a nucleotide sequence (SEQ ID N0:174) designated herein as
DNA43363.
Figure 175 shows a nucleotide sequence (SEQ ID N0:175) designated herein as
DNA43420.
Figure 176 shows a nucleotide sequence (SEQ ID N0:176) designated herein as
DNA443479.
Figure 177 shows a nucleotide sequence (SEQ ID N0:177) designated herein as
DNA43489.
Figure 178 shows a nucleotide sequence (SEQ ID N0:178) designated herein as
DNA43498.
Figure 179 shows a nucleotide sequence (SEQ ID N0:179) designated herein as
DNA43509.
Figure 180 shows a nucleotide sequence (SEQ ID N0:180) designated herein as
DNA43512.
Figure 181 shows a nucleotide sequence (SEQ ID N0:181) designated herein as
DNA43531.
Figure 182 shows a nucleotide sequence (SEQ ID N0:182) designated herein as
DNA43546.
Figure 183 shows a nucleotide sequence (SEQ ID N0:183) designated herein as
DNA43586.
Figure 184 shows a nucleotide sequence (SEQ ID N0:184) designated herein as
DNA43862.
Figure 185 shows a nucleotide sequence (SEQ ID N0:185) designated herein as
DNA43887.
Figure 186 shows a nucleotide sequence (SEQ ID N0:186) designated herein as
DNA43936.
Figure 187 shows a nucleotide sequence (SEQ ID N0:187) designated herein as
DNA43961.
Figure 188 shows a nucleotide sequence (SEQ ID N0:188) designated herein as
DNA43971.
Figure 189 shows a nucleotide sequence (SEQ ID N0:189) designated herein as
DNA44048.
Figure 190 shows a nucleotide sequence (SEQ ID N0:190) designated herein as
DNA44920.
Figure 191 shows a nucleotide sequence (SEQ ID N0:191) designated herein as
DNA44922.
Figure 192 shows a nucleotide sequence (SEQ ID N0:192) designated herein as
DNA44934.
Figure 193 shows a nucleotide sequence (SEQ ID N0:193) designated herein as
DNA44987.
Figure 194 shows a nucleotide sequence (SEQ ID N0:194) designated herein as
DNA45014.
Figure 195 shows a nucleotide sequence (SEQ ID N0:195) designated herein as
DNA45030.
Figure 196 shows a nucleotide sequence (SEQ ID N0:196) designated herein as
DNA45051.
Figure 197 shows a nucleotide sequence (SEQ ID N0:197) designated herein as
DNA45064.
Figure 198 shows a nucleotide sequence (SEQ ID N0:198) designated herein as
DNA45282.
Figure 199 shows a nucleotide sequence (SEQ ID N0:199) designated herein as
DNA45288.
Figure 200 shows a nucleotide sequence (SEQ ID N0:200) designated herein as
DNA45300.
Figure 201 shows a nucleotide sequence (SEQ ID N0:201) designated herein as
DNA45740.
Figure 202 shows a nucleotide sequence (SEQ ID N0:202) designated herein as
DNA45759.
Figure 203 shows a nucleotide sequence (SEQ ID N0:203) designated herein as
DNA45784.
Figure 204 shows a nucleotide sequence (SEQ ID N0:204) designated herein as
DNA45789.
9
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Figure 205 shows a nucleotide sequence (SEQ ID N0:205) designated herein as
DNA45816.
Figure 206 shows a nucleotide sequence (SEQ ID N0:206) designated herein as
DNA45944.
Figure 207 shows a nucleotide sequence (SEQ ID N0:207) designated herein as
DNA45954.
Figure 208 shows a nucleotide sequence (SEQ ID N0:208) designated herein as
DNA45964.
Figure 209 shows a nucleotide sequence (SEQ ID N0:209) designated herein as
DNA45993.
Figure 210 shows a nucleotide sequence (SEQ ID N0:210) designated herein as
DNA46092.
Figure 211 shows a nucleotide sequence (SEQ ID N0:211) designated herein as
DNA46213.
Figure 212 shows a nucleotide sequence (SEQ ID N0:212) designated herein as
DNA46215.
Figure 213 shows a nucleotide sequence (SEQ ID N0:213) designated herein as
DNA46226.
Figure 214 shows a nucleotide sequence (SEQ ID N0:214) designated herein as
DNA46328.
Figure 215 shows a nucleotide sequence (SEQ ID N0:215) designated herein as
DNA47580.
Figure 216 shows a nucleotide sequence (SEQ ID N0:216) designated herein as
DNA47691.
Figure 217 shows a nucleotide sequence (SEQ ID N0:217) designated herein as
DNA47751.
Figure 218 shows a nucleotide sequence (SEQ ID N0:218) designated herein as
DNA47835.
Figure 219 shows a nucleotide sequence (SEQ ID N0:219) designated herein as
DNA47858.
Figure 220 shows a nucleotide sequence (SEQ ID N0:220) designated herein as
DNA47890.
Figure 221 shows a nucleotide sequence (SEQ ID N0:221) designated herein as
DNA47930.
Figure 222 shows a nucleotide sequence (SEQ ID N0:222) designated herein as
DNA47990.
Figure 223 shows a nucleotide sequence (SEQ ID N0:223) designated herein as
DNA48054.
Figure 224 shows a nucleotide sequence (SEQ ID N0:224) designated herein as
DNA48124.
Figure 225 shows a nucleotide sequence (SEQ ID N0:225) designated herein as
DNA48131.
Figure 226 shows a nucleotide sequence (SEQ ID N0:226) designated herein as
DNA48162.
Figure 227 shows a nucleotide sequence (SEQ ID N0:227) designated herein as
DNA48209.
Figure 228 shows a nucleotide sequence (SEQ ID N0:228) designated herein as
DNA48389.
Figure 229 shows a nucleotide sequence (SEQ ID N0:229) designated herein as
DNA48446.
Figure 230 shows a nucleotide sequence (SEQ ID N0:230) designated herein as
DNA48466.
Figure 231 shows a nucleotide sequence (SEQ ID N0:231) designated herein as
DNA48576.
Figure 232 shows a nucleotide sequence (SEQ ID N0:232) designated herein as
DNA48598.
Figure 233 shows a nucleotide sequence (SEQ ID N0:233) designated herein as
DNA48666.
Figure 234 shows a nucleotide sequence (SEQ ID N0:234) designated herein as
DNA48748.
Figure 235 shows a nucleotide sequence (SEQ ID N0:235) designated herein as
DNA48777.
Figure 236 shows a nucleotide sequence (SEQ ID N0:236) designated herein as
DNA48830.
Figure 237 shows a nucleotide sequence (SEQ ID N0:237) designated herein as
DNA49352.
Figure 238 shows a nucleotide sequence (SEQ ID N0:238) designated herein as
DNA49407.
Figure 239 shows a nucleotide sequence (SEQ ID N0:239) designated herein as
DNA49448.
Figure 240 shows a nucleotide sequence (SEQ ID N0:240) designated herein as
DNA49528.
Figure 241 shows a nucleotide sequence (SEQ ID N0:241) designated herein as
DNA49529.
Figure 242 shows a nucleotide sequence (SEQ ID N0:242) designated herein as
DNA49948.
CA 02378403 2002-O1-04
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Figure 243 shows a nucleotide sequence (SEQ ID N0:243) designated herein as
DNA49956.
Figure 244 shows a nucleotide sequence (SEQ ID N0:244) designated herein as
DNA49992.
Figure 245 shows a nucleotide sequence (SEQ ID N0:245) designated herein as
DNA50307.
Figure 246 shows a nucleotide sequence (SEQ ID N0:246) designated herein as
DNA50319.
Figure 247 shows a nucleotide sequence (SEQ ID N0:247) designated herein as
DNA50346.
Figure 248 shows a nucleotide sequence (SEQ ID N0:248) designated herein as
DNA50354.
Figure 249 shows a nucleotide sequence (SEQ ID N0:249) designated herein as
DNA50356.
Figure 250 shows a nucleotide sequence (SEQ ID N0:250) designated herein as
DNA50405.
Figure 251 shows a nucleotide sequence (SEQ ID N0:251) designated herein as
DNA50421.
Figure 252 shows a nucleotide sequence (SEQ ID N0:252) designated herein as
DNA50423.
Figure 253 shows a nucleotide sequence (SEQ ID N0:253) designated herein as
DNA50527.
Figure 254 shows a nucleotide sequence (SEQ ID N0:254) designated herein as
DNA50584.
Figure 255 shows a nucleotide sequence (SEQ ID N0:255) designated herein as
DNA50626.
Figure 256 shows a nucleotide sequence (SEQ ID N0:256) designated herein as
DNA50637.
Figure 257 shows~a nucleotide sequence (SEQ ID N0:257) designated herein as
DNA50650.
Figure 258 shows a nucleotide sequence (SEQ ID N0:258) designated herein as
DNA50674.
Figure 259 shows a nucleotide sequence (SEQ ID N0:259) designated herein as
DNA50675.
Figure 260 shows a nucleotide sequence (SEQ ID N0:260) designated herein as
DNA50698.
Figure 261 shows a nucleotide sequence (SEQ ID N0:261) designated herein as
DNA50730.
Figure 262 shows a nucleotide sequence (SEQ ID N0:262) designated herein as
DNA50737.
Figure 263 shows a nucleotide sequence (SEQ ID N0:263) designated herein as
DNA51003.
Figure 264 shows a nucleotide sequence (SEQ ID N0:264) designated herein as
DNA51010.
Figure 265 shows a nucleotide sequence (SEQ ID N0:265) designated herein as
DNA51059.
Figure 266 shows a nucleotide sequence (SEQ ID N0:266) designated herein as
DNA51413.
Figure 267 shows a nucleotide sequence (SEQ ID N0:267) designated herein as
DNA51712.
Figure 268 shows a nucleotide sequence (SEQ ID N0:268) designated herein as
DNA51795.
Figure 269 shows a nucleotide sequence (SEQ ID N0:269) designated herein as
DNA52199.
Figure 270 shows a nucleotide sequence (SEQ ID N0:270) designated herein as
DNA52218.
Figure 271 shows a nucleotide sequence (SEQ ID N0:271) designated herein as
DNA52352.
Figure 272 shows a nucleotide sequence (SEQ ID N0:272) designated herein as
DNA54446.
Figure 273 shows a nucleotide sequence (SEQ ID N0:273) designated herein as
DNA54552.
Figure 274 shows a nucleotide sequence (SEQ ID N0:274) designated herein as
DNA54580.
Figure 275 shows a nucleotide sequence (SEQ ID N0:275) designated herein as
DNA54623.
Figure 276 shows a nucleotide sequence (SEQ ID N0:276) designated herein as
DNA54672.
Figure 277 shows a nucleotide sequence (SEQ ID N0:277) designated herein as
DNA54840.
Figure 278 shows a nucleotide sequence (SEQ ID N0:278) designated herein as
DNA54856.
Figure 279 shows a nucleotide sequence (SEQ ID N0:279) designated herein as
DNA54882.
Figure 280 shows a nucleotide sequence (SEQ ID N0:280) designated herein as
DNA54943.
11
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Figure 281 shows a nucleotide sequence (SEQ ID N0:281) designated herein as
DNA54970.
Figure 282 shows a nucleotide sequence (SEQ ID N0:282) designated herein as
DNA55134.
Figure 283 shows a nucleotide sequence (SEQ ID N0:283) designated herein as
DNA55198.
Figure 284 shows a nucleotide sequence (SEQ ID N0:284) designated herein as
DNA55199.
Figure 285 shows a nucleotide sequence (SEQ ID N0:285) designated herein as
DNA55292.
Figure 286 shows a nucleotide sequence (SEQ ID N0:286) designated herein as
DNA55646.
Figure 287 shows a nucleotide sequence (SEQ ID N0:287) designated herein as
DNA56553.
Figure 288 shows a nucleotide sequence (SEQ ID N0:288) designated herein as
DNA56554.
Figure 289 shows a nucleotide sequence (SEQ ID N0:289) designated herein as
DNA56556.
Figure 290 shows a nucleotide sequence (SEQ ID N0:290) designated herein as
DNA56587.
Figure 291 shows a nucleotide sequence (SEQ ID N0:291) designated herein as
DNA56590.
Figure 292 shows a nucleotide sequence (SEQ ID N0:292) designated herein as
DNA56600.
Figure 293 shows a nucleotide sequence (SEQ ID N0:293) designated herein as
DNA56648.
Figure 294 shows a nucleotide sequence (SEQ ID N0:294) designated herein as
DNA56650.
Figure 295 shows a nucleotide sequence (SEQ ID N0:295) designated herein as
DNA56707.
Figure 296 shows a nucleotide sequence (SEQ ID N0:296) designated herein as
DNA56717.
Figure 297 shows a nucleotide sequence (SEQ ID N0:297) designated herein as
DNA58387.
Figure 298 shows a nucleotide sequence (SEQ ID N0:298) designated herein as
DNA58414.
Figure 299 shows a nucleotide sequence (SEQ ID N0:299) designated herein as
DNA58529.
Figure 300 shows a nucleotide sequence (SEQ ID N0:300) designated herein as
DNA59385.
Figure 301 shows a nucleotide sequence (SEQ ID N0:301) designated herein as
DNA59789.
Figure 302 shows a nucleotide sequence (SEQ ID N0:302) designated herein as
DNA60321.
Figure 303 shows a nucleotide sequence (SEQ ID N0:303) designated herein as
DNA60370.
Figure 304 shows a nucleotide sequence (SEQ ID N0:304) designated herein as
DNA60406.
Figure 305 shows a nucleotide sequence (SEQ ID N0:305) designated herein as
DNA60438.
Figure 306 shows a nucleotide sequence (SEQ ID N0:306) designated herein as
DNA60460.
Figure 307 shows a nucleotide sequence (SEQ ID N0:307) designated herein as
DNA60466.
Figure 308 shows a nucleotide sequence (SEQ ID N0:308) designated herein as
DNA60508.
Figure 309 shows a nucleotide sequence (SEQ ID N0:309) designated herein as
DNA60542.
Figure 310 shows a nucleotide sequence (SEQ ID N0:310) designated herein as
DNA60590.
Figure 311 shows a nucleotide sequence (SEQ ID N0:311) designated herein as
DNA61350.
Figure 312 shows a nucleotide sequence (SEQ ID N0:312) designated herein as
DNA61356.
Figure 313 shows a nucleotide sequence (SEQ ID N0:313) designated herein as
DNA61478.
Figure 314 shows a nucleotide sequence (SEQ ID N0:314) designated herein as
DNA61513.
Figure 315 shows a nucleotide sequence (SEQ ID N0:315) designated herein as
DNA61561.
Figure 316 shows a nucleotide sequence (SEQ ID N0:316) designated herein as
DNA61895.
Figure 317 shows a nucleotide sequence (SEQ ID N0:317) designated herein as
DNA61930.
Figure 318 shows a nucleotide sequence (SEQ ID N0:318) designated herein as
DNA61953.
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Figure 319 shows a nucleotide sequence (SEQ ID N0:319) designated herein as
DNA62011.
Figure 320 shows a nucleotide sequence (SEQ ID N0:320) designated herein as
DNA62080.
Figure 321 shows a nucleotide sequence (SEQ ID N0:321) designated herein as
DNA62126.
Figure 322 shows a nucleotide sequence (SEQ ID N0:322) designated herein as
DNA62154.
Figure 323 shows a nucleotide sequence (SEQ ID N0:323) designated herein as
DNA62170.
Figure 324 shows a nucleotide sequence (SEQ ID N0:324) designated herein as
DNA62193.
Figure 325 shows a nucleotide sequence (SEQ ID N0:325) designated herein as
DNA62261.
Figure 326 shows a nucleotide sequence (SEQ ID N0:326) designated herein as
DNA62291.
Figure 327 shows a nucleotide sequence (SEQ ID N0:327) designated herein as
DNA62422.
Figure 328 shows a nucleotide sequence (SEQ ID N0:328) designated herein as
DNA62436.
Figure 329 shows a nucleotide sequence (SEQ ID N0:329) designated herein as
DNA62524.
Figure 330 shows a nucleotide sequence (SEQ ID N0:330) designated herein as
DNA62589.
Figure 331 shows a nucleotide sequence (SEQ ID N0:331) designated herein as
DNA63878.
Figure 332 shows a nucleotide sequence (SEQ ID N0:332) designated herein as
DNA64017.
Figure 333 shows a nucleotide sequence (SEQ ID N0:333) designated herein as
DNA64045.
Figure 334 shows a nucleotide sequence (SEQ ID N0:334) designated herein as
DNA64101.
Figure 335 shows a nucleotide sequence (SEQ ID N0:335) designated herein as
DNA64183.
Figure 336 shows a nucleotide sequence (SEQ ID N0:336) designated herein as
DNA64193.
Figure 337 shows a nucleotide sequence (SEQ ID N0:337) designated herein as
DNA64199.
Figure 338 shows a nucleotide sequence (SEQ ID N0:338) designated herein as
DNA64268.
Figure 339 shows a nucleotide sequence (SEQ ID N0:339) designated herein as
DNA64304.
Figure 340 shows a nucleotide sequence (SEQ ID N0:340) designated herein as
DNA64453.
Figure 341 shows a nucleotide sequence (SEQ ID N0:341) designated herein as
DNA64458.
Figure 342 shows a nucleotide sequence (SEQ ID N0:342) designated herein as
DNA64512.
Figure 343 shows a nucleotide sequence (SEQ ID N0:343) designated herein as
DNA64540.
Figure 344 shows a nucleotide sequence (SEQ ID N0:344) designated herein as
DNA64552.
Figure 345 shows a nucleotide sequence (SEQ ID N0:345) designated herein as
DNA64557.
Figure 346 shows a nucleotide sequence (SEQ ID N0:346) designated herein as
DNA64569.
Figure 347 shows a nucleotide sequence (SEQ ID N0:347) designated herein as
DNA64627.
Figure 348 shows a nucleotide sequence (SEQ ID N0:348) designated herein as
DNA64745.
Figure 349 shows a nucleotide sequence (SEQ ID N0:349) designated herein as
DNA64784.
Figure 350 shows a nucleotide sequence (SEQ ID N0:350) designated herein as
DNA65609.
Figure 351 shows a nucleotide sequence (SEQ ID N0:351) designated herein as
DNA65644.
Figure 352 shows a nucleotide sequence (SEQ ID N0:352) designated herein as
DNA65720.
Figure 353 shows a nucleotide sequence (SEQ ID N0:353) designated herein as
DNA65752.
Figure 354 shows a nucleotide sequence (SEQ ID N0:354) designated herein as
DNA65771.
Figure 355 shows a nucleotide sequence (SEQ ID N0:355) designated herein as
DNA65833.
Figure 356 shows a nucleotide sequence (SEQ ID N0:356) designated herein as
DNA65836.
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Figure 357 shows a nucleotide sequence (SEQ ID N0:357) designated herein as
DNA65864.
Figure 358 shows a nucleotide sequence (SEQ ID N0:358) designated herein as
DNA65869.
Figure 359 shows a nucleotide sequence (SEQ ID N0:359) designated herein as
DNA65928.
Figure 360 shows a nucleotide sequence (SEQ ID N0:360) designated herein as
DNA66065.
Figure 361 shows a nucleotide sequence (SEQ ID N0:361) designated herein as
DNA66095.
Figure 362 shows a nucleotide sequence (SEQ ID N0:362) designated herein as
DNA66197.
Figure 363 shows a nucleotide sequence (SEQ ID N0:363) designated herein as
DNA66217.
Figure 364 shows a nucleotide sequence (SEQ ID N0:364) designated herein as
DNA66231.
Figure 365 shows a nucleotide sequence (SEQ ID N0:365) designated herein as
DNA66404.
Figure 366 shows a nucleotide sequence (SEQ ID N0:366) designated herein as
DNA66432.
Figure 367 shows a nucleotide sequence (SEQ ID N0:367) designated herein as
DNA67076.
Figure 368 shows a nucleotide sequence (SEQ ID N0:368) designated herein as
DNA68013.
Figure 369 shows a nucleotide sequence (SEQ ID N0:369) designated herein as
DNA68018.
Figure 370 shows a nucleotide sequence (SEQ ID N0:370) designated herein as
DNA68034.
Figure 371 shows a nucleotide sequence (SEQ ID N0:371) designated herein as
DNA68119.
Figure 372 shows a nucleotide sequence (SEQ ID N0:372) designated herein as
DNA68248.
Figure 373 shows a nucleotide sequence (SEQ ID N0:373) designated herein as
DNA68383.
Figure 374 shows a nucleotide sequence (SEQ ID N0:374) designated herein as
DNA68423.
Figure 375 shows a nucleotide sequence (SEQ ID N0:375) designated herein as
DNA68441.
Figure 376 shows a nucleotide sequence (SEQ ID N0:376) designated herein as
DNA68459.
Figure 377 shows a nucleotide sequence (SEQ ID N0:377) designated herein as
DNA68509.
Figure 378 shows a nucleotide sequence (SEQ ID N0:378) designated herein as
DNA68514.
Figure 379 shows a nucleotide sequence (SEQ ID N0:379) designated herein as
DNA68521.
Figure 380 shows a nucleotide sequence (SEQ ID N0:380) designated herein as
DNA68532.
Figure 381 shows a nucleotide sequence (SEQ ID N0:381) designated herein as
DNA68540.
Figure 382 shows a nucleotide sequence (SEQ ID N0:382) designated herein as
DNA68561.
Figure 383 shows a nucleotide sequence (SEQ ID N0:383) designated herein as
DNA68585.
Figure 384 shows a nucleotide sequence (SEQ ID N0:384) designated herein as
DNA69491.
Figure 385 shows a nucleotide sequence (SEQ ID N0:385) designated herein as
DNA70222.
Figure 386 shows a nucleotide sequence (SEQ ID N0:386) designated herein as
DNA70239.
Figure 387 shows a nucleotide sequence (SEQ ID N0:387) designated herein as
DNA70244.
Figure 388 shows a nucleotide sequence (SEQ ID N0:388) designated herein as
DNA70349.
Figure 389 shows a nucleotide sequence (SEQ ID N0:389) designated herein as
DNA70400.
Figure 390 shows a nucleotide sequence (SEQ ID N0:390) designated herein as
DNA70413.
Figure 391 shows a nucleotide sequence (SEQ ID N0:391) designated herein as
DNA70526.
Figure 392 shows a nucleotide sequence (SEQ ID N0:392) designated herein as
DNA70685.
Figure 393 shows a nucleotide sequence (SEQ ID N0:393) designated herein as
DNA70732.
Figure 394 shows a nucleotide sequence (SEQ ID N0:394) designated herein as
DNA72634.
14
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Figure 395 shows a nucleotide sequence (SEQ ID N0:395) designated herein as
DNA72683.
Figure 396 shows a nucleotide sequence (SEQ ID N0:396) designated herein as
DNA72695.
Figure 397 shows a nucleotide sequence (SEQ ID N0:397) designated herein as
DNA72864.
Figure 398 shows a nucleotide sequence (SEQ ID N0:398) designated herein as
DNA73156.
Figure 399 shows a nucleotide sequence (SEQ ID N0:399) designated herein as
DNA73275.
Figure 400 shows a nucleotide sequence (SEQ ID N0:400) designated herein as
DNA74052.
Figure 401 shows a nucleotide sequence (SEQ ID N0:401) designated herein as
DNA74063.
Figure 402 shows a nucleotide sequence (SEQ ID N0:402) designated herein as
DNA74072.
Figure 403 shows a nucleotide sequence (SEQ ID N0:403) designated herein as
DNA74140.
Figure 404 shows a nucleotide sequence (SEQ ID N0:404) designated herein as
DNA74216.
Figure 405 shows a nucleotide sequence (SEQ ID N0:405) designated herein as
DNA74218.
Figure 406 shows a nucleotide sequence (SEQ ID N0:406) designated herein as
DNA74228.
Figure 407 shows a nucleotide sequence (SEQ ID N0:407) designated herein as
DNA74256.
Figure 408 shows a nucleotide sequence (SEQ ID N0:408) designated herein as
DNA75062.
Figure 409 shows a nucleotide sequence (SEQ ID N0:409) designated herein as
DNA76137.
Figure 410 shows a nucleotide sequence (SEQ ID N0:410) designated herein as
DNA76158.
Figure 411 shows a nucleotide sequence (SEQ ID N0:411) designated herein as
DNA77098.
Figure 412 shows a nucleotide sequence (SEQ ID N0:412) designated herein as
DNA77791.
Figure 413 shows a nucleotide sequence (SEQ ID N0:413) designated herein as
DNA77968.
Figure 414 shows a nucleotide sequence (SEQ ID N0:414) designated herein as
DNA77976.
Figure 415 shows a nucleotide sequence (SEQ ID N0:415) designated herein as
DNA78017.
Figure 416 shows a nucleotide sequence (SEQ ID N0:416) designated herein as
DNA78095.
Figure 417 shows a nucleotide sequence (SEQ ID N0:417) designated herein as
DNA78103.
Figure 418 shows a nucleotide sequence (SEQ ID N0:418) designated herein as
DNA78113.
Figure 419 shows a nucleotide sequence (SEQ ID N0:419) designated herein as
DNA78746.
Figure 420 shows a nucleotide sequence (SEQ ID N0:420) designated herein as
DNA78759.
Figure 421 shows a nucleotide sequence (SEQ ID N0:421) designated herein as
DNA78796.
Figure 422 shows a nucleotide sequence (SEQ ID N0:422) designated herein as
DNA79561.
Figure 423 shows a nucleotide sequence (SEQ ID N0:423) designated herein as
DNA79602.
Figure 424 shows a nucleotide sequence (SEQ ID N0:424) designated herein as
DNA79617.
Figure 425 shows a nucleotide sequence (SEQ ID N0:425) designated herein as
DNA79628.
Figure 426 shows a nucleotide sequence (SEQ ID N0:426) designated herein as
DNA79640.
Figure 427 shows a nucleotide sequence (SEQ ID N0:427) designated herein as
DNA79661.
Figure 428 shows a nucleotide sequence (SEQ ID N0:428) designated herein as
DNA79684.
Figure 429 shows a nucleotide sequence (SEQ ID N0:429) designated herein as
DNA79717.
Figure 430 shows a nucleotide sequence (SEQ ID N0:430) designated herein as
DNA79733.
Figure 431 shows a nucleotide sequence (SEQ ID N0:431) designated herein as
DNA79970.
Figure 432 shows a nucleotide sequence (SEQ ID N0:432) designated herein as
DNA80050.
CA 02378403 2002-O1-04
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Figure 433 shows a nucleotide sequence (SEQ ID N0:433) designated herein as
DNA80247.
Figure 434 shows a nucleotide sequence (SEQ ID N0:434) designated herein as
DNA80265.
Figure 435 shows a nucleotide sequence (SEQ ID N0:435) designated herein as
DNA80615.
Figure 436 shows a nucleotide sequence (SEQ ID N0:436) designated herein as
DNA80623.
Figure 437 shows a nucleotide sequence (SEQ ID N0:437) designated herein as
DNA80627.
Figure 438 shows a nucleotide sequence (SEQ ID N0:438) designated herein as
DNA81896.
Figure 439 shows a nucleotide sequence (SEQ ID N0:439) designated herein as
DNA81918.
Figure 440 shows a nucleotide sequence (SEQ ID N0:440) designated herein as
DNA81976.
Figure 441 shows a nucleotide sequence (SEQ ID N0:441) designated herein as
DNA82017.
Figure 442 shows a nucleotide sequence (SEQ ID N0:442) designated herein as
DNA82024.
Figure 443 shows a nucleotide sequence (SEQ ID N0:443) designated herein as
DNA82027.
Figure 444 shows a nucleotide sequence (SEQ ID N0:444) designated herein as
DNA82115.
Figure 445 shows a nucleotide sequence (SEQ ID N0:445) designated herein as
DNA82154.
Figure 446 shows a nucleotide sequence (SEQ ID N0:446) designated herein as
DNA82157.
Figure 447 shows a nucleotide sequence (SEQ ID N0:447) designated herein as
DNA82166.
Figure 448 shows a nucleotide sequence (SEQ ID N0:448) designated herein as
DNA82182.
Figure 449 shows a nucleotide sequence (SEQ ID N0:449) designated herein as
DNA82212.
Figure 450 shows a nucleotide sequence (SEQ ID N0:450) designated herein as
DNA82498.
Figure 451 shows a nucleotide sequence (SEQ ID N0:451) designated herein as
DNA82499.
Figure 452 shows a nucleotide sequence (SEQ ID N0:452) designated herein as
DNA82504.
Figure 453 shows a nucleotide sequence (SEQ ID N0:453) designated herein as
DNA82531.
Figure 454 shows a nucleotide sequence (SEQ ID N0:454) designated herein as
DNA82693.
Figure 455 shows a nucleotide sequence (SEQ ID N0:455) designated herein as
DNA82702.
Figure 456 shows a nucleotide sequence (SEQ ID N0:456) designated herein as
DNA82786.
Figure 457 shows a nucleotide sequence (SEQ ID N0:457) designated herein as
DNA82851.
Figure 458 shows a nucleotide sequence (SEQ ID N0:458) designated herein as
DNA82898.
Figure 459 shows a nucleotide sequence (SEQ ID N0:459) designated herein as
DNA82935.
Figure 460 shows a nucleotide sequence (SEQ ID N0:460) designated herein as
DNA82977.
Figure 461 shows a nucleotide sequence (SEQ ID N0:461) designated herein as
DNA82989.
Figure 462 shows a nucleotide sequence (SEQ ID N0:462) designated herein as
DNA83628.
Figure 463 shows a nucleotide sequence (SEQ ID N0:463) designated herein as
DNA83630.
Figure 464 shows a nucleotide sequence (SEQ ID N0:464) designated herein as
DNA83749.
Figure 465 shows a nucleotide sequence (SEQ ID N0:465) designated herein as
DNA83772.
Figure 466 shows a nucleotide sequence (SEQ ID N0:466) designated herein as
DNA83800.
Figure 467 shows a nucleotide sequence (SEQ ID N0:467) designated herein as
DNA83950.
Figure 468 shows a nucleotide sequence (SEQ ID N0:468) designated herein as
DNA84027.
Figure 469 shows a nucleotide sequence (SEQ ID N0:469) designated herein as
DNA84076.
Figure 470 shows a nucleotide sequence (SEQ ID N0:470) designated herein as
DNA84109.
16
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Figure 471 shows a nucleotide sequence (SEQ ID as DNA85072.
N0:471) designated herein
Figure 472 shows a nucleotide sequence (SEQ ID as DNA85154.
N0:472) designated herein
Figure 473 shows a nucleotide sequence (SEQ ID as DNA85193.
N0:473) designated herein
Figure 474 shows a nucleotide sequence (SEQ ID as DNA85224.
N0:474) designated herein
Figure 475 shows a nucleotide sequence (SEQ ID as DNA85237.
N0:475) designated herein
Figure 476 shows a nucleotide sequence (SEQ ID as DNA85289.
N0:476) designated herein
Figure 477 shows a nucleotide sequence (SEQ ID as DNA85357.
N0:477) designated herein
Figure 478 shows a nucleotide sequence (SEQ ID as DNA85361.
N0:478) designated herein
Figure 479 shows a nucleotide sequence (SEQ ID as DNA85371.
N0:479) designated herein
Figure 480 shows a nucleotide sequence (SEQ ID as DNA86875.
N0:480) designated herein
Figure 481 shows a nucleotide sequence (SEQ ID as DNA86876.
N0:481) designated herein
Figure 482 shows a nucleotide sequence (SEQ ID as DNA86905.
N0:482) designated herein
Figure 483 shows a nucleotide sequence (SEQ ID as DNA86945.
N0:483) designated herein
Figure 484 shows a nucleotide sequence (SEQ ID as DNA86969.
N0:484) designated herein
Figure 485 shows a nucleotide sequence (SEQ ID as DNA87050.
N0:485) designated herein
Figure 486 shows a nucleotide sequence (SEQ ID as DNA87094.
N0:486) designated herein
Figure 487 shows a nucleotide sequence (SEQ ID as DNA87126.
N0:487) designated herein
Figure 488 shows a nucleotide sequence (SEQ ID as DNA87493.
N0:488) designated herein
Figure 489 shows a nucleotide sequence (SEQ ID as DNA87494.
N0:489) designated herein
Figure 490 shows a nucleotide sequence (SEQ ID as DNA87505.
N0:490) designated herein
Figure 491 shows a nucleotide sequence (SEQ ID as DNA87566.
N0:491) designated herein
Figure 492 shows a nucleotide sequence (SEQ ID as DNA87586.
N0:492) designated herein
Figure 493 shows a nucleotide sequence (SEQ ID as DNA87649.
N0:493) designated herein
Figure 494 shows a nucleotide sequence (SEQ ID as DNA89340.
N0:494) designated herein
Figure 495 shows a nucleotide sequence (SEQ ID as DNA89355.
N0:495) designated herein
Figure 496 shows a nucleotide sequence (SEQ ID as DNA89365.
N0:496) designated herein
Figure 497 shows a nucleotide sequence (SEQ ID as DNA89419.
N0:497) designated herein
Figure 498 shows a nucleotide sequence (SEQ ID as DNA89470.
N0:498) designated herein
Figure 499 shows a nucleotide sequence (SEQ ID as DNA89480.
N0:499) designated herein
Figure 500 shows a nucleotide sequence (SEQ ID as DNA89549.
N0:500) designated herein
Figure 501 shows a nucleotide sequence (SEQ ID as DNA89606.
N0:501) designated herein
Figure 502 shows a nucleotide sequence (SEQ ID as DNA89615.
N0:502) designated herein
Figure 503 shows a nucleotide sequence (SEQ ID as DNA89669.
N0:503) designated herein
Figure 504 shows a nucleotide sequence (SEQ ID as DNA89760.
N0:504) designated herein
Figure 505 shows a nucleotide sequence (SEQ ID as DNA89766.
N0:505) designated herein
Figure 506 shows a nucleotide sequence (SEQ ID as DNA89772.
N0:506) designated herein
Figure 507 shows a nucleotide sequence (SEQ ID as DNA89773.
N0:507) designated herein
Figure 508 shows a nucleotide sequence (SEQ ID as DNA89774.
N0:508) designated herein
17
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Figure 509 shows a nucleotide sequence (SEQ ID N0:509) designated herein as
DNA89872.
Figure 510 shows a nucleotide sequence (SEQ ID NO:510) designated herein as
DNA89918.
Figure 511 shows a nucleotide sequence (SEQ ID NO:511) designated herein as
DNA89928.
Figure S 12 shows a nucleotide sequence (SEQ ID N0:512) designated herein as
DNA89930.
Figure 513 shows a nucleotide sequence (SEQ ID N0:513) designated herein as
DNA91463.
Figure 514 shows a nucleotide sequence (SEQ ID N0:514) designated herein as
DNA91507.
Figure 515 shows a nucleotide sequence (SEQ ID NO:515) designated herein as
DNA93615.
Figure 516 shows a nucleotide sequence (SEQ ID N0:516) designated herein as
DNA94011.
Figure 517 shows a nucleotide sequence (SEQ ID N0:517) designated herein as
DNA94043.
Figure 518 shows a nucleotide sequence (SEQ ID N0:518) designated herein as
DNA94050.
Figure 519 shows a nucleotide sequence (SEQ ID N0:519) designated herein as
DNA94097.
Figure 520 shows a nucleotide sequence (SEQ ID N0:520) designated herein as
DNA94098.
Figure 521 shows a nucleotide sequence (SEQ ID N0:521) designated herein as
DNA94100.
Figure 522 shows a nucleotide sequence (SEQ ID N0:522) designated herein as
DNA94126.
Figure 523 shows a nucleotide sequence (SEQ ID N0:523) designated herein as
DNA94136.
Figure 524 shows a nucleotide sequence (SEQ ID N0:524) designated herein as
DNA94156.
Figure 525 shows a nucleotide sequence (SEQ ID N0:525) designated herein as
DNA94219.
Figure 526 shows a nucleotide sequence (SEQ ID N0:526) designated herein as
DNA94254.
Figure 527 shows a nucleotide sequence (SEQ ID N0:527) designated herein as
DNA94274.
Figure 528 shows a nucleotide sequence (SEQ ID N0:528) designated herein as
DNA94292.
Figure 529 shows a nucleotide sequence (SEQ ID N0:529) designated herein as
DNA94360.
Figure 530 shows a nucleotide sequence (SEQ ID N0:530) designated herein as
DNA94377.
Figure 531 shows a nucleotide sequence (SEQ ID N0:531) designated herein as
DNA94477.
Figure 532 shows a nucleotide sequence (SEQ ID N0:532) designated herein as
DNA94518.
Figure 533 shows a nucleotide sequence (SEQ ID N0:533) designated herein as
DNA94533.
Figure 534 shows a nucleotide sequence (SEQ ID N0:534) designated herein as
DNA95370.
Figure 535 shows a nucleotide sequence (SEQ ID N0:535) designated herein as
DNA97358.
Figure 536 shows a nucleotide sequence (SEQ ID N0:536) designated herein as
DNA97374.
Figure 537 shows a nucleotide sequence (SEQ ID N0:537) designated herein as
DNA97470.
Figure 538 shows a nucleotide sequence (SEQ ID N0:538) designated herein as
DNA97581.
Figure 539 shows a nucleotide sequence (SEQ ID N0:539) designated herein as
DNA97767.
Figure 540 shows a nucleotide sequence (SEQ ID N0:540) designated herein as
DNA97842.
Figure 541 shows a nucleotide sequence (SEQ ID N0:541) designated herein as
DNA97949.
Figure 542 shows a nucleotide sequence (SEQ ID N0:542) designated herein as
DNA97987.
Figure 543 shows a nucleotide sequence (SEQ ID N0:543) designated herein as
DNA97995.
Figure 544 shows a nucleotide sequence (SEQ ID N0:544) designated herein as
DNA98293.
Figure 545 shows a nucleotide sequence (SEQ ID N0:545) designated herein as
DNA98294.
Figure 546 shows a nucleotide sequence (SEQ ID N0:546) designated herein as
DNA98346.
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Figure 547 shows a nucleotide sequence (SEQ ID N0:547) designated herein as
DNA98360.
Figure 548 shows a nucleotide sequence (SEQ ID N0:548) designated herein as
DNA98829.
Figure 549 shows a nucleotide sequence (SEQ ID N0:549) designated herein as
DNA 101514.
Figure 550 shows a nucleotide sequence (SEQ ID NO:550) designated herein as
DNA 101572.
Figure 551 shows a nucleotide sequence (SEQ ID N0:551) designated herein as
DNA101580.
Figure 552 shows a nucleotide sequence (SEQ ID N0:552) designated herein as
DNA101595.
Figure 553 shows a nucleotide sequence (SEQ ID N0:553) designated herein as
DNA101633.
Figure 554 shows a nucleotide sequence (SEQ ID N0:554) designated herein as
DNA101717.
Figure 555 shows a nucleotide sequence (SEQ ID N0:555) designated herein as
DNA101768.
Figure 556 shows a nucleotide sequence (SEQ ID N0:556) designated herein as
DNA107332.
Figure 557 shows a nucleotide sequence (SEQ ID N0:557) designated herein as
DNA43499.
Figure 558 shows a nucleotide sequence (SEQ ID N0:558) designated herein as
DNA45713.
Figure 559 shows a nucleotide sequence (SEQ ID N0:559) designated herein as
DNA46089.
Figure 560 shows a nucleotide sequence (SEQ ID N0:560) designated herein as
DNA68256.
Figure 561 shows a nucleotide sequence (SEQ ID N0:561) designated herein as
DNA70305.
Figure 562 shows a nucleotide sequence (SEQ ID N0:562) designated herein as
DNA82953.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
The term "SRT polypeptide" when used herein encompasses "native sequence SRT
polypeptides" and
"SRT polypeptide variants" (which are further defined herein). "SRT" is a
designation given to those
polypeptides which are encoded by the nucleic acid molecules shown in the
accompanying figures and variants
thereof, nucleic acid molecules comprising the sequence shown in the
accompanying figures and variants thereof
as well as fragments of the above. The SRT polypeptides of the invention may
be isolated from a variety of
sources, such as from human tissue types or from another source, or prepared
by recombinant and/or synthetic
methods.
A "native sequence" SRT polypeptide comprises a polypeptide having the same
amino acid sequence
as the corresponding SRT polypeptide derived from nature. Such native sequence
SRT polypeptides can be
isolated from nature or can be produced by recombinant and/or synthetic means.
The term "native sequence
SRT polypeptide" specifically encompasses naturally-occurring truncated or
secreted forms (e.g. , an extracellular
domain sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring
allelic variants of the polypeptide.
An SRT polypeptide "extracellular domain" or "ECD" refers to a form of the SRT
polypeptide which
is essentially free of the transmembrane and cytoplasmic domains. Ordinarily,
an SRT polypeptide ECD will
have less than about 1 % of such transmembrane and/or cytoplasmic domains and
preferably, will have less than
about 0.5 % of such domains. It will be understood that any transmembrane
domains) identified for the SRT
polypeptides of the present invention are identified pursuant to criteria
routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries of a
transmembrane domain may vary but
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most likely by no more than about 5 amino acids at either end of the domain as
initially identified.
"Variant SRT polypeptide" means an active SRT polypeptide as defined below
having at least about
80% amino acid sequence identity with the amino acid sequence of a
specifically derived fragment of any other
polypeptide which will be specifically recited. Such variant SRT polypeptides
include, for instance, SRT
polypeptides wherein one or more amino acid residues are added, or deleted, at
the N- and/or C-terminus, as
well as within one or more internal domains, of the full-length amino acid
sequence. Ordinarily, a variant SRT
polypeptide will have at least about 80 % amino acid sequence identity, more
preferably at least about 81 % amino
acid sequence identity, more preferably at least about 82 % amino acid
sequence identity, more preferably at least
about 83 % amino acid sequence identity, more preferably at least about 84 %
amino acid sequence identity, more
preferably at least about 85% amino acid sequence identity, more preferably at
least about 86% amino acid
sequence identity, more preferably at least about 87 % amino acid sequence
identity, more preferably at least
about 88 % amino acid sequence identity, more preferably at least about 89 %
amino acid sequence identity, more
preferably at least about 90 % amino acid sequence identity, more preferably
at least about 91 % amino acid
sequence identity, more preferably at least about 92% amino acid sequence
identity, more preferably at least
about 93 % amino acid sequence identity, more preferably at least about 94 %
amino acid sequence identity, more
preferably at least about 95 % amino acid sequence identity, more preferably
at least about 96 % amino acid
sequence identity, more preferably at least about 97 % amino acid sequence
identity, more preferably at least
about 98 % amino acid sequence identity and yet more preferably at least about
99 % amino acid sequence identity
with an SRT polypeptide encoded by a nucleic acid molecule shown in one of the
accompanying figures or a
specified fragment thereof. SRT variant polypeptides do not encompass the
native SRT polypeptide sequence.
Ordinarily, SRT variant polypeptides are at least about 10 amino acids in
length, often at least about 20 amino
acids in length, more often at least about 30 amino acids in length, more
often at least about 40 amino acids in
length, more often at least about 50 amino acids in length, more often at
least about 60 amino acids in length,
more often at least about 70 amino acids in length, more often at least about
80 amino acids in length, more often
at least about 90 amino acids in length, more often at least about 100 amino
acids in length, more often at least
about 150 amino acids in length, more often at least about 200 amino acids in
length, more often at least about
250 amino acids in length, more often at least about 300 amino acids in
length, or more.
"Percent ( % ) amino acid sequence identity" with respect to the SRT
polypeptide sequences identified
herein is defined as the percentage of amino acid residues in a candidate
sequence that are identical with the
amino acid residues in a SRT sequence, after aligning the sequences and
introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering any
conservative substitutions as part of
the sequence identity. Alignment for purposes of determining percent amino
acid sequence identity can be
achieved in various ways that are within the skill in the art, for instance,
using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)
software. Those skilled in
the art can determine appropriate parameters for measuring alignment,
including any algorithms needed to
achieve maximal alignment over the full-length of the sequences being
compared. For purposes herein, however,
amino acid sequence identity values are obtained as described below by using
the sequence comparison
computer program ALIGN-2, wherein the complete source code for the ALIGN-2
program is provided in Table
CA 02378403 2002-O1-04
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1. The ALIGN-2 sequence comparison computer program was authored by Genentech,
Inc. and the source code
shown in Table 1 has been filed with user documentation in the U.S. Copyright
Office, Washington D.C.,
20559, where it is registered under U.S. Copyright Registration No. TXU510087.
The ALIGN-2 program is
publicly available through Genentech, Inc. , South San Francisco, California
or may be compiled from the source
code provided in Table 1. The ALIGN-2 program should be compiled for use on a
UNIX operating system,
preferably digital UNIX V4.OD. All sequence comparison parameters are set by
the ALIGN-2 program and
do not vary.
For purposes herein, the % amino acid sequence identity of a given amino acid
sequence A to, with,
or against a given amino acid sequence B (which can alternatively be phrased
as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity to, with, or
against a given amino acid
sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues in
B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of
B to A. As examples of % amino acid sequence identity calculations, Tables 2
and 3 demonstrate how to
calculate the % amino acid sequence identity of the amino acid sequence
designated "Comparison Protein" to
the amino acid sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained
as described above using the ALIGN-2 sequence comparison computer program.
However, % amino acid
sequence identity may also be determined using the sequence comparison program
NCBI-BLAST2 (Altschul et
al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be
downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all
of those search parameters are set to default values including, for example,
unmask = yes, strand = all,
expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-
value = 0.01, constant for
multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix =
BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino
acid sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence A that has
or comprises a certain % amino
acid sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues
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in B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will not equal
the % amino acid sequence identity
of B to A.
"SRT variant polynucleotide" or "SRT variant nucleic acid sequence" means a
nucleic acid molecule
which has at least about 80% nucleic acid sequence identity with~any of the
nucleic acid sequences shown in the
accompanying figures or a specified fragment thereof. Ordinarily, a SRT
variant polynucleotide will have at
least about 80% nucleic acid sequence identity, more preferably at least about
81 % nucleic acid sequence
identity, more preferably at least about 82 % nucleic acid sequence identity,
more preferably at least about 83
nucleic acid sequence identity, more preferably at least about 84 % nucleic
acid sequence identity, more
preferably at least about 85% nucleic acid sequence identity, more preferably
at least about 86% nucleic acid
sequence identity, more preferably at least about 87 % nucleic acid sequence
identity, more preferably at least
about 88 % nucleic acid sequence identity, more preferably at least about 89 %
nucleic acid sequence identity,
more preferably at least about 90% nucleic acid sequence identity, more
preferably at least about 91 % nucleic
acid sequence identity, more preferably at least about 92% nucleic acid
sequence identity, more preferably at
least about 93 % nucleic acid sequence identity, more preferably at least
about 94 % nucleic acid sequence
identity, more preferably at least about 95 % nucleic acid sequence identity,
more preferably at least about 96
nucleic acid sequence identity, more preferably at least about 97 % nucleic
acid sequence identity, more
preferably at least about 98% nucleic acid sequence identity and yet more
preferably at least about 99% nucleic
acid sequence identity with any of the nucleic acid sequences shown in the
accompanying figures or a specified
fragment thereof. SRT polynucleotide variants do not encompass the native SRT
nucleotide sequence.
Ordinarily, SRT variant polynucleotides are at least about 10 nucleotides in
length, often at least about
15 nucleotides in length, often at least about 20 nucleotides in length, often
at least about 25 nucleotides in
length, often at least about 30 nucleotides in length, often at least about 35
nucleotides in length, often at least
about 40 nucleotides in length, often at least about 45 nucleotides in length,
often at least about 50 nucleotides
in length, often at least about 55 nucleotides in length, often at least about
60 nucleotides in length, often at least
about 65 nucleotides in length, often at least about 65 nucleotides in length,
often at least about 70 nucleotides
in length, often at least about 75 nucleotides in length, often at least about
80 nucleotides in length, often at least
about 85 nucleotides in length, often at least about 90 nucleotides in length,
often at least about 95 nucleotides
in length, often at least about 100 nucleotides in length, or more.
"Percent ( % ) nucleic acid sequence identity" with respect to the SRT
polypeptide-encoding nucleic acid
sequences identified herein is defined as the percentage of nucleotides in a
candidate sequence that are identical
with the nucleotides in a SRT polypeptide-encoding nucleic acid sequence,
after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence
identity. Alignment for purposes of
determining percent nucleic acid sequence identity can be achieved in various
ways that are within the skill in
the art, for instance, using publicly available computer software such as
BLAST, BLAST-2, ALIGN, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring
alignment, including any algorithms needed to achieve maximal alignment over
the full-length of the sequences
being compared. For purposes herein, however, % nucleic acid sequence identity
values are obtained as
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described below by using the sequence comparison computer program ALIGN-2,
wherein the complete source
code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence
comparison computer program
was authored by Genentech, Inc. and the source code shown in Table 1 has been
filed with user documentation
in the U.S. Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration
No. TXU510087. The ALIGN-2 program is publicly available through Genentech,
Inc., South San Francisco,
California or may be compiled from the source code provided in Table 1. The
ALIGN-2 program should be
compiled for use on a UNIX operating system, preferably digital UNIX V4.OD.
All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % nucleic acid sequence identity of a given nucleic
acid sequence C to, with,
or against a given nucleic acid sequence D (which can alternatively be phrased
as a given nucleic acid sequence
C that has or comprises a certain % nucleic acid sequence identity to, with,
or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-2
in that program's alignment of C and D, and where Z is the total number of
nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence
D, the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to
C. As examples of % nucleic acid sequence identity calculations, Tables 4 and
5 demonstrate how to calculate
the % nucleic acid sequence identity of the nucleic acid sequence designated
"Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA".
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are obtained
as described above using the ALIGN-2 sequence comparison computer program.
However, % nucleic acid
sequence identity may also be determined using the sequence comparison program
NCBI-BLAST2 (Altschul et
al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be
downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all
of those search parameters are set to default values including, for example,
unmask = yes, strand = all,
expected occurrences = 10, minimum low complexity length = 15/5, mufti-pass e-
value = 0.01, constant for
mufti-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix =
BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence
identity of a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which can
alternatively be phrased as a given nucleic acid sequence C that has or
comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid sequence D) is
calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program NCBI-
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BLAST2 in that program's alignment of C and D, and where Z is the total number
of nucleotides in D. It will
be appreciated that where the length of nucleic acid sequence C is not equal
to the length of nucleic acid sequence
D, the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to
C.
In other embodiments, SRT variant polynucleotides are nucleic acid molecules
that encode an active
SRT polypeptide and which are capable of hybridizing, preferably under
stringent hybridization conditions, to
any of the nucleotide sequences shown in the accompanying figures or their
complements. SRT variant
polypeptides may be those that are encoded by a SRT variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity
comparisons performed as
described above, includes amino acid residues in the sequences compared that
are not only identical, but also
those that have similar properties. Amino acid residues that score a positive
value to an amino acid residue of
interest are those that are either identical to the amino acid residue of
interest or are a preferred substitution (as
defined in Table 6 below) of the amino acid residue of interest.
For purposes herein, the % value of positives of a given amino acid sequence A
to, with, or against a
given amino acid sequence B (which can alternatively be phrased as a given
amino acid sequence A that has or
comprises a certain % positives to, with, or against a given amino acid
sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as
defined above by the sequence
alignment program ALIGN-2 in that program's alignment of A and B, and where Y
is the total number of amino
acid residues in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the
length of amino acid sequence B, the % positives of A to B will not equal the
% positives of B to A.
"Isolated, " when used to describe the various polypeptides disclosed herein,
means polypeptide that has
been identified and separated and/or recovered from a component of its natural
environment. Preferably, the
isolated polypeptide is free of association with all components with which it
is naturally associated. Contaminant
components of its natural environment are materials that would typically
interfere with diagnostic or therapeutic
uses for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least
15 residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions using
Coomassie blue or, preferably,
silver stain. Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one
component of the SRT natural environment will not be present. Ordinarily,
however, isolated polypeptide will
be prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a SRT polypeptide is a nucleic
acid molecule that is
identified and separated from at least one contaminant nucleic acid molecule
with which it is ordinarily associated
in the natural source of the SRT-encoding nucleic acid. Preferably, the
isolated nucleic is free of association
with all components with which it is naturally associated. An isolated SRT-
encoding nucleic acid molecule is
24
CA 02378403 2002-O1-04
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other than in the form or setting in which it is found in nature. Isolated
nucleic acid molecules therefore are
distinguished from the SRT-encoding nucleic acid molecule as it exists in
natural cells. However, an isolated
nucleic acid molecule encoding a SRT polypeptide includes SRT-encoding nucleic
acid molecules contained in
cells that ordinarily express SRT where, for example, the nucleic acid
molecule is in a chromosomal location
different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the
expression of an operably
linked coding sequence in a particular host organism. The control sequences
that are suitable for prokaryotes,
for example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic
acid sequence. For example, DNA for a presequence or secretory leader is
operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is positioned so as
to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are contiguous,
and, in the case of a secretory
leader, contiguous and in reading phase. However, enhancers do not have to be
contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-SRT
monoclonal antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-SRT antibody
compositions with polyepitopic specificity, single chain anti-SRT antibodies,
and fragments of anti-SRT
antibodies (see below). The term "monoclonal antibody" as used herein refers
to an antibody obtained from a
population of substantially homogeneous antibodies, i. e. , the individual
antibodies comprising the population are
identical except for possible naturally-occurring mutations that may be
present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when
complementary strands are present in an environment below their melting
temperature. The higher the degree
of desired homology between the probe and hybridizable sequence, the higher
the relative temperature which
can be used. As a result, it follows that higher relative temperatures would
tend to make the reaction conditions
more stringent, while lower temperatures less so. For additional details and
explanation of stringency of
hybridization reactions, see Ausubel et al., Current Protocols in Molecular
Biolo~y, Wiley Interscience
Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may
be identified by those
that: (1) employ low ionic strength and high temperature for washing, for
example 0.015 M sodium
chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C;
(2) employ during hybridization a
denaturing agent, such as formamide, for example, 50 % (v/v) formamide with
0.1 % bovine serum
CA 02378403 2002-O1-04
WO 01/07611 PCT/US00/20006
albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer
at pH 6.5 with 750 mM sodium
chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5
x SSC (0.75 M NaCI, 0.075 M
sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate,
5 x Denhardt's solution,
sonicated salmon sperm DNA (50 ~.g/ml), 0.1 % SDS, and 10% dextran sulfate at
42°C, with washes at 42°C
in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55
°C, followed by a high-stringency wash
consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately stringent conditions" may be identified as described by Sambrook
et al., Molecular
Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and
include the use of washing
solution and hybridization conditions (e.g., temperature, ionic strength and
%SDS) less stringent that those
described above. An example of moderately stringent conditions is overnight
incubation at 37°C in a solution
comprising: 20 % formamide, 5 x SSC ( 150 mM NaCI, 15 mM trisodium citrate),
50 mM sodium phosphate (pH
7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured
sheared salmon sperm DNA,
followed by washing the filters in 1 x SSC at about 37-50°C. The
skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate factors such
as probe length and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide
comprising a SRT
polypeptide fused to a "tag polypeptide" . The tag polypeptide has enough
residues to provide an epitope against
which an antibody can be made, yet is short enough such that it does not
interfere with activity of the polypeptide
to which it is fused. The tag polypeptide preferably also is fairly unique so
that the antibody does not
substantially cross-react with other epitopes. Suitable tag polypeptides
generally have at least six amino acid
residues and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino
acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules
which combine the
binding specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin
constant domains. Structurally, the immunoadhesins comprise a fusion of an
amino acid sequence with the
desired binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence. The adhesin
part of an immunoadhesin
molecule typically is a contiguous amino acid sequence comprising at least the
binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin may
be obtained from any
immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including
IgA-1 and IgA-2), IgE, IgD
or IgM.
"Active" or "activity" for the purposes herein refers to forms) of SRT which
retain a biological and/or
an immunological activity of native or naturally-occurring SRT, wherein
"biological" activity refers to a
biological function (either inhibitory or stimulatory) caused by a native or
naturally-occurring SRT other than
the ability to induce the production of an antibody against an antigenic
epitope possessed by a native or naturally
occurring SRT and an "immunological" activity refers to the ability to induce
the production of an antibody
against an antigenic epitope possessed by a native or naturally-occurring SRT.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully
blocks, inhibits, or neutralizes a biological activity of a native SRT
polypeptide disclosed herein. In a similar
26
CA 02378403 2002-O1-04
WO 01/07611 PCT/US00/20006
manner, the term "agonist" is used in the broadest sense and includes any
molecule that mimics a biological
activity of a native SRT polypeptide disclosed herein. Suitable agonist or
antagonist molecules specifically
include agonist or antagonist antibodies or antibody fragments, fragments or
amino acid sequence variants of
native SRT polypeptides, peptides, small organic molecules, etc. Methods for
identifying agonists or
antagonists of a SRT polypeptide may comprise contacting a SRT polypeptide
with a candidate agonist or
antagonist molecule and measuring a detectable change in one or more
biological activities normally associated
with the SRT polypeptide.
"Treatment" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein
the object is to prevent or slow down (lessen) the targeted pathologic
condition or disorder. Those in need of
treatment include those already with the disorder as well as those prone to
have the disorder or those in whom
the disorder is to be prevented.
"Chronic" administration refers to administration of the agents) in a
continuous mode as opposed to
an acute mode, so as to maintain the initial therapeutic effect (activity) for
an extended period of time.
"Intermittent" administration is treatment that is not consecutively done
without interruption, but rather is cyclic
m nature.
"Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as dogs,
cats, cattle, horses, sheep, pigs, goats,
rabbits, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which
are nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically
acceptable carriers include buffers such as phosphate, citrate, and other
organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols
such as mannitol or sorbitol; salt-
forming counterions such as sodium; and/or nonionic surfactants such as
TWEEN~, polyethylene glycol (PEG),
and PLURONICST"'.
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen binding or
variable region of the intact antibody. Examples of antibody fragments include
Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies (Zapata et al., Protein En~. 8(10):
1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from antibody
fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab"
fragments, each with a single antigen-binding site, and a residual "Fc"
fragment, a designation reflecting the
ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment
that has two antigen-combining sites
and is still capable of cross-linking antigen.
27
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"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding
site. This region consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent
association. It is in this configuration that the three CDRs of each variable
domain interact to define an antigen-
binding site on the surface of the VH-VL dimer. Collectively, the six CDRs
confer antigen-binding specificity
to the antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific
for an antigen) has the ability to recognize and bind antigen, although at a
lower affinity than the entire binding
site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain
(CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the
addition of a few residues at the
carboxy terminus of the heavy chain CH1 domain including one or more cysteines
from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the cysteine
residues) of the constant domains bear
a free thiol group. F(ab')Z antibody fragments originally were produced as
pairs of Fab' fragments which have
hinge cysteines between them. Other chemical couplings of antibody fragments
are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one
of two clearly distinct types, called kappa and lambda, based on the amino
acid sequences of their constant
domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several of these may be further divided into subclasses (isotypes),
e.g., IgGI, IgG2, IgG3, IgG4, IgA,
and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the sFv to form
the desired structure for
antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable domain (VL) in the
same polypeptide chain (VH - VL). By using a linker that is too short to allow
pairing between the two domains
on the same chain, the domains are forced to pair with the complementary
domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in, for example,
EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or
recovered from a
component of its natural environment. Contaminant components of its natural
environment are materials which
would interfere with diagnostic or therapeutic uses for the antibody, and may
include enzymes, hormones, and
other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the
antibody will be purified (1)
to greater than 95 % by weight of antibody as determined by the Lowry method,
and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-
terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-
PAGE under reducing or
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nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of the
antibody's natural environment will not be
present. Ordinarily, however, isolated antibody will be prepared by at least
one purification step.
An antibody that "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on
a particular polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide
without substantially binding to any other polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound or
composition which is conjugated
directly or indirectly to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself
(e.g. radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical
alteration of a substrate compound or composition which is detectable.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the
present invention can
adhere. Examples of solid phases encompassed herein include those formed
partially or entirely of glass (e.g.,
controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides,
polystyrene, polyvinyl alcohol and
silicones. In certain embodiments, depending on the context, the solid phase
can comprise the well of an assay
plate; in others it is a purification column (e.g., an affinity chromatography
column). This term also includes
a discontinuous solid phase of discrete particles, such as those described in
U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant
which is useful for delivery of a drug (such as a SRT polypeptide or antibody
thereto) to a mammal. The
components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of
biological membranes.
A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
An "oligonucleotide" or "oligomer" is a stretch of nucleotide residues which
has a sufficient number
of bases to be used in a polymerase chain reaction (PCR). These sequences are
based on (or designed from)
genomic or cDNA sequences and may be used to amplify, confirm, or reveal the
presence of an identical, similar
or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides
or oligomers comprise portions
of a DNA sequence having at least about 10 nucleotides as described above.
Oligonucleotides may be chemically
synthesized and may be used as probes.
"Probes" are nucleic acid sequences of variable length, preferably between
about 10 and as many as
about 6000 nucleotides, depending upon use. They are used in the detection of
identical, similar or
complementary nucleic acid sequences. Longer length probes are usually
obtained from a natural or recombinant
source, are highly specific and are often much slower to hybridize to a target
nucleic acid than are oligomers.
Probes may be single- or double-stranded and may be carefully designaed to
have specificity in PCR,
hybridization membrane-based, or ELISA-like technologies.
"Detectably labeled" with regard to a nucleic acid molecule of the present
invention means that the
molecule has attached thereto, either covalently or non-covalently, a compound
which is detectable such as, for
example, radionuclides, enzymes, fluorescent, chemi-luminescent, or
chromogenic agents. Detectable labels
associate with, establish the presence of, and may allow quantification of a
particular nucleic or amino acid
sequence.
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A "portion" or "fragment" of a polynucleotide or nucleic acid molecule
comprises all or any part of the
nucleotide sequence having fewer nucleotides than about 6 kb, preferably fewer
than about 1 kb which can be
used as a probe. Such probes may be labelled with detectable labels using nick
translation, Klenow fill-in
reaction, PCR or other methods well known in the art. After pretesting to
optimize reaction conditions and to
eliminate false positives, nucleic acid probes may be used in Southern,
Northern or in situ hybridizations to
determine whether DNA or RNA encoding the protein is present in a biological
sample, cell type, tissue, organ
or organism.
CA 02378403 2002-O1-04
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Table 1
/*
* C-C
increased
from
12
to
15
* Z
is
average
of
EQ
* B
is
average
of
ND
* match with stop is M; stop-stop = 0; J (joker) match = 0
* %
IldefineM -8 /* value of a match with a stop */
int _day[26][26] _ {
/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/* { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-I,-2,-1, O, M, 1, 0,-2,
A I, 1, 0, 0,-6, 0,-3, 0},
*/
/* { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2, M,-1, 1, 0,
B 0, 0, 0,-2,-5, 0,-3, 1},
*/
/* {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4, M,-3,-5,-4,
C 0,-2, 0,-2,-8, 0, 0,-5},
*/
1$ /* { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1, 2,-1,
D 0, 0, 0,-2,-7, 0,-4, 2},
*/
/* { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1, M,-1, 2,-1,
E 0, 0, 0,-2,-7, 0,-4, 3},
*/
/* {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-5,-4,-3,-3,
F 0,-1, 0, 0, 7,-5},
*/
/* { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, O, M,-1,-1,-3,
G 1, 0, 0,-1,-7, 0,-5, 0},
*/
/* {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2,-1,-1,
H 0,-2,-3, 0, 0, 2},
*/
/* {-1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2, M,-2,-2,-2,-1,
I 0, 0, 4,-5, 0,-1,-2},
*/
/* { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, O, M, 0, 0, 0,
J 0, 0, 0, 0, 0, 0, 0, 0},
*/
/* {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1, M,-1, 1, 3,
K 0, 0, 0,-2,-3, 0,-4, 0},
*/
/* {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1,
L 0, 2,-2, 0,-1,-2},
*/
/* {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, 0,-2,-1,
M 0, 2,-4, 0,-2,-1},
*/
/* { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1, 1, 0,
N 1, 0, 0,-2,-4, 0,-2, 1},
*/
/* { M,_M,_M,_M, M, M, M, M, M, M, M, M, M, M, O, M, M, M,
O M, M, M, M, M, M, M, M},
*/
/* { 1,-I,-3,-1,-1,-5,-1, 0,-2, 0,-1,-3,-2,-1, M, 6, 0, 0,
P 1, 0, 0,-1,-6, 0,-5, 0},
*/
/* { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1, M, 0, 4, 1,-1,-1,
Q 0,-2,-5, 0,-4, 3},
*/
/* {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, O, M, 0, 1, 6,
R 0,-1, 0,-2, 2, 0,-4, 0},
*/
/* { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M, 1,-1, 0,
S 2, 1, 0,-1,-2, 0,-3, 0},
*/
/* { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, O, M, 0,-1,-1,
T 1, 3, 0, 0,-5, 0,-3, 0},
*/
/*U*/ {0,0,0,0,0,0,0,0,0,0,0,0,0,0, M,0,0,0,0,0,0,0,0,0,0,0},
/* { 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2, M,-1,-2,-2,-1,
V 0, 0, 4,-6, 0,-2,-2},
*/
/* {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4, M,-6,-5, 2,-2,-5,
W 0,-6,17, 0, 0,-6},
*/
/* { o, o, o, o, o, o, o, o, o, o, o, o, o, o,_M, o, o, o,
x o, o, o, o, o, o, o, o},
*/
/* {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4,-3,-3,
Y 0,-2, 0, 0,10,-4},
*/
/* { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1, M, 0, 3, 0,
Z 0, 0, 0,-2,-6, 0,-4, 4}
*/
};
45
55
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Table 1 (cont'~
/*
*/
#include
< stdio.h
>
#include
< ctype.
h >
#defineMAXJMP 16 /* max jumps in a diag */
#defineMAXGAP 24 /* don't continue to penalize
gaps larger than this */
#defineJMPS 1024/* max jmps in an path */
#defineMX 4 /* save if there's at least
MX-1 bases since last jmp
*/
#defineDMAT 3 /* value of matching bases
*/
#defineDMIS 0 /* penalty for mismatched
bases */
#defineDINSO 8 /* penalty for a gap */
#defineDINS1 1 /* penalty per base */
IS #definePINSO 8 /* penalty for a gap */
#definePINS1 4 /* penalty per residue */
struct
jmp
{
short n[MAXJMP];
/*
size
of
jmp
(neg
for
dely)
*/
unsigned x[MAXJMP];
short /*
base
no.
of
jmp
in
seq
x
*/
}; /* limits seq to 2"16 -1 */
struct ag
di {
int score;/* score at last jmp */
long offset;/* offset of prev block */
short ijmp;/* current jmp index */
struct jp; /* list of jmps */
jmp
};
struct
path
{
int spc; /* number of leading spaces
*/
shortn[JMPS]; /* size of jmp (gap) */
int x[JMPS]; /* loc of jmp (last elem before
gap) */
}>
char *ofile; /* output file name */
char *namex[2]; /* seq names: getseqsQ */
char *prog; /* prog name for err msgs
*/
char *seqx[2]; /* seqs: getseqsQ */
int dmax; /* best diag: nwQ */
int dmax0; /* final diag */
int dna; /* set if dna: main() */
int endgaps; /* set if penalizing end gaps
*/
int gapx, /* total gaps in seqs */
gapy;
int len0, /* seq lens */
lenl;
int ngapx, /* total size of gaps */
ngapy;
int smax; /* max score: nwQ */
int *xbm; /* bitmap for matching */
long offset; /* current offset in jmp file
*/
$0 struct diag*dx; /* holds diagonals */
struct pathpp[2]; /* holds path for seqs */
char *calloc(),*mallocQ, *indexQ, *strcpyQ;
char *getseqQ,
*g
callocQ;
5 5
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Table 1 (cont'1
/* Needleman-Wunsch alignment program
* usage: progs file 1 filet
* where filel and filet are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with '; ' >' or ' <' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*/
#include "nw.h"
IS #include "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
static ~bval[26] _ {
1, 2~(1< <('D'-'A'))~(1< <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14,
1«15, 1«16, 1«17, 1«18, 1«19, 1«20, 1«21, 1«22,
1«23, 1«24, 1«25(1«('E'-'A'))~(1«('Q'-'A'))
main(ac, av) main
int ac;
char *av[];
{
prog = av[0];
if (ac ! = 3) {
fprintf(stderr, "usage: % s filet filet\n", prog);
fprintf(stderr,"where filet and filet are two dna or two protein
sequences.\n");
fprintf(stderr,"The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignored\n");
fprintf(stderr, "Output is in the file \"align.out\"\n");
exit( 1 );
namex[0] = av[1];
namex[1] = av[2];
seqx[0] = getseq(namex[0], &len0);
seqx[1] = getseq(namex[1], &lenl);
4$ xbm = (dna)? dbval : ~bval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; /* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps */
printQ; /* print stars, alignment */
cleanup(0); /* unlink any tmp files */
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Table 1 (cony)
/* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
$ * a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
*/
nw() riW
{
char *px, *py; /* seqs and ptrs */
int *ndely, *dely; /* keep track of dely */
int ndelx, delx; /* keep track of delx */
int *tmp; /* for swapping row0, rowl */
int mis; /* score for each type */
IS int ins0, insl; /* insertion penalties */
register id; /* diagonalindex */
register ij; 1* jmp index */
register *col0, *coll; /* score for curr, last row */
register xx, yy; /* index into seqs */
dx = (struct diag *)g calloc("to get diags", len0+lenl+1, sizeof(struct
diag));
ndely = (int *)g calloc("to get ndely", lenh+1, sizeof(int));
dely = (int *)g calloc("to get defy", lenl + 1, sizeof(int));
2$ col0 = (int *)g calloc("to get col0", lent + 1, sizeof(int));
toll = (int *)g calloc("to get coil", lent+1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
insl = (dna)? DINS1 : PINS1;
smax = -10000;
if (endgaps) {
for (col0[0] = dely[0] _ -ins0, yy = 1; yy < = lenl; yy++) {
col0[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yy] = yy;
col0[0] = 0; /* Waterman Bull Math Biol 84 */
else
for (yy = 1; yy < = lenl; yy++)
dely[yy] _ -ins0;
/* fill in match matrix
*/
for (px = seqx[0], xx = 1; xx < = len0; px++, xx++) {
/* initialize first entry in col
*/
if (endgaps) {
~(~ _= 1)
coil[0] = delx = -(ins0+insl);
else
coi l [0] = delx = col0[0] - ins l ;
ndelx = xx;
else {
toll[0] = 0;
delx = -ins0;
ndelx = 0;
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Table 1 (cont'1
for (py = seqx[1], yy = 1; yy < = lenl; py++,
yy++) {
mis = col0[yy-1];
if (dna)
$ mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT :
DMIS;
else
mis += day[*px-'A'][*py-'A'l;
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
*/
if (endgaps ~ ~ ndely[yy] < MAXGAP) {
if (col0[yy] - ins0 > = defy[yy]) {
IS defy[yy] = col0[yy] - (ins0+insl);
ndely[yy] = 1;
} else {
dely[yy] -= insl;
ndely[yy] + +;
}
} else {
if (col0[yy] - (ins0+insl) > = defy[yy]) {
dely[yy] = col0[yy] - (ins0+insl);
ndely[yy] = l;
} else
ndely[yy] + +;
}
/* update penalty for del in y seq;
* favor new del over ongong del
*/
if (endgaps ~ ~ ndelx < MAXGAP) {
if (coll[yy-1] - ins0 > = delx) {
delx = coll[yy-1] - (ins0+insl);
ndelx = 1;
} else {
delx -= insl;
ndelx+ +;
}
} else {
if (coll[yy-1] - (ins0+insl) > = delx) {
delx = coll[yy-1] - (ins0+insl);
ndelx = 1;
} else
ndelx+ +;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over defy
*/
60
...nw
CA 02378403 2002-O1-04
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Table 1 cony)
id=xx-yy+lenl-1;
if (mis > = delx && mis > = dely[yy])
toll [yy] = mis;
else if (delx > = dely[yy]) {
toll[yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id] . ijmp+ + ;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
}
else {
toll[yy] = dely[yy];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id]. ijmp+ +;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
.}
dx[id].jp.n[ij] _ -ndely[yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
}
if (xx == len0 && yy < lenl) {
/* last col
*/
if (endgaps)
toll[yy] -= ins0+insl*(lenl-yy);
if (cot l [yy] > smax) {
smax = toll[yy];
dmax = id;
}
}
$0 if (endgaps && xx < len0)
toll[yy-1] -= ins0+insl*(len0-xx);
if (toll[yy-1] > smax) {
smax = toll[yy-1];
dmax = id;
}
tmp = col0; col0 = coil; toll = tmp;
}
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)coll); }
...nw
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Table 1 (cony)
/*
* print() -- only routine visible outside this module
* static:
* getmatQ -- trace back best path, count matches: print()
* pr align() -- print alignment of described in array p[]: print()
* dumpblockQ -- dump a block of lines with numbers, stars: pr align()
* numsQ -- put out a number line: dumpblockQ
* putlineQ -- put out a line (name, [num], seq, [num]): dumpblockQ
* stars() - -put a line of stars: dumpblockQ
* stripnameQ -- strip any path and prefix from a seqname
*/
IS #include "nw.h"
#define SPC 3
#define P LINE 256 /* maximum output line */
#define P SPC 3 /* space between name or num and seq */
extern _day[26][26];
int olen; /* set output line length */
FILE *fx; /* output file */
print
print()
{
int lx, 1y, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr, " % s: can't write % s\n", prog, ofile);
cleanup(1);
fprintf(fx, " < first sequence: % s (length = % d)\n", namex[0], len0);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[1], lenl);
olen = 60;
lx = IenO;
1y = lenl;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x */
pp[0].spc = firstgap = lent - dmax - 1;
1y -= pp[0].spc;
else if (dmax > lenl - 1) { /* leading gap in y */
pp[1].spc = firstgap = dmax - (lent - 1);
4$ Ix -= pp[1].spc;
if (dmax0 < len0 - 1) { /* trailing gap in x */
lastgap = len0 - dmax0 -1;
lx -= lastgap;
)
else if (dmax0 > len0 - 1) { /* trailing gap in y */
lastgap = dmax0 - (len0 - 1);
1y -= lastgap;
getmat(lx, 1y, firstgap, lastgap);
pr align();
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Table 1 (cony)
/*
* trace back the best path, count matches
*/
static
$ getmat(lx, 1y, firstgap, lastgap) getIriat
int Ix, 1y; /* "core" (minus endgaps) */
int firstgap, lastgap; /* leading trailing overlap */ '
{
int rnn, i0, i 1, siz0, siz 1;
char outx[32];
double pct;
register n0, n1;
register char *p0, *pl;
1$ /* get total matches, score
*/
i0 = i1 = siz0 = sizl = 0;
p0 = seqx[0] + pp[1].spc;
p1 = seqx[1] + pp[0].spc; '
n0 = pp[1].spc + 1;
n1 = pp[0].spc + 1;
= p;
while ( *p0 && *pl ) {
if (siz0) {
p1++;
n1++;
siz0--;
else if (sizl) {
p0++;
n0++;
siz 1--;
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm+ +;
if (n0++ _= pp[0].x[i0])
siz0 = pp[O].n[i0++];
if (n1++ _= pp[1].x[il])
sizl = pp[1].n[il++];
p0++;
p1++;
/* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
SO */
if (endgaps)
Ix = (len0 < lenl)? len0 : lenl;
else
lx = (lx < 1y)? lx : 1y;
pct = 100. *(double)nm/(double)Ix;
fprintf(fx, "\n");
fprintf(fx, " < % d match% s in an overlap of % d: % .2f percent
similarity\n",
~, (~ _= I)? ".. . "es" lx, pct);
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Table 1 (cony)
fprintf(fx, " < gaps in first sequence: %d", gapx); ... getrilat
if (gapx) {
(void) sprintf(outx, " ( % d % s % s)",
ngapx, (dna)? "base":"residue", (ngapx = = 1)? "':"s");
fprintf(fx,"%s", outx);
fprintf(fx, ", gaps in second sequence: % d", gapy);
if (gapY) {
(void) sprintf(outx, " ( % d % s % s)",
ngapy, (dna)? "base":"residue", (ngapy = = 1)? "':"s");
fprintf(fx,"%s", outx);
1
if (dna)
I S fprintf(fx,
"\n < score: % d (match = % d, mismatch = % d, gap penalty = % d + % d per
base)\n",
smax, DMAT, DMIS, DINSO, DINSI);
else
fprintf(fx,
"\n < score: % d (Dayhoff PAM 250 matrix, gap penalty = % d + % d per
residue)\n" ,
smax, PINSO, PINSI);
if (endgaps)
fprintf(fx,
" < endgaps penalized. left endgap: % d % s % s, right endgap: % d % s % s\n"
,
firstgap, (dna)? "base" : "residue", (firstgap == 1)? "" . "s",
lastgap, (dna)? "base" : "residue", (lastgap == 1)? "' . "s");
else
fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core -- for checking */
static lmax; /* lengths of stripped file names */
static ij[2]; /* jmp index for a path */
static nc[2]; /* number at start of current line */
static ni[2]; /* current elem number -- for gapping */
static siz[2];
static char*ps[2]; /* ptr to current element */
static char*po[2]; /* ptr to next output char slot */
static charout[2][P LINE]; /* output line */
static charstar[P LINE]; /* set by stars() */
/*
* print of described in struct path pp[]
alignment
*/
static
pr align() pr allgri
{
int nn; /* char count */
int more;
register i;
for (i = 0, Imax = 0; i < 2; i++) {
nn = stripname(namex[i]);
if (nn > lmax)
$$ lmax = nn;
nc[i] = 1;
ni[i] = I;
siz[i] = ij[ij = 0;
ps[i] = seqx[i];
po[i] = out[i]; }
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Table 1 (cony)
for (nn = nm = 0, more = l; more; ) { ...pr align
for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence?
*/
if (!*ps[i])
continue;
- more++;
if (pp[i].spc) { /* leading space */
*po[i]++ _ ,
pp[i] . spc--;
)
else if (siz[i]) { /* in a gap */
*po[i]++ _ ,
siz[i]--;
else { /* we're putting a seq element
*/
*Pofil = *Ps[i];
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i] + +;
ps[i]++;
/*
* are we at next gap for this seq?
*/
if (ni[i] _= pp[i].x[ij[i]]) {
/*
* we need to merge all gaps
* at this location
*/
siz[i] = pp[i].n[ij[i]++];
while (ni[i] _= pp[i].x[ij[i]])
siz[i] += pp[i].n[ij[i]++];
ni[i]++;
if (++nn == olen ~ ~ !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++)
po[i] = out[i];
nn = 0;
}
/*
* dump a block of lines, including numbers, stars: pr align()
*/
static
dumpblockQ dumpblock
{
register i;
for (i = 0; i < 2; i++)
*po[i]__ _ '\0'~
CA 02378403 2002-O1-04
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Table 1 (cony)
...dumpblock
(void) putc('\n', fx);
for (i = 0; i < 2; i++) {
if (*out[i] && (*out[i] ! _ ' ' I I *(Po[iD ! _ ' ')) {
if (i == 0)
nums(i);
if (i == 0 && *out[1])
starsQ;
putline(i);
if (i == 0 && *out[1])
fprintf(fx, star);
if (i == 1)
nums(i);
1
/*
* put out a number line: dumpblockQ
*/
static
nums(ix) nums
int ix; /* index in out[] holding seq line */
{
char mine[P LINE];
register i, j;
register char *pn, *px, *py;
for (pn = mine, i = 0; i < lmax+P SPC; i++, pn++)
*Pn = ,
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
if (*py =- ' I I *PY =_ -')
*Pn = ,
else {
if (i% 10 == 0 I I (i == 1 && nc[ix] != 1)) {
j = (i < 0)? -i : i;
for (px = pn; j; j /= 10, px--)
*px=j%10+'0';
if (i < 0)
*px = ,
1
else
*Pn = >
i++;
1
1
*Pn = '\0';
nc[ix] = i;
$0 for (pn = nline; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
1
5$ /*
* put out a line (name, [num], seq, [num]): dumpblockQ
*/
static
putline(ix) puthrie
60 int ix; {
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Table 1 cont'~
. . . putline
int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px !_ '''; px++, i++)
(void) putc(*px, fx);
for (; i < lmax+P SPC; i++)
(void) putc(' ', fx);
/* these count from 1:
* ni[] is current element (from 1)
* nc[] is number at start of current line
*/
for (px = out[ix]; *px; px++)
(void) putc(*px&Ox7F, fx);
(void) putc('\n', fx);
/*
* put line of stars (seqs always in out[0], out[1]): dumpblockQ
a
*/
static
stars()stars
{
int i;
register char *p0, *pl, cx, *px;
if (!*out[0] ~ ~ (*out[0] _- ' && *(po(0]) _- ' ') ~ ~
!*out[1] ~ ~ (*out[1] _- ' && *(po[1]) _- '))
return;
px = star;
for (i = lmax+P SPC; i; i--)
*px++ _ ,
for (p0 = out[0], p1 = out[1]; *p0 && *pl; p0++, p1++) {
if (isalpha(*p0) && isalpha(*pl)) {
if (xbm[*p0-'A']&xbm[*pl-'A']) {
cx = '*';
nm++;
else if (!dna && day[*p0-'A'][*pl-'A'] > 0)
cx = ,
else
cx = ,
else
cx = ,
*px++ = cx;
*px++ _ '\n';
*Px = ~\~~;
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Table 1 (cony)
/*
* strip path or prefix from pn, return len: pr align()
*/ _
static
S stripname(pn) stripname
char *pn; /* file name (may be path) */
register char *px, *py;
IO py = 0;
for (px = pn; *px; px++)
~(*Px =_ '/')
py=px+1;
(PY)
IS (void) strcpy(pn, py);
return(strlen(pn));
2S
3S
4S
SO
SS
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Table 1 (cony)
/*
* cleanup() -- cleanup any tmp file
* getseq() -- read in seq, set dna, len, maxlen
* g callocQ -- callocQ with error checkin
* readjmpsQ -- get the good jmps, from tmp file if necessary
* writejmpsQ -- write a filled array of jmps to a tmp file: nwQ
*/
#include "nw.h"
#include < sys/file.h >
char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */
FILE *fj;
int cleanup(); /* cleanup tmp file */
I S long lseek();
/*
* remove any tmp file if we blow
*/
cleanup(i) Cleanup
int i;
{
if (fj)
(void) unlink(jname);
exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with '; , ' <', or ' >'
* seq in upper or lower case
*/
char
getseq(file, len) getSeQ
3$ char *file; /* file name */
int *len; /* seq len */
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp; ,
if ((fp = fopen(file, "r")) _ = 0) {
fprintf(stderr," % s: can't read % s\n", prog, file);
exit(1);
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =- ' ~ ~ *line =- ' <' ~ ~ *line =- ' >')
continue;
for (px = line; *px ! _ '\n'; px++)
if (isupper(*px) ~ ~ islower(*px))
tlen+ +;
if ((pseq = malloc((unsigned)(tlen+6))) _ = 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for %s\n", prog, tlen+6,
file);
exit( 1 );
pseq[0] = pseq[1] = pseq[2] = pseq[3] _ '\0';
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Table 1 (cony)
... getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line =- ' ~ ~ *line =_ ' <' ~ ~ *line =_ ' >')
continue;
for (px = line; *px ! _ '\n'; px++) {
if (isupper(*px))
*py++ _ *px;
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU",*(py-1)))
1$ natgc + +;
*py++ _ '\0';
*pY = ~ \0' ;
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
2$ char
g calloc(msg, nx, sz) ' g_Ca110C
char *msg; /* program, calling routine */
int nx, sz; /* number and size of elements */
{
char *px, *callocQ;
if ((px = calloc((unsigned)nx, (unsigned)sz)) _ = 0)
(*msg) {
fprintf(stderr, " % s: g callocQ failed % s (n= % d, sz= % d)\n", prog, msg,
nx, sz);
3S exit(1);
)
)
return(px);
/*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main()
*/
readjmpsQ readjmps
4$ {
int fd = -1;
int siz, i0, i1;
register i, j, xx;
$0 if (fj) {
(void) fclose(fj);
if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintf(stderr, " % s: can't open() % s\n", prog, jname);
cleanup( 1 );
$$ }
for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j--)
60 ,
4$
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Table 1 (cony)
...readjmps
if (j < 0 && dx[dmax].offset && fj) {
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
}
else
break;
}
if (i > = JMPS) {
fprintf(stderr, " % s: too many gaps in alignment\n" , prog);
cleanup(1);
}
if (j >=o){
siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x[j];
dmax + = siz;
if (siz < 0) { /* gap in second seq */
pp[1].n[il] _ -siz;
xx + = siz;
/*id=xx-yy+lenl-1
*/
pp[1].x[il] = xx - dmax + lent - 1;
gapy+ +;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~ ~ endgaps)? -siz : MAXGAP;
i1++;
}
else if (siz > 0) { /* gap in first seq */
pp(0].n[io] = siz;
pp[0].x[io] = xx;
gapx+ +;
3S ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
}
else
)
break;
}
4$ /* reverse the order of jmps
*/
for (j = 0, i0--; j < i0; j + +, i0--) {
i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i;
i = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[io] = i;
}
for (j = 0, i1--; j < i1; j++, i1--) {
i = pp[1].n[j]; pp[1].n[j] = pp[1].n[il]; pp[1].n[il] = i;
i = pp[1].x[j]; pp[1].x[j] = pp[1].x[il]; pp[1].x[il] = i;
}
if (fa > = o)
(void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0;
} }
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Table 1 (cony)
/*
* write a filled jmp struct offset of the prev one (if any): nwQ
*/
writejmps(ix) writejmps
iut ix;
char *mktempQ;
if (!fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, " % s: can't mktempQ % s\n", prog, jname);
cleanup(1);
1$ if ((F = fopen(jname, "w")) _ = 0) {
fprintf(stderr, " % s: can't write % s\n", prog, jname);
exit( 1 );
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
30
40
50
60
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Table 2
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 15 = 33.3
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Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 10 = 50%
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Table 4
PRO-DNA NNNNNNNNNNNNNN
(Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
6 divided by 14 = 42.9
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Table 5
PRO-DNA NNNNNNNNNNNN
(Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
4 divided by 12 = 33.3
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II. Compositions and Methods of the Invention
A. Full-length SRT Polvpeptides
The present invention provides newly identified and isolated polynucleotide
sequences encoding at least
a portion of full-length human polypeptides referred to in the present
application as SRT polypeptides. In
particular, cDNAs encoding at least a portion of SRT polypeptides have been
identified and isolated, as disclosed
in further detail in the Examples below. For sake of simplicity, in the
present specification the polypeptides
encoded by nucleic acid molecules disclosed herein as well as all further
native homologues and variants included
in the foregoing definition of SRT, will be referred to as "SRT", regardless
of their origin or mode of
preparation.
B. SRT Polypeptide Variants
In addition to the native sequence SRT polypeptides described herein, it is
contemplated that SRT
variants can be prepared. SRT variants can be prepared by introducing
appropriate nucleotide changes into the
SRT DNA, and/or by synthesis of the desired SRT polypeptide. Those skilled in
the art will appreciate that
amino acid changes may alter post-translational processes of the SRT, such as
changing the number or position
of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native sequence SRT or in various domains of the SRT
described herein, can be made,
for example, using any of the techniques and guidelines for conservative and
non-conservative mutations set
forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a
substitution, deletion or insertion of one
or more codons encoding the SRT that results in a change in the amino acid
sequence of the SRT as compared
with the native sequence SRT. Optionally the variation is by substitution of
at least one amino acid with any
other amino acid in one or more of the domains of the SRT. Guidance in
determining which amino acid residue
may be inserted, substituted or deleted without adversely affecting the
desired activity may be found by
comparing the sequence of the SRT with that of homologous known protein
molecules and minimizing the
number of amino acid sequence changes made in regions of high homology. Amino
acid substitutions can be
the result of replacing one amino acid with another amino acid having similar
structural and/or chemical
properties, such as the replacement of a leucine with a serine, i.e.,
conservative amino acid replacements.
Insertions or deletions may optionally be in the range of about 1 to 5 amino
acids. The variation allowed may
be determined by systematically making insertions, deletions or substitutions
of amino acids in the sequence and
testing the resulting variants for activity exhibited by the full-length or
mature native sequence.
SRT polypeptide fragments are provided herein. Such fragments may be truncated
at the N-terminus
or C-terminus, or may lack internal residues, for example, when compared with
a full-length native protein.
Certain fragments lack amino acid residues that are not essential for a
desired biological activity of the SRT
polypeptide.
SRT fragments may be prepared by any of a number of conventional techniques.
Desired peptide
fragments may be chemically synthesized. An alternative approach involves
generating SRT fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme known to
cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with suitable
restriction enzymes and isolating the
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desired fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding
a desired polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
Preferably, SRT polypeptide
fragments share at least one biological and/or immunological activity with the
corresponding native SRT
polypeptide.
In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the heading
of preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial
changes, denominated exemplary substitutions in Table 6, or as further
described below in reference to amino
acid classes, are introduced and the products screened.
Table 6
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys
Asn (N) gln; his; lys; arg gln
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gln; lys; arg arg
Ile (I) leu; val; met; ala; phe;
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
ala; norleucine leu
Substantial modifications in function or immunological identity of the SRT
polypeptide are accomplished
by selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain. Naturally occurring residues
are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
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(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
Such substituted residues also may be introduced into the conservative
substitution sites or, more preferably, into
the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonueleotide-mediated (site-
direeted) mutagenesis, alanirie scanning, and PCR mutagenesis. Site-directed
mutagenesis [Carter et al., Nucl.
Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)],
cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA,
317:415 (1986)] or other known techniques can be performed on the cloned DNA
to produce the SRT variant
DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids along a
contiguous sequence. Among the preferred scanning amino acids are relatively
small, neutral amino acids. Such
amino acids include alanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the beta-carbon
and is less likely to alter the main-
chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-
1085 (1989)]. Alanine is also
typically preferred because it is the most common amino acid. Further, it is
frequently found in both buried and
exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1
(1976)]. If alanine substitution does not yield adequate amounts of variant,
an isoteric amino acid can be used.
C. Modifications of SRT Polvpeptides
Covalent modifications of SRT polypeptides are included within the scope of
this invention. One type
of covalent modification includes reacting targeted amino acid residues of a
SRT polypeptide with an organic
derivatizing agent that is capable of reacting with selected side chains or
the N- or C- terminal residues of the
SRT. Derivatization with bifunctional agents is useful, for instance, for
crosslinking SRT to a water-insoluble
support matrix or surface for use in the method for purifying anti-SRT
antibodies, and vice-versa. Commonly
used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-
hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid,
homobifunctional imidoesters,
including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate), bifunctional maleimides such as
bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-
azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding
glutamyl and aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
groups of seryl or threonyl residues, methylation of the a-amino groups of
lysine, arginine, and histidine side
chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco,
pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-
terminal carboxyl group.
Another type of covalent modification of the SRT polypeptide included within
the scope of this invention
comprises altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern"
is intended for purposes herein to mean deleting one or more carbohydrate
moieties found in native sequence
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WO 01/07611 PCT/US00/20006
SRT (either by removing the underlying glycosylation site or by deleting the
glycosylation by chemical and/or
enzymatic means), and/or adding one or more glycosylation sites that are not
present in the native sequence SRT.
In addition, the phrase includes qualitative changes in the glycosylation of
the native proteins, involving a change
in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the SRT polypeptide may be accomplished by
altering the amino acid
sequence. The alteration may be made, for example, by the addition of, or
substitution by, one or more serine
or threonine residues to the native sequence SRT (for O-linked glycosylation
sites). The SRT amino acid
sequence may optionally be altered through changes at the DNA level,
particularly by mutating the DNA
encoding the SRT polypeptide at preselected bases such that codons are
generated that will translate into the
desired amino acids.
Another means of increasing the number of carbohydrate moieties on the SRT
polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide. Such methods
are described in the art, e.g.,
in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC
Crit. Rev. Biochem., pp. 259-
306 (1981).
Removal of carbohydrate moieties present on the SRT polypeptide may be
accomplished chemically or
enzymatically or by mutational substitution of codons encoding for amino acid
residues that serve as targets for
glycosylation. Chemical deglycosylation techniques are lrnown in the art and
described, for instance, by
Hakimuddin et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al.,
Anal. Biochem. 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety
of endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138:350 (1987).
Another type of covalent modification of SRT comprises linking the SRT
polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene
glycol, or polyoxyalkylenes, in
the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
The SRT polypeptides of the present invention may also be modified in a way to
form chimeric
molecules comprising SRT fused to another, heterologous polypeptide or amino
acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the SRT with
a tag polypeptide
which provides an epitope to which an anti-tag antibody can selectively bind.
The epitope tag is generally placed
at the amino- or carboxyl- terminus of the SRT. The presence of such epitope-
tagged forms of the SRT can be
detected using an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the SRT to
be readily purified by affinity purification using an anti-tag antibody or
another type of affinity matrix that binds
to the epitope tag. Various tag polypeptides and their respective antibodies
are well known in the art. Examples
include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its
antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc
tag and the 8F9, 3C7, 6E10,
G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biolo~y, 5:3610-3616 (1985)]; and the
Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein En inQ Bering, 3(6):547-
553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,
BioTechnolo~y, 6:1204-1210
(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)];
an a-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10
protein peptide tag [Lutz-
CA 02378403 2002-O1-04
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Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the SRT with an
immunoglobulin or a particular region of an immunoglobulin. For a bivalent
form of the chimeric molecule (also
referred to as an "immunoadhesin"), such a fusion could be to the Fc region of
an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane domain deleted
or inactivated) form of a SRT
polypeptide in place of at least one variable region within an Ig molecule. In
a particularly preferred
embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3
regions of an IgGI molecule. For the production of immunoglobulin fusions see
also US Patent No. 5,428,130
issued June 27, 1995.
D. Preparation of SRT Polypeptides
The description below relates primarily to production of SRT by culturing
cells transformed or
transfected with a vector containing SRT nucleic acid. It is, of course,
contemplated that alternative methods,
which are well known in the art, may be employed to prepare SRT. For instance,
the SRT sequence, or portions
thereof, may be produced by direct peptide synthesis using solid-phase
techniques [see, e.g., Stewart et al.,
Solid-Phase Peptide S nt~, W.H. Freeman Co., San Francisco, CA (1969);
Merrifield, J. Am. Chem. Soc.,
85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual
techniques or by automation.
Automated synthesis may be accomplished, for instance, using an Applied
Biosystems Peptide Synthesizer
(Foster City, CA) using manufacturer's instructions. Various portions of the
SRT may be chemically
synthesized separately and combined using, chemical or enzymatic methods to
produce the full-length SRT.
1. Isolation of DNA Encoding SRT
DNA encoding SRT may be obtained from a cDNA library prepared from tissue
believed to possess
the SRT mRNA and to express it at a detectable level. Accordingly, human SRT
DNA can be conveniently
obtained from a cDNA library prepared from human tissue, such as described in
the Examples. The SRT-
encoding gene may also be obtained from a genomic library or by known
synthetic procedures (e.g., automated
nucleic acid synthesis).
Libraries can be screened with probes (such as antibodies to the SRT or
oligonucleotides of at least
about 20-80 bases) designed to identify the gene of interest or the protein
encoded by it, wherein those probes
may be based upon the polynucleotide sequences shown in the accompanying
figures. Screening the cDNA or
genomic library with the selected probe may be conducted using standard
procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring
Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding SRT is to use PCR
methodology [Sambrook et al.,
supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The
oligonucleotide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon
hybridization to DNA in the library
being screened. Methods of labeling are well known in the art, and include the
use of radiolabels like 32P-labeled
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ATP, biotinylation or enzyme labeling. Hybridization conditions, including
moderate stringency and high
stringency, are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known
sequences deposited and available in public databases such as GenBank or other
private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across
the full-length sequence can be determined using methods known in the art and
as described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al.,
suura, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for SRT
production and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired sequences. The
culture conditions, such as media,
temperature, pH and the like, can be selected by the skilled artisan without
undue experimentation. In general,
principles, protocols, and practical techniques for maximizing the
productivity of cell cultures can be found in
Mammalian Cell BiotechnoloQV: a Practical Approach, M. Butler, ed. (IRL Press,
1991) and Sambrook et al.,
Supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCl2, CaP04, liposome-mediated and
electroporation. Depending on the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra, or
electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as
described by Shaw et al. , Gene, 23:315 ( 1983) and WO 89/05859 published 29
June 1989. For mammalian cells
without such cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, Virolo~y,
52:456-457 (1978) can be employed. General aspects of mammalian cell host
system transfections have been
described in U.S. Patent No. 4,399,216. Transformations into yeast are
typically carried out according to the
method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al.,
Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such as by
nuclear microinjection,
electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene, polyornithine, may
also be used. For various techniques for transforming mammalian cells, see
Keown et al., Methods in
Enzymolo;y, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein
include prokaryote, yeast,
or higher eukaryote cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative
or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
Various E. coli strains are
publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli
X1776 (ATCC 31,537); E. coli
strain W3110 (ATCC 27,325) and KS 772 (ATCC 53,635). Other suitable
prokaryotic host cells include
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Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia,
Klebsiella, Proteus, Salmonella,
e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B.
subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 April 1989),
Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are
illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host because it is a
common host strain for recombinant
DNA product fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For
example, strain W3110 may be modified to effect a genetic mutation in the
genes encoding proteins endogenous
to the host, with examples of such hosts including E. coli W3110 strain 1A2,
which has the complete genotype
tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E.
coli W3110 strain 27C7
(ATCC 55,244), which has the complete genotype tonA ptr3 phoA EI S (argF-
lac)169 degP ompT kan ; E. coli
W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-
lac)169 degP ompT rbs7
ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-
kanamycin resistant degP deletion
mutation; and an E. coli strain having mutant periplasmic protease disclosed
in U. S. Patent No. 4,946,783 issued
7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase
reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning
or expression hosts for SRT-encoding vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic
host microorganism. Others include Schizosaccharomycespombe (Beach and Nurse,
Nature, 290: 140 [1981];
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No.
4,943,529; Fleer et al.,
Bio/Technoloey, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683,
CBS4574; Louveneourt et
al., J. Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus
(ATCC 16,045), K. wickeramii (ATCC
24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg
et al., Bio/Technolo~y,
8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);
Pichia pastoris (EP 183,070;
Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida;
Trichoderma reesia (EP 244,234);
Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263
[1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and
filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January
1991), andAspergillus hosts such
as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289
[1983]; Tilburn et al., Gene,
26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and
Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein
and include, but are not limited
to, yeast capable of growth on methanol selected from the genera consisting of
Hansenula, Candida, Kloeckera,
Pichia, Saccharomyces, Torulopsis, and Ilhodotorula. A list of specific
species that are exemplary of this class
of yeasts may be found in C. Anthony, The Biochemistr~of Meth l~phs, 269
(1982).
Suitable host cells for the expression of glycosylated SRT are derived from
multieellular organisms.
Examples of invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf9, as well as plant
cells. Examples of useful mammalian host cell lines include Chinese hamster
ovary (CHO) and COS cells.
More specific examples include monkey kidney CV1 line transformed by SV40 (COS-
7, ATCC CRL 1651);
human embryonic kidney line (293 or 293 cells subeloned for growth in
suspension culture, Graham et al., J.
58
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Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and
Chasin, Proc. Natl. Acad.
Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Re~rod.,
23:243-251 (1980)); human lung
cells (W 138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
mammary tumor (MMT
060562, ATCC CCL51). The selection of the appropriate host cell is deemed to
be within the skill in the art.
3. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding SRT may be inserted into
a replicable vector
for cloning (amplification of the DNA) or for expression. Various vectors are
publicly available. The vector
may, for example, be in the form of a plasmid, cosmid, viral particle, or
phage. The appropriate nucleic acid
sequence may be inserted into the vector by a variety of procedures. In
general, DNA is inserted into an
appropriate restriction endonuclease sites) using techniques known in the art.
Vector components generally
include, but are not limited to, one or more of a signal sequence, an origin
of replication, one or more marker
genes, an enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable
vectors containing one or more of these components employs standard ligation
techniques which are known to
the skilled artisan.
The SRT may be produced recombinantly not only directly, but also as a fusion
polypeptide with a
heterologous polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site
at the N-terminus of the mature protein or polypeptide. In general, the signal
sequence may be a component of
the vector, or it may be a part of the SRT-encoding DNA that is inserted into
the vector. The signal sequence
may be a prokaryotic signal sequence selected, for example, from the group of
the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion
the signal sequence may be, e.g.,
the yeast invertase leader, alpha factor leader (including Saccharomyces and
Kluyveromyces a-factor leaders,
the latter described in U.S. Patent No. 5,010,182), or acid phosphatase
leader, the C. albicans glucoamylase
leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/
13646 published 15 November
1990. In mammalian cell expression, mammalian signal sequences may be used to
direct secretion of the
protein, such as signal sequences from secreted polypeptides of the same or
related species, as well as viral
secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate
in one or more selected host cells. Such sequences are well known for a
variety of bacteria, yeast, and viruses.
The origin of replication from the plasmid pBR322 is suitable for most Gram-
negative bacteria, the 2~, plasmid
origin is suitable for yeast, and various viral origins (5V40, polyoma,
adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, e.g., the gene encoding D-alanine racemase
for Bacilli.
An example of suitable selectable markers for mammalian cells are those that
enable the identification
of cells competent to take up the SRT-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate
59
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WO 01/07611 PCT/US00/20006
host cell when wild-type DHFR is employed is the CHO cell line deficient in
DHFR activity, prepared and
propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216
(1980). A suitable selection
gene for use in yeast is the trill gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39
(1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157
(1980)]. The trill gene provides
a selection marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example, ATCC No.
44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to
the SRT-encoding nucleic
acid sequence to direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well
known. Promoters suitable for use with prokaryotic hosts include the ~3-
lactamase and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544
(1979)], alkaline phosphatase, a
tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776], and hybrid
promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA,
80:21-25 (1983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding SRT.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-
phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or
other glycolytic enzymes [Hess
et al., J. Adv. Enzyme Red, 7:149 (1968); Holland, Biochemistry, 17:4900
(1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-
6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription
controlled by growth conditions, are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism,
metallothionein, glyceraldehyde-3-
phosphate dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP 73,657.
SRT transcription from vectors in mammalian host cells is controlled, for
example, by promoters
obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 July
1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous
mammalian promoters, e.g., the
actin promoter or an immunoglobulin promoter, and from heat-shock promoters,
provided such promoters are
compatible with the host cell systems.
Transcription of a DNA encoding the SRT by higher eukaryotes may be increased
by inserting an
enhancer sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300
bp, that act on a promoter to increase its transcription. Many enhancer
sequences are now known from
mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin).
Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on
the late side of the replication
origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the
replication origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or
CA 02378403 2002-O1-04
WO 01/07611 PCT/US00/20006
3' to the SRT coding sequence, but is preferably located at a site 5' from the
promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated
cells from other multicellular organisms) will also contain sequences
necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly
available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated portion
of the mRNA encoding SRT.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of SRT in recombinant
vertebrate cell culture are described in Gething et al., Nature, 293:620-625
(1981); Mantei et al., Nature,
281:40-46 (1979); EP 117,060; and EP 117,058.
4. Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample directly, for
example, by
conventional Southern blotting, Northern blotting to quantitate the
transcription of mRNA [Thomas, Proc. Natl.
Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ
hybridization, using an
appropriately labeled probe, based on the sequences provided herein.
Alternatively, antibodies may be employed
that can recognize specific duplexes, including DNA duplexes, RNA duplexes,
and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and the assay
may be carried out where the
duplex is bound to a surface, so. that upon the formation of duplex on the
surface, the presence of antibody bound
to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as
immunohistochemical staining of cells or tissue sections and assay of cell
culture or body fluids, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be prepared in
any mammal. Conveniently, the
antibodies may be prepared against a native sequence SRT polypeptide or
against a synthetic peptide based on
the DNA sequences provided herein or against exogenous sequence fused to SRT
DNA and encoding a specific
antibody epitope.
5. Purification of Polvpeptide
Forms of SRT may be recovered from culture medium or from host cell lysates. -
If membrane-bound,
it can be released from the membrane using a suitable detergent solution (e.g.
Triton-X 100) or by enzymatic
cleavage. Cells employed in expression of SRT can be disrupted by various
physical or chemical means, such
as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing
agents.
It may be desired to purify SRT from recombinant cell proteins or
polypeptides. The following
procedures are exemplary of suitable purification procedures: by fractionation
on an ion-exchange column;
ethanol precipitation; reverse phase HPLC; chromatography on silica or on a
canon-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel
filtration using, for example,
Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG;
and metal chelating columns
to bind epitope-tagged forms of the SRT. Various methods of protein
purification may be employed and such
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methods are known in the art and described for example in Deutscher, Methods
in Enzymolo~y, 182 (1990);
Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New
York (1982). The purification
steps) selected will depend, for example, on the nature of the production
process used and the particular SRT
produced.
E. Uses for SRT Polynucleotides and Polvpeptides
SRT nucleotide sequences (and/or their complements) disclosed herein have
various applications in the
art of molecular biology, including for example uses as hybridization probes,
in chromosome and gene mapping,
in tissue typing, disease tissue detection, in PCR technologies, in screening
for new therapeutic molecules and
in the generation of anti-sense RNA and DNA. SRT nucleic acid will also be
useful for the preparation of SRT
polypeptides by the recombinant techniques described herein.
The SRT polynucleotides disclosed herein, or portions thereof, may be used as
hybridization probes for
a cDNA library to isolate the full-length SRT cDNA or to isolate still other
cDNAs (for instance, those encoding
naturally-occurring variants of SRT or SRT from other species) which have a
desired sequence identity to the
SRT sequence of interest. Optionally, the length of the probes will be about
20 to about 50 bases. The
hybridization probes may be derived from at least partially novel regions of
the nucleotide sequences disclosed
herein wherein those regions may be determined without undue experimentation
or from genomic sequences
including promoters, enhancer elements and introns of native sequence SRT. By
way of example, a screening
method will comprise isolating the coding region of the SRT gene using the
known DNA sequence to synthesize
a selected probe of about 40 bases. Hybridization probes may be labeled by a
variety of labels, including
radionucleotides such as 32P or 355, or enzymatic labels such as alkaline
phosphatase coupled to the probe via
avidin/biotin coupling systems. Labeled probes having a sequence complementary
to that of the SRT gene of
the present invention can be used to screen libraries of human cDNA, genomic
DNA or mRNA to determine
which members of such libraries the probe hybridizes to. Hybridization
techniques are described in further detail
in the Examples below.
PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188
provides additional uses for
oligonucleotides based upon the polynucleotide sequences disclosed in the
accompanying figures. Such
oligomers are generally chemically synthesized, but they may be of recombinant
origin or a mixture of both.
Oligomers generally comprise two nucleotide sequences, one with sense
orientation (5' to 3') and one with
antisense (3' to 5') employed under optimized conditions for identification of
a specific gene or diagnostic use.
The same two oligomers, nested sets of oligomers, or even a degenerate pool of
oligomers may be employed
under less stringent conditions for identification and/or quantitation of
closely related DNA or RNA sequences.
Full length genes may be cloned utilizing partial nucleotide sequence and
various methods known in the
art. Gobinda et al. PCR Methods Applic. 2:318-322 (1993) disclose "restriction-
site PCR" as a direct method
which uses universal primers to retrieve unknown sequence adjacent to a known
locus. First, genomic DNA is
amplified in the presence of primer to linker and a primer specific to the
known region. The amplified sequences
are subjected to a second round of PCR with the same linker primer and another
specific primer internal to the
first one. Products of each round of PCR are transcribed with an appropriate
RNA polymerase and sequenced
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using reverse transcriptase. Gobinda et al present data concerning Factor IX
for which they identified a
conserved stretch of 20 nucleotides in the 3' noncoding region of the gene.
Inverse PCR is the first method to report successful acquisition of unknown
sequences starting with
primers based on a known region (Triglia et al., Nucleic Acids Res. 16:8186
(1988). The method uses several
restriction enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then
circularized by intramolecular ligation and used as a PCR template. Divergent
primers are designed from the
known region. The multiple rounds of restriction enzyme digestions and
ligations that are necessary prior to PCR
make the procedure slow and expensive (Gobinda et al, supra).
Capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-119 (1991) is a
method for PCR
amplification of DNA fragments adjacent to a known sequence inhuman and YAC
DNA. As noted by Gobinda
et al. (supra), capture PCR also requires multiple restriction enzyme
digestions and ligations to place an
engineered double-stranded sequence into an unknown portion of the DNA
molecule before PCR. Although the
restriction and ligation reactions are carried out simultaneously, the
requirements for extension, immobilization
and two rounds of PCR and purification prior to sequencing render the method
cumbersome and time consuming.
Parker et al., Nucleic Acids Res. 19:3055-3060 (1991) teach walking PCR, a
method for targeted gene
walking which permits retrieval of unknown sequence. PromoterFinderTM is a new
kit available from Clontech
(Palo Alto, Calif.) which uses PCR and primers derived from p53 to walk in
genomic DNA. Nested primers
and special PromoterFinder libraries are used to detect upstream sequences
such as promoters and regulatory
elements. This process avoids the need to screen libraries and is useful in
finding intron/exon junctions.
Another new PCR method, "Improved Method for Obtaining Full Length cDNA
Sequences" (see U.S.
Patent No. 5,817,479, issued October 6, 1998), employs XL-PCR (Perkin-Elmer,
Foster City, Calif.) to amplify
and extend partial nucleotide sequence into longer pieces of DNA. This method
was developed to allow a single
researcher to process multiple genes (up to 20 or more) at one time and to
obtain an extended (possibly
full-length) sequence within 6-10 days. This new method replaces methods which
use labelled probes to screen
plasmid libraries and allow one researcher to process only about 3-5 genes in
14-40 days.
In the first step, which can be performed in about two days, any two of a
plurality of primers are
designed and synthesized based on a known partial sequence. In step 2, which
takes about six to eight hours,
the sequence is extended by PCR amplification of a selected library. Steps 3
and 4, which take about one day,
are purification of the amplified cDNA and its ligation into an appropriate
vector. Step 5, which takes about one
day, involves transforming and growing up host bacteria. In step 6, which
takes approximately five hours, PCR
is used to screen bacterial clones for extended sequence. The final steps,
which take about one day, involve the
preparation and sequencing of selected clones.
If the full length cDNA has not been obtained, the entire procedure is
repeated using either the original
library or some other preferred library. The preferred library may be one that
has been size-selected to include
only larger cDNAs or may consist of single or combined commercially available
libraries, eg. lung, liver, heart
and brain from Gibco/BRL (Gaithersburg, Md.). The cDNA library may have been
prepared with oligo (dT)
or random priming. Random primed libraries are preferred in that they will
contain more sequences which
contain 5' ends of genes. A randomly primed library may be particularly useful
if an oligo (dT) library does not
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yield a complete gene.
The nucleotide sequence for any particular polynucleotide shown in the
accompanying figures can also
be used to generate probes for mapping the native genomic sequence. The
sequence may be mapped to a
particular chromosome or to a specific region of the chromosome using well
known techniques. These include
in situ hybridization to chromosomal spreads (Verma et al., "Human
Chromosomes: A Manual of Basic
Techniques", Pergamon Press, New York City, 1988), flow-sorted chromosomal
preparations, or artificial
chromosome constructions such as yeast artificial chromosomes (YACs),
bacterial artificial chromosomes
(BACs), bacterial P1 constructions or single chromosome cDNA libraries.
In situ hybridization of chromosomal preparations and physical mapping
techniques such as linkage
analysis using established chromosomal markers are invaluable in extending
genetic maps. Examples of genetic
maps can be found in the 1994 Genome Issue of Science (265:1981f). Often the
placement of a gene on the
chromosome of another mammalian species may reveal associated markers even if
the number or arm of a
particular human chromosome is not known. New partial nucleotide sequences can
be assigned to chromosomal
arms, or parts thereof, by physical mapping. This provides valuable
information to investigators searching for
disease genes using positional cloning or other gene discovery techniques.
Once a disease or syndrome, such
as ataxia telangiectasia (AT), has been crudely localized by genetic linkage
to a particular genomic region, for
example, AT to l 1q22-23 (Gatti et al., Nature 336:577-580 (1988), any
sequences mapping to that area may
represent genes for further investigation. The nucleotide sequences of the
subject invention may also be used to
detect differences in the chromosomal location of nucleotide sequences due to
translocation, inversion, etc.,
between normal and carrier or affected individuals.
The partial nucleotide sequence encoding a particular SRT polypeptide may be
used to produce an amino
acid sequence using well known methods of recombinant DNA technology. The
amino acid or peptide may be
expressed in a variety of host cells, either prokaryotic or eukaryotic. Host
cells may be from the same species
from which the nucleotide sequence was derived or from a different species.
Advantages of producing an amino
acid sequence or peptide by recombinant DNA technology include obtaining
adequate amounts for
purification and the availability of simplified purification procedures.
Cells transformed with an SRT nucleotide sequence may be cultured under
conditions suitable for the
expression and recovery of peptide from cell culture as described above. The
peptide produced by a recombinant
cell may be secreted or may be contained intracellularly depending on the
sequence itself and/or the vector used.
In general, it is more convenient to prepare recombinant proteins in secreted
form, and this is accomplished by
ligating SRT to a recombinant nucleotide sequence which directs its movement
through a particular prokaryotic
or eukaryotic cell membrane. Other recombinant constructions may join SRT to
nucleotide sequence encoding
a polypeptide domain which will facilitate protein purification (Knoll et al.,
DNA Cell Biol. 12:441-53 (1993).
Other useful fragments of the SRT nucleic acids include antisense or sense
oligonucleotides comprising
a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding
to target SRT mRNA (sense)
or SRT DNA (antisense) sequences. Antisense or sense oligonucleotides,
according to the present invention,
comprise a fragment of the coding region of SRT DNA. Such a fragment generally
comprises at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive
an antisense or a sense
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oligonucleotide, based upon a cDNA sequence encoding a given protein is
described in, for example, Stein and
Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechm9ues 6:958,
1988).
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation
of duplexes that block transcription or translation of the target sequence by
one of several means, including
enhanced degradation of the duplexes, premature termination of transcription
or translation, or by other means.
The antisense oligonucleotides thus may be used to block expression of SRT
proteins. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified sugar-
phosphodiester backbones (or other
sugar linkages, such as those described in WO 91/06629) and wherein such sugar
linkages are resistant to
endogenous nucleases. Such oligonucleotides with resistant sugar linkages are
stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to be able to
bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those
oligonucleotides which are
covalently linked to organic moieties, such as those described in WO 90/10048,
and other moieties that increases
affinity of the oligonucleotide for a target nucleic acid sequence, such as
poly-(L-lysine). Further still,
intercalating agents, such as ellipticine, and alkylating agents or metal
complexes may be attached to sense or
antisense oligonucleotides to modify binding specificities of the antisense or
sense oligonucleotide for the target
nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing
the target nucleic acid
sequence by any gene transfer method, including, for example, CaP04 mediated
DNA transfection,
electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
In a preferred procedure, an
antisense or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic
acid sequence is contacted with the recombinant retroviral vector, either in
vivo or ex vivo. Suitable retroviral
vectors include, but are not limited to, those derived from the murine
retrovirus M-MuLV, N2 (a retrovirus
derived from M-MuLV), or the double copy vectors designated DCTSA, DCTSB and
DCTSC (see WO
90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide
sequence by formation of a conjugate with a ligand binding molecule, as
described in WO 91/04753. Suitable
ligand binding molecules include, but are not limited to, cell surface
receptors, growth factors, other cytokines,
or other ligands that bind to cell surface receptors. Preferably, conjugation
of the ligand binding molecule does
not substantially interfere with the ability of the ligand binding molecule to
bind to its corresponding molecule
or receptor, or block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into
a cell containing the target
nucleic acid sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The
sense or antisense oligonucleotide-lipid complex is preferably dissociated
within the cell by an endogenous lipase.
The probes may also be employed in PCR techniques to generate a pool of
sequences for identification
of closely related SRT coding sequences.
Nucleotide sequences encoding an SRT can also be used to construct
hybridization probes for mapping
the gene which encodes that SRT and for the genetic analysis of individuals
with genetic disorders. The
nucleotide sequences provided herein may be mapped to a chromosome and
specific regions of a chromosome
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using known techniques, such as in situ hybridization, linkage analysis
against known chromosomal markers,
and hybridization screening with libraries.
When the coding sequences for SRT encode a protein which binds to another
protein (example, where
the SRT is a receptor), the SRT can be used in assays to identify the other
proteins or molecules involved in the
binding interaction. By such methods, inhibitors of the receptor/ligand
binding interaction can be identified.
Proteins involved in such binding interactions can also be used to screen for
peptide or small molecule inhibitors
or agonists of the binding interaction. Also, the receptor SRT can be used to
isolate correlative ligand(s).
Screening assays can be designed to fmd lead compounds that mimic the
biological activity of a native SRT or
a receptor for SRT. Such screening assays will include assays amenable to high-
throughput screening of
chemical libraries, making them particularly suitable for identifying small
molecule drug candidates. Small
molecules contemplated include synthetic organic or inorganic compounds. The
assays can be performed in a
variety of formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and
cell based assays, which are well characterized in the art.
Nucleic acids which encode SRT or its modified forms can also be used to
generate either transgenic
animals or "knock out" animals which, in turn, are useful in the development
and screening of therapeutically
useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal
having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of the animal at
a prenatal, e.g., an embryonic
stage. A transgene is a DNA which is integrated into the genome of a cell from
which a transgenic animal
develops. In one embodiment, cDNA encoding SRT can be used to clone genomic
DNA encoding SRT in
accordance with established techniques and the genomic sequences used to
generate transgenic animals that
contain cells which express DNA encoding SRT. Methods for generating
transgenic animals, particularly
animals such as mice or rats, have become conventional in the art and are
described, for example, in U.S. Patent
Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted
for SRT transgene incorporation
with tissue-specific enhancers. Transgenic animals that include a copy of a
transgene encoding SRT introduced
into the germ line of the animal at an embryonic stage can be used to examine
the effect of increased expression
of DNA encoding SRT. Such animals can be used as tester animals for reagents
thought to confer protection
from, for example, pathological conditions associated with its overexpression.
In accordance with this facet of
the invention, an animal is treated with the reagent and a reduced incidence
of the pathological condition,
compared to untreated animals bearing the transgene, would indicate a
potential therapeutic intervention for the
pathological condition.
Alternatively, non-human homologues of SRT can be used to construct a SRT
"knock out" animal which
has a defective or altered gene encoding SRT as a result of homologous
recombination between the endogenous
gene encoding SRT and altered genomic DNA encoding SRT introduced into an
embryonic stem cell of the
animal. For example, cDNA encoding SRT can be used to clone genomic DNA
encoding SRT in accordance
with established techniques. A portion of the genomic DNA encoding SRT can be
deleted or replaced with
another gene, such as a gene encoding a selectable marker which can be used to
monitor integration. Typically,
several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are
included in the vector [see e.g.,
Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous
recombination vectors]. The vector
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is introduced into an embryonic stem cell line (e.g., by electroporation) and
cells in which the introduced DNA
has homologously recombined with the endogenous DNA are selected [see e.g., Li
et al., Cell, 69:915 (1992)].
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse or rat) to form aggregation
chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J.
Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then
be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term to create a
"knock out" animal. Progeny
harboring the homologously recombined DNA in their germ cells can be
identified by standard techniques and
used to breed animals in which all cells of the animal contain the
homologously recombined DNA. Knockout
animals can be characterized for instance, for their ability to defend against
certain pathological conditions and
for their development of pathological conditions due to absence of the SRT
polypeptide.
Nucleic acid encoding the SRT polypeptides may also be used in gene therapy.
In gene therapy
applications, genes are introduced into cells in order to achieve in vivo
synthesis of a therapeutically effective
genetic product, for example for replacement of a defective gene. "Gene
therapy" includes both conventional
gene therapy where a lasting effect is achieved by a single treatment, and the
administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for blocking the
expression of certain genes in
vivo. It has already been shown that short antisense oligonucleotides can be
imported into cells where they act
as inhibitors, despite their low intracellular concentrations caused by their
restricted uptake by the cell
membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]).
The oligonucleotides can be
modified to enhance their uptake, e.g. by substituting their negatively
charged phosphodiester groups by
uncharged groups.
There are a variety of techniques available for introducing nucleic acids into
viable cells. The
techniques vary depending upon whether the nucleic acid is transferred into
cultured cells in vitro, or in vivo in
the cells of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro
include the use of liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene transfer
techniques include transfection with viral
(typically retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in
Biotechnoloey 11, 205-210 [1993]). In some situations it is desirable to
provide the nucleic acid source with
an agent that targets the target cells, such as an antibody specific for a
cell surface membrane protein or the
target cell, a ligand for a receptor on the target cell, etc. Where liposomes
are employed, proteins which bind
to a cell surface membrane protein associated with endocytosis may be used for
targeting and/or to facilitate
uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell
type, antibodies for proteins which
undergo internalization in cycling, proteins that target intracellular
localization and enhance intracellular half life.
The technique of receptor-mediated endocytosis is described, for example, by
Wu et al., J: Biol. Chem. 262,
4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene
marking and gene therapy protocols see Anderson et al., Science 256, 808-813
(1992).
The SRT polypeptides described herein may also be employed as molecular weight
markers for protein
electrophoresis purposes.
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The nucleic acid molecules encoding the SRT polypeptides or fragments thereof
described herein are
useful for chromosome identification. In this regard, there exists an ongoing
need to identify new chromosome
markers, since relatively few chromosome marking reagents, based upon actual
sequence data are presently
available. Each SRT nucleic acid molecule of the present invention can be used
as a chromosome marker.
The SRT polypeptides and nucleic acid molecules of the present invention may
also be used for tissue
typing, wherein the SRT polypeptides of the present invention may be
differentially expressed in one tissue as
compared to another, for example in a diseased tissue versus a normal tissue.
SRT nucleic acid molecules will
fmd use for generating probes for PCR, Northern analysis, Southern analysis
and Western analysis.
The SRT polypeptides described herein and antibodies thereagainst may also be
employed as therapeutic
agents. The SRT polypeptides of the present invention can be formulated
according to known methods to
prepare pharmaceutically useful compositions, whereby the SRT product hereof
is combined in admixture with
a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are
prepared for storage by mixing the
active ingredient having the desired degree of purity with optional
physiologically acceptable carriers, excipients
or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers, excipients
or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include buffers
such as phosphate, citrate and other
organic acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins;
hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine,
arginine or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA;
sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as
sodium; and/or nonionic surfactants
such as TWEENT"', PLURONICST"' or PEG.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by
filtration through sterile filtration membranes, prior to or following
lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a
sterile access port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
The route of administration is in accord with known methods, e.g. injection or
infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical
administration, or by sustained release systems.
Dosages and desired drug concentrations of pharmaceutical compositions of the
present invention may
vary depending on the particular use envisioned. The determination of the
appropriate dosage or route of
administration is well within the skill of an ordinary physician. Animal
experiments provide reliable guidance
for the determination of effective doses for human therapy. Interspecies
scaling of effective doses can be
performed following the principles laid down by Mordenti, J. and Chappell, W.
"The use of interspecies scaling
in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al.,
Eds., Pergamon Press, New
York 1989, pp. 42-96.
When in vivo administration of a SRT polypeptide or agonist or antagonist
thereof is employed, normal
dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body
weight or more per day,
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preferably about 1 P.g/kg/day to 10 mg/kg/day, depending upon the route of
administration. Guidance as to
particular dosages and methods of delivery is provided in the literature; see,
for example, U.S. Pat. Nos.
4,657,760; 5,206,344; or 5,225,212. It is anticipated that different
formulations will be effective for different
treatment compounds and different disorders, that administration targeting one
organ or tissue, for example, may
necessitate delivery in a manner different from that to another organ or
tissue.
Where sustained-release administration of a SRT polypeptide is desired in a
formulation with release
characteristics suitable for the treatment of any disease or disorder
requiring administration of the SRT
polypeptide, microencapsulation of the SRT polypeptide is contemplated.
Microencapsulation of recombinant
proteins for sustained release has been successfully performed with human
growth hormone (rhGH), interferon-
(rhIFN- ), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799
(1996); Yasuda, Biomed.
Ther., 27:1221-1223 (1993); Hora et al., Bio/Technolo~v. 8:755-758 (1990);
Cleland, "Design and Production
of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere
Systems," in Vaccine Design:
The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New
York, 1995), pp. 439-462;
WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.
The sustained-release formulations of these proteins were developed using poly-
lactic-coglycolic acid
15' (PLGA) polymer due to its biocompatibility and wide range of biodegradable
properties. The degradation
products of PLGA, lactic and glycolic acids, can be cleared quickly within the
human body. Moreover, the
degradability of this polymer can be adjusted from months to years depending
on its molecular weight and
composition. Lewis, "Controlled release of bioactive agents from
lactide/glycolide polymer," in: M. Chasm
and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel
Dekker: New York, 1990),
pp.l-41.
This invention encompasses methods of screening compounds to identify those
that mimic the SRT
polypeptide (agonists) or prevent the effect of the SRT polypeptide
(antagonists). Screening assays for antagonist
drug candidates are designed to identify compounds that bind or complex with
the SRT polypeptides encoded
by the genes identified herein, or otherwise interfere with the interaction of
the encoded polypeptides with other
cellular proteins. Such screening assays will include assays amenable to high-
throughput screening of chemical
libraries, making them particularly suitable for identifying small molecule
drug candidates.
The assays can be performed in a variety of formats, including protein-protein
binding assays,
biochemical screening assays, immunoassays, and cell-based assays, which are
well characterized in the art.
All assays for antagonists are common in that they call for contacting the
drug candidate with a SRT
polypeptide encoded by a nucleic acid identified herein under conditions and
for a time sufficient to allow these
two components to interact.
In binding assays, the interaction is binding and the complex formed can be
isolated or detected in the
reaction mixture. In a particular embodiment, the SRT polypeptide encoded by
the gene identified herein or the
drug candidate is immobilized on a solid phase, e.g., on a microtiter plate,
by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by coating the
solid surface with a solution of
the SRT polypeptide and drying. Alternatively, an immobilized antibody, e.g.,
a monoclonal antibody, specific
for the SRT polypeptide to be immobilized can be used to anchor it to a solid
surface. The assay is performed
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by adding the non-immobilized component, which may be labeled by a detectable
label, to the immobilized
component, e.g., the coated surface containing the anchored component. When
the reaction is complete, the
non-reacted components are removed, e.g., by washing, and complexes anchored
on the solid surface are
detected. When the originally non-immobilized component carries a detectable
label, the detection of label
immobilized on the surface indicates that complexing occurred. Where the
originally non-immobilized
component does not carry a label, complexing can be detected, for example, by
using a labeled antibody
specifically binding the immobilized complex.
If the candidate compound interacts with but does not bind to a particular SRT
polypeptide encoded by
a gene identified herein, its interaction with that polypeptide can be assayed
by methods well known for detecting
protein-protein interactions. Such assays include traditional approaches, such
as, e.g., cross-linking, co-
immunoprecipitation, and co-purification through gradients or chromatographic
columns. In addition, protein-
protein interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc.
Natl. Acad. Sci. USA, 88:9578-
9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA,
89: 5789-5793 (1991). Many
transcriptional activators, such as yeast GAL4, consist of two physically
discrete modular domains, one acting
as the DNA-binding domain, the other one functioning as the transcription-
activation domain. The yeast
expression system described in the foregoing publications (generally referred
to as the "two-hybrid system")
takes advantage of this property, and employs two hybrid proteins, one in
which the target protein is fused to
the DNA-binding domain of GAL4, and another, in which candidate activating
proteins are fused to the
activation domain. The expression of a GALL-lacZ reporter gene under control
of a GAL4-activated promoter
depends on reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting
polypeptides are detected with a chromogenic substrate for (3-galactosidase. A
complete kit
(MATCHMAKERT"') for identifying protein-protein interactions between two
specific proteins using the two-
hybrid technique is commercially available from Clontech. This system can also
be extended to map protein
domains involved in specific protein interactions as well as to pinpoint amino
acid residues that are crucial for
these interactions.
Compounds that interfere with the interaction of a gene encoding a SRT
polypeptide identified herein
and other intra- or extracellular components can be tested as follows: usually
a reaction mixture is prepared
containing the product of the gene and the intra- or extracellular component
under conditions and for a time
allowing for the interaction and binding of the two products. To test the
ability of a candidate compound to
inhibit binding, the reaction is run in the absence and in the presence of the
test compound. In addition, a
placebo may be added to a third reaction mixture, to serve as positive
control. The binding (complex formation)
between the test compound and the intra- or extracellular component present in
the mixture is monitored as
described hereinabove. The formation of a complex in the control reactions)
but not in the reaction mixture
containing the test compound indicates that the test compound interferes with
the interaction of the test compound
and its reaction partner.
To assay for antagonists, the SRT polypeptide may be added to a cell along
with the compound to be
screened for a particular activity and the ability of the compound to inhibit
the activity of interest in the presence
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of the SRT polypeptide indicates that the compound is an antagonist to the SRT
polypeptide. Alternatively,
antagonists may be detected by combining the SRT polypeptide and a potential
antagonist with membrane-bound
SRT polypeptide receptors or recombinant receptors under appropriate
conditions for a competitive inhibition
assay. The SRT polypeptide can be labeled, such as by radioactivity, such that
the number of SRT polypeptide
molecules bound to the receptor can be used to determine the effectiveness of
the potential antagonist. The gene
encoding the receptor can be identified by numerous methods known to those of
skill in the art, for example,
ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun.,
1(2): Chapter 5 (1991).
Preferably, expression cloning is employed wherein polyadenylated RNA is
prepared from a cell responsive to
the SRT polypeptide and a cDNA library created from this RNA is divided into
pools and used to transfect COS
cells or other cells that are not responsive to the SRT polypeptide.
Transfected cells that are grown on glass
slides are exposed to labeled SRT polypeptide. The SRT polypeptide can be
labeled by a variety of means
including iodination or inclusion of a recognition site for a site-specific
protein lcinase. Following fixation and
incubation, the slides are subjected to autoradiographic analysis. Positive
pools are identified and sub-pools are
prepared and re-transfected using an interactive sub-pooling and re-screening
process, eventually yielding a
single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled SRT
polypeptide can be photoaffinity
linked with cell membrane or extract preparations that express the receptor
molecule. Cross-linked material is
resolved by PAGE and exposed to X-ray film. The labeled complex containing the
receptor can be excised,
resolved into peptide fragments, and subjected to protein micro-sequencing.
The amino acid sequence obtained
from micro- sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA
library to identify the gene encoding the putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor
would be incubated with labeled SRT polypeptide in the presence of the
candidate compound. The ability of the
compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide
that binds to the fusions of
immunoglobulin with SRT polypeptide, and, in particular, antibodies including,
without limitation, poly- and
monoclonal antibodies and antibody fragments, single-chain antibodies, anti-
idiotypic antibodies, and chimeric
or humanized versions of such antibodies or fragments, as well as human
antibodies and antibody fragments.
Alternatively, a potential antagonist may be a closely related protein, for
example, a mutated form of the SRT
polypeptide that recognizes the receptor but imparts no effect, thereby
competitively inhibiting the action of the
SRT polypeptide.
Another potential SRT polypeptide antagonist is an antisense RNA or DNA
construct prepared using
antisense technology, where, e.g., an antisense RNA or DNA molecule acts to
block directly the translation of
mRNA by hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used
to control gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are
based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding
portion of the polynucleotide
sequence, which encodes the mature SRT polypeptides herein, is used to design
an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be
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complementary to a region of the gene involved in transcription (triple helix -
see Lee et al., Nucl. Acids Res.,
6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al.,
Science, 251:1360 (1991)), thereby
preventing transcription and the production of the SRT polypeptide. The
antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into the SRT polypeptide
(antisense - Okano, Neurochem., 56:560 (1991); Oli og-deoxvnucleotides as
Antisense Inhibitors of Gene
Expression (CRC Press: Boca Raton, FL, 1988). The oligonucleotides described
above can also be delivered
to cells such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of the SRT
polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived
from the translation-initiation site,
e.g., between about -10 and +10 positions of the target gene nucleotide
sequence, are preferred.
Potential antagonists include small molecules that bind to the active site,
the receptor binding site, or
growth factor or other relevant binding site of the SRT polypeptide, thereby
blocking the normal biological
activity of the SRT polypeptide. Examples of small molecules include, but are
not limited to, small peptides or
peptide-like molecules, preferably soluble peptides, and synthetic non-
peptidyl organic or inorganic compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA.
Ribozymes act by sequence-specific hybridization to the complementary target
RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential
RNA target can be identified by
known techniques. For further details see, e.g., Rossi, Current BioloQV, 4:469-
471 (1994), and PCT publication
No. WO 97/33551 (published September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded
and composed of deoxynucleotides. The base composition of these
oligonucleotides is designed such that it
promotes triple-helix formation via Hoogsteen base-pairing rules, which
generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further details see,
e.g., PCT publication No. WO
97/33551, supra.
These small molecules can be identified by any one or more of the screening
assays discussed
hereinabove and/or by any other screening techniques well known for those
skilled in the art.
F. Anti-SRT Polypentide Antibodies
The present invention further provides anti-SRT antibodies. Exemplary
antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate antibodies.
1. Polvclonal Antibodies
The anti-SRT antibodies may comprise polyclonal antibodies. Methods of
preparing polyclonal
antibodies are known to the skilled artisan. Polyclonal antibodies can be
raised in a mammal, for example, by
one or more injections of an immunizing agent and, if desired, an adjuvant.
Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The
immunizing agent may include the SRT polypeptide or a fusion protein thereof.
It may be useful to conjugate
the immunizing agent to a protein known to be immunogenic in the mammal being
immunized. Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine
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thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may
be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic
trehalose dicorynomycolate).
The immunization protocol may be selected by one skilled in the art without
undue experimentation.
Monoclonal Antibodies
The anti-SRT antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be
prepared using hybridoma methods, such as those described by Kohler and
Milstein, Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically
bind to the immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
The immunizing agent will typically include the SRT polypeptide or a fusion
protein thereof. Generally,
either peripheral blood lymphocytes ("PBLs") are used if cells of human origin
are desired, or spleen cells or
lymph node cells are used if non-human mammalian sources are desired. The
lymphocytes are then fused with
an immortalized cell line using a suitable fusing agent, such as polyethylene
glycol, to form a hybridoma cell
[Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized
cell lines are usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine and human
origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma
cells may be cultured in a
suitable culture medium that preferably contains one or more substances that
inhibit the growth or survival of
the unfused, immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances
prevent the growth of HGPRT-
deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of
antibody by the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More
preferred immortalized cell lines are murine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the
production of human monoclonal antibodies [Kozbor, J. Immunol. , 133:3001 (
1984); Brodeur et al . , Monoclonal
Antibody Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of
monoclonal antibodies directed against SRT. Preferably, the binding
specificity of monoclonal antibodies
produced by the hybridoma cells is determined by immunoprecipitation or by an
in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques and assays are
known in the art. The binding affinity of the monoclonal antibody can, for
example, be determined by the
Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution
procedures and grown by standard methods [coding, su ra . Suitable culture
media for this purpose include,
for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells
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may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture
medium or ascites fluid by conventional immunoglobulin purification procedures
such as, for example, protein
A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described
in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the
invention can be readily isolated
and sequenced using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding
specifically to.genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression
vectors, which are then transfected into host cells such as simian COS cells,
Chinese hamster ovary (CHO) cells,
or myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be modified, for
example, by substituting the
coding sequence for human heavy and light chain constant domains in place of
the homologous murine sequences
[U.5. Patent No. 4,816,567; Morrison et al., su ra or by covalently joining to
the immunoglobulin coding
sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent
antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well
known in the art. For example, one method involves recombinant expression of
immunoglobulin light chain and
modified heavy chain. The heavy chain is truncated generally at any point in
the Fc region so as to prevent
heavy chain crosslinking. Alternatively, the relevant cysteine residues are
substituted with another amino acid
residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to
produce fragments thereof, particularly, Fab fragments, can be accomplished
using routine techniques known
in the art.
3. Human and Humanized Antibodies
The anti-SRT antibodies of the invention may further comprise humanized
antibodies or human
antibodies. Humanized forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient antibody) in
which residues from a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In
some instances, Fv framework residues of the human immunoglobulin are replaced
by corresponding non-human
residues. Humanized antibodies may also comprise residues which are found
neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the humanized
antibody will comprise
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substantially all of at least one, and typically two, variable domains, in
which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise
at least a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin [Jones
et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329
(1988); and Presta, Curr. On.
Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human. These non-
human amino acid residues are often referred to as "import" residues, which
are typically taken from an "import"
variable domain. Humanization can be essentially performed following the
method of Winter and co-workers
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-
327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences
for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U. S. Patent
No. 4,816,567), wherein substantially less than an intact human variable
domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from
analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage
display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks
et al., J. Mol. Biol., 222:581
(1991)]. The techniques of Cole et al. and Boerner et al. are also available
for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Theranv,
Alan R. Liss, p. 77 (1985) and
Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies
can be made by introducing
of human immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin
genes have been partially or completely inactivated. Upon challenge, human
antibody production is observed,
which closely resembles that seen in humans in all respects, including gene
rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in U.S. Patent
Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al.,
Bio/Technolo~y 10, 779-783 (1992); Lonberg etal., Nature 368 856-859 (1994);
Morrison, Nature 368, 812-13
(1994); Fishwild et al., Nature Biotechnolosv 14, 845-51 (1996); Neuberger,
Nature Biotechnoloev 14, 826
(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
4. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding
specificities for at least two different antigens. In the present case, one of
the binding specificities is for the
SRT, the other one is for any other antigen, and preferably for a cell-surface
protein or receptor or receptor
subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant
production of bispecific antibodies is based on the co-expression of two
immunoglobulin heavy-chain/light-chain
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pairs, where the two heavy chains have different specificities [Milstein and
Cuello, Nature, 305:537-539 (1983)].
Because of the random assortment of immunoglobulin heavy and light chains,
these hybridomas (quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the correct bispecific
structure. The purification of the correct molecule is usually accomplished by
affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in
Traunecker et al., EMBO
J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
be fused to immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin
heavy-chain constant domain, comprising at least part of the hinge, CH2, and
CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the site necessary
for light-chain binding present in
at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain
fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-transfected into a suitable
host organism. For further details of generating bispecific antibodies see,
for example, Suresh et al., Methods
in Enzvmoloev, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers which
are recovered from recombinant
cell culture. The preferred interface comprises at least a part of the CH3
region of an antibody constant domain.
In this method, one or more small amino acid side chains from the interface of
the first antibody molecule are
replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory
"cavities" of identical or similar
size to the large side chains) are created on the interface of the second
antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or threonine). This
provides a mechanism for increasing
the yield of the heterodimer over other unwanted end-products such as
homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')Z
bispecific antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared can be prepared using chemical
linkage. Brennan et al. , Science 229:81 ( 1985) describe a procedure wherein
intact antibodies are proteolytically
cleaved to generate F(ab')2 fragments. These fragments are reduced in the
presence of the dithiol complexing
agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an
equimolar amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies
produced can be used as agents for the selective immobilization of enzymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected
to directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
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Various technique for making and isolating bispecific antibody fragments
directly from recombinant cell
culture have also been described. For example, bispecific antibodies have been
produced using leucine zippers.
Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun
proteins were linked to the Fab' portions of two different antibodies by gene
fusion. The antibody homodimers
were reduced at the hinge region to form monomers and then re-oxidized to form
the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody" technology
described by Hollinger et al. , Proc. Nat!. Acad. Sci. USA 90:6444-6448 (
1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain
(VH) connected to a light-chain variable domain (V~) by a linker which is too
short to allow pairing between the
two domains on the same chain. Accordingly, the VH and V~ domains of one
fragment are forced to pair with
the complementary V~ and V" domains of another fragment, thereby forming two
antigen-binding sites. Another
strategy for making bispecific antibody fragments by the use of single-chain
Fv (sFv) dimers has also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared.
Tutt et al. , J. ~Immunol. 147:60 ( 1991 ).
Exemplary bispecific antibodies may bind to two different epitopes on a given
SRT polypeptide herein.
Alternatively, an anti-SRT polypeptide arm may be combined with an arm which
binds to a triggering molecule
on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or
B7), or Fc receptors for IgG
(FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus
cellular defense mechanisms
to the cell expressing the particular SRT polypeptide. Bispecific antibodies
may also be used to localize cytotoxic
agents to cells which express a particular SRT polypeptide. These antibodies
possess a SRT-binding arm and
an arm which binds a cytotoxic agent or a radionuclide chelator, such as
EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the SRT polypeptide and further
binds tissue factor (TF).
5. HeteroconLQate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate
antibodies are composed of two covalently joined antibodies. Such antibodies
have, for example, been proposed
to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980],
and for treatment of HIV infection
[WO 91/00360; WO 92!200373; EP 03089]. It is contemplated that the antibodies
may be prepared in vitro
using known methods in synthetic protein chemistry, including those involving
crosslinking agents. For
example, immunotoxins may be constructed using a disulfide exchange reaction
or by forming a thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and
those disclosed, for example, in U.S. Patent No. 4,676,980.
6. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to
enhance, e.g., the effectiveness of the antibody in treating cancer. For
example, cysteine residues) may be
introduced into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
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homodimeric antibody thus generated may have improved internalization
capability and/or increased complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See
Caron et al. , J. Exp Med. , 176:
1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric
antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional cross-
linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be
engineered that has dual Fc regions
and may thereby have enhanced complement lysis and ADCC capabilities. See
Stevenson et al., Anti-Cancer
Drug Design. 3: 219-230 (1989).
7. Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent
such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin
of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are
available for the production of radioconjugated antibodies. Examples include
Z'ZBi, '3'I, '3'In, ~°Y, and '86Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters
(such as disuccinimidyl suberate),
aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al. , Science, 238:
1098 (1987). Carbon-14-labeled 1-
isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is
an exemplary chelating agent
for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such
streptavidin) for
utilization in tumor pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed
by removal of unbound conjugate from the circulation using a clearing agent
and then administration of a
"ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
8. Immunolinosomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing
the antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad.
Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos.
4,485,045 and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Patent No.
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5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-
PE). Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired diameter.
Fab' fragments of the antibody of the present invention can be conjugated to
the liposomes as described in Martin
et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent
(such as Doxorubicin) is optionally contained within the liposome. See Gabizon
et al. , J. National Cancer Inst. ,
81(19): 1484 (1989).
9. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a SRT polypeptide identified herein, as well
as other molecules identified
by the screening assays disclosed hereinbefore, can be administered for the
treatment of various disorders in the
form of pharmaceutical compositions.
If the SRT polypeptide is intracellular and whole antibodies are used as
inhibitors, internalizing
antibodies are preferred. However, lipofections or liposomes can also be used
to deliver the antibody, or an
antibody fragment, into cells. Where antibody fragments are used, the smallest
inhibitory fragment that
specifically binds to the binding domain of the target protein is preferred.
For example, based upon the variable-
region sequences of an antibody, peptide molecules can be designed that retain
the ability to bind the target
protein sequence. Such peptides can be synthesized chemically and/or produced
by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-
7893 (1993). The fommlation
herein may also contain more than one active compound as necessary for the
particular indication being treated,
preferably those with complementary activities that do not adversely affect
each other. Alternatively, or in
addition, the composition may comprise an agent that enhances its function,
such as, for example, a cytotoxic
agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such
molecules are suitably present in
combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for
example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal drug
delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles, and
nanocapsules) or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by
filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in
the form of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include
polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U. S.
Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate,
non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT TM (injectable
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microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-D-(-)-3-
hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins for shorter
time periods. When encapsulated
antibodies remain in the body for a long time, they may denature or aggregate
as a result of exposure to moisture
at 37°C, resulting in a loss of biological activity and possible
changes in immunogenicity. Rational strategies
can be devised for stabilization depending on the mechanism involved. For
example, if the aggregation
mechanism is discovered to be intermolecular S-S bond formation through thio-
disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture
content, using appropriate additives, and developing specific polymer matrix
compositions.
G. Uses for anti-SRT Antibodies
The anti-SRT antibodies of the invention have various utilities. For example,
anti-SRT antibodies may
be used in diagnostic assays for SRT, e.g., detecting its expression in
specific cells, tissues, or serum. Various
diagnostic assay techniques known in the art may be used, such as competitive
binding assays, direct or indirect
sandwich assays and immunoprecipitation assays conducted in either
heterogeneous or homogeneous phases
[Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)
pp. 147-158]. The antibodies
used in the diagnostic assays can be labeled with a detectable moiety. The
detectable moiety should be capable
of producing, either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a
radioisotope, such as 3H,'°C,'zP, 355, or'zSI, a fluorescent or
chemiluminescent compound, such as fluorescein
isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or
horseradish peroxidase. Any method known in the art for conjugating the
antibody to the detectable moiety may
be employed, including those methods described by Hunter et al., Nature,
144:945 (1962); David et al.,
Biochemistrv, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981);
and Nygren, J. Histochem. and
Cytochem., 30:407 (1982).
Anti-SRT antibodies also are useful for the affinity purification of SRT from
recombinant cell culture
or natural sources. In this process, the antibodies against SRT are
immobilized on a suitable support, such a
Sephadex resin or filter paper, using methods well known in the art. The
immobilized antibody then is contacted
with a sample containing the SRT to be purified, and thereafter the support is
washed with a suitable solvent that
will remove substantially all the material in the sample except the SRT, which
is bound to the immobilized
antibody. Finally, the support is washed with another suitable solvent that
will release the SRT from the
antibody.
The following examples are offered for illustrative purposes only, and are not
intended to limit the scope
of the present invention in any way.
All patent and literature references cited in the present specification are
hereby incorporated by reference
in their entirety.
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EXAMPLES
Commercially available reagents referred to in the examples were used
according to manufacturer's
instructions unless otherwise indicated. The source of those cells identified
in the following examples, and
throughout the specification, by ATCC accession numbers is the American Type
Culture Collection, Manassas,
VA.
EXAMPLE 1
Isolation of SRT cDNAs
Preparation of oli.~o dT primed cDNA library
mRNA was isolated from human tissue using reagents and protocols from
Invitrogen, San Diego, CA
(Fast Track 2). This RNA was used to generate an oligo dT primed cDNA library
in the vector pRKSD using
reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script
Plasmid System). In this
procedure, the double stranded cDNA was sized to greater than 1000 by and the
SaII/NotI linkered cDNA was
cloned into XhoI/NotI cleaved vector. pRKSD is a cloning vector that has an
sp6 transcription initiation site
followed by an SfiI restriction enzyme site preceding the XhoI/NotI cDNA
cloning sites.
2. Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent
the 5' ends of the primary
cDNA clones. Sp6 RNA was generated from the primary library (described above),
and this RNA was used
to generate a random primed cDNA library in the vector pSST-AMY.O using
reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In this
procedure the double stranded cDNA
was sized to S00-1000 bp, tinkered with blunt to NotI adaptors, cleaved with
SfiI, and cloned into SfiI/NotI
cleaved vector. pSST-AMY.O is a cloning vector that has a yeast alcohol
dehydrogenase promoter preceding
the cDNA cloning sites and the mouse amylase sequence (the mature sequence
without the secretion signal)
followed by the yeast alcohol dehydrogenase terminator, after the cloning
sites. Thus, cDNAs cloned into this
vector that are fused in frame with the amylase sequence will lead to the
secretion of amylase from appropriately
transfected yeast colonies.
3. Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to
which was added
electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and
vector mixture was then
electroporated as recommended by the manufacturer. Subsequently, SOC media
(Life Technologies, 1 ml) was
added and the mixture was incubated at 37°C for 30 minutes. The
transformants were then plated onto 20
standard 150 mm LB plates containing ampicillin and incubated for 16 hours
(37°C). Positive colonies were
scraped off the plates and the DNA was isolated from the bacterial pellet
using standard protocols, e.g. CsCI-
gradient. The purified DNA was then carried on to the yeast protocols below.
The yeast methods were divided into three categories: (1) Transformation of
yeast with the
plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones
secreting amylase; and (3) PCR
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amplification of the insert directly from the yeast colony and purification of
the DNA for sequencing and further
analysis.
The yeast strain used was HD56-SA (ATCC-90785). This strain has the following
genotype: MAT
alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL+, SUC+, GAL+.
Preferably, yeast mutants can be
employed that have deficient post-translational pathways. Such mutants may
have translocation deficient alleles
in sec7l, sec72, sec62, with truncated sec71 being most preferred.
Alternatively, antagonists (including
antisense nucleotides and/or ligands) which interfere with the normal
operation of these genes, other proteins
implicated in this post translation pathway (e.g., SEC6lp, SEC72p, SEC62p,
SEC63p, TDJIp or SSAlp-4p)
or the complex formation of these proteins may also be preferably employed in
combination with the amylase-
expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al.,
Nucl. Acid. Res.,
20:1425 (1992). Transformed cells were then inoculated from agar into YEPD
complex media broth (100 ml)
and grown overnight at 30°C. The YEPD broth was prepared as described
in Kaiser et al., Methods in Yeast
Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The
overnight culture was then
diluted to about 2 x 106 cells/ml (approx. ODD=0.1) into fresh YEPD broth (500
ml) and regrown to 1 x 10'
cells/ml (approx. ODD=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into
GS3 rotor bottles in a
Sorval GS3 rotor at 5,000 rpm for S minutes, the supernatant discarded, and
then resuspended into sterile water,
and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant
was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10
mM Tris-HCI, 1 mM EDTA
pH 7.5, 100 mM Li200CCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells (100 p,1) with freshly
denatured single stranded
salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1
~.g, vol. < 10 ~,1) in
microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE
(600 ~1, 40% polyethylene
glycol-4000, 10 mM Tris-HCI, 1 mM EDTA, 100 mM Li200CCH3, pH 7.5) was added.
This mixture was
gently mixed and incubated at 30°C while agitating for 30 minutes. The
cells were then heat shocked at 42°C
for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000
rpm for 5-10 seconds, decanted and
resuspended into TE (500 ~,1, 10 mM Tris-HCI, 1 mM EDTA pH 7.5) followed by
recentrifugation. The cells
were then diluted into TE (1 ml) and aliquots (200 ~1) were spread onto the
selective media previously prepared
in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was
performed using a single, large
scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil
(SCD-Ura) prepared as
described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor
Press, Cold Spring Harbor, NY, p.
208-210 (1994). Transformants were grown at 30°C for 2-3 days.
The detection of colonies secreting amylase was performed by including red
starch in the selective
growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as
per the procedure described by
Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starch was
incorporated into the SCD-Ura agar
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plates at a final concentration of 0.15 % (w/v), and was buffered with
potassium phosphate to a pH of 7.0 (50-
100 mM final concentration).
The positive colonies were picked and streaked across fresh selective media
(onto 150 mm plates) in
order to obtain well isolated and identifiable single colonies. Well isolated
single colonies positive for amylase
secretion were detected by direct incorporation of red starch into buffered
SCD-Ura agar. Positive colonies were
determined by their ability to break down starch resulting in a clear halo
around the positive colony visualized
directly.
4. Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick
and diluted into sterile
water (30 ~1) in a 96 well plate. At this time, the positive colonies were
either frozen and stored for subsequent
analysis or immediately amplified. An aliquot of cells (5 ~cl) was used as a
template for the PCR reaction in a
25 p1 volume containing: 0.5 ~,1 Klentaq (Clontech, Palo Alto, CA); 4.0 ~,1 10
mM dNTP's (Perkin Elmer-
Cetus); 2.5 ~,1 Klentaq buffer (Clontech); 0.25 ~,1 forward oligo 1; 0.25 ~,1
reverse oligo 2; 12.5 ~.1 distilled
water. The sequence of the forward oligonucleotide 1 was:
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID N0:563)
The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID N0:564)
PCR was then performed as follows:
a. Denature 92°C, 5 minutes
b. 3 cycles of: Denature 92°C, 30 seconds
Anneal 59°C, 30 seconds
Extend 72°C, 60 seconds
c. 3 cycles of: Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds
Extend 72°C, 60 seconds
d. 25 cycles of: Denature 92°C, 30 seconds
Anneal 55 °C, 30 seconds
Extend 72°C, 60 seconds
e. Hold 4°C
The underlined regions of the oligonucleotides disclosed above annealed to the
ADH promoter region
and the amylase region, respectively, and amplified a 307 by region from
vector pSST-AMY.O when no insert
was present. Typically, the first 18 nucleotides of the 5' end of these
oligonucleotides contained annealing sites
for the sequencing primers. Thus, the total product of the PCR reaction from
an empty vector was 343 bp.
However, signal sequence-fused cDNA resulted in considerably longer nucleotide
sequences.
Following the PCR, an aliquot of the reaction (5 ~1) was examined by agarose
gel electrophoresis in
a 1 % agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described
by Sambrook et al., supra.
Clones resulting in a single strong PCR product larger than 400 by were
further analyzed by DNA sequencing
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after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, CA).
cDNA molecules isolated from this amylase screen are shown in Figures 1-562
(SEQ ID NOS:1-562,
respectively), wherein the nucleotides "N" and "X" represent any nucleotide.
The cDNA libraries from which
these cDNA molecules were obtained are as follows:
(a) Human liver tissue
Figures 1-19, 124 and 130.
(b) Human placenta tissue
Figures 20-73.
(c) Human retina tissue
Figures 74-75, 81, 107-108, 139-140 and 340-341.
(d) Human salivar~gland tissue
Figures 76-78.
(e) Human umbilical vein endothelial cells
Figures 79-80, 97, 110, 245-252, 254-260, 263-265, 413-421,
433-437, 444-449, 454-456, 462-467,
477-478, 480-485, 492-493, 515 and 548.
(f) Human thyroid tissue
Figures 82-84, 90-91, 96, 109, 141-143 and 268.
(g) Human small intestine tissue
Figures 85-86, 144-161 and 267.
(h) Human colon carcinoma tissue
Figure 87.
(i) Human lung~ndothelial cells
Figures 88 and 93-95.
(j) Human hypothalamus tissue
Figure 89.
(k) Human breast carcinoma tissue
Figures 92, 111-115, 206-213, 228-232, 269-270, 450-453, 534-547, 556 and 559.
(1) Human aortic endothelial cells
Figures 98-102, 125-129, 136-138, 216-217, 253, 261-262, 300-301, 327-330, 365-
367 and 385-387.
(m) Human uterus tissue
Figures 103-106, 170-173, 176-183, 233-235, 238, 242-244, 266, 311-312 and
557.
(n) Human lung_carcinoma tissue
Figures 106-108, 201-205, 221-227, 271-274, 334-339, 342-348, 350-351, 360-
364, 372, 388-408,
411, 431-432, 479, 558 and 560-561.
(o) Human mammary epithelial cells
Figures 119-121, 214 and 316-320.
(p) Human chronic myelo~enous leukemia tissue
Figures 122-123 and 131-135.
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(q) Human spinal cord tissue
Figures 162, 167-169, 198-200, 236 and 315.
(r) Human fetal brain tissue
Figures 163-166, 174-175, 332-333, 422-430 and 494-502.
(s) Human fetal kidney
tissue
Figures 184-197, 409-410
and 412.
(t) Human prostate tissue
Figures 215, 237, 239-241
and 349.
(u) Human mammary_,,gland
tissue
Figures 218-220, 275-276
and 331.
(v) Human adenocarcinoma
tissue
Figures 277-299 and
302-310.
(w) Human fetal small intestine
tissue
Figures 313-314.
(x) Human fetal lung_tissue
Figures 321-326.
(y) Human testis tissue
Figures 352-359, 368-371, 377-384, 438-443, 457-461, 486-491, 513-514, 516-527
and 562.
(z) Human MCF-7 cells
Figures 373-376, 468-476, 503-512, 528-533 and 549-555.
EXAMPLE 2
Identification of full-length cDNA molecules
Oligonucleotide probes may be generated from the sequence of any of the SRT
polynucleotide sequences
disclosed herein, including those shown in Figures 1 to 562 and used to screen
human cDNA libraries prepared
as described in paragraph 1 of Example 1 above. The cloning vector may be
pRKSB (pRKSB is a precursor of
pRKSD that does not contain the SfiI site; see, Holmes et al., Science
253:1278-1280 (1991)), and the cDNA
size cut may be less than 2800 bp. The oligonucleotides probes may be
synthesized: 1) to identify by PCR a
cDNA library that contained the sequence of interest, and 2) for use as probes
to isolate a clone of the full-length
coding sequence for SRT. Forward and reverse PCR primers generally range from
20 to 30 nucleotides and are
often designed to give a PCR product of about 100-1000 by in length. The probe
sequences are typically 40-55
by in length. In order to screen several libraries for a full-length clone,
DNA from the libraries may be screened
by PCR amplification, as per Ausubel et al., Current Protocols in Molecular
Biolo~v, supra, with the PCR
primer pair. A positive library may then be used to isolate clones encoding
the gene of interest using the probe
oligonucleotide and one of the primer pairs.
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EXAMPLE 3
Use of SRT~olynucleotides as hybridization probes
The following method describes use of a nucleotide sequence encoding SRT as a
hybridization probe.
DNA comprising the coding sequence of full-length or mature SRT is employed as
a probe to screen
for homologous DNAs (such as those encoding naturally-occurring variants of
SRT) in human tissue cDNA
libraries or human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the following
high stringency conditions. Hybridization of radiolabeled SRT-derived probe to
the filters is performed in a
solution of 50% formamide, Sx SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50
mM sodium phosphate, pH
6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20
hours. Washing of the filters is performed
in an aqueous solution of O.lx SSC and 0.1 % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence SRT can
then be identified using standard techniques known in the art.
EXAMPLE 4
E~ression of SRT in E. coli
This example illustrates preparation of an unglycosylated form of SRT by
recombinant expression in
E. coli.
The DNA sequence encoding SRT is initially amplified using selected PCR
primers. The primers
should contain restriction enzyme sites which correspond to the restriction
enzyme sites on the selected
expression vector. A variety of expression vectors may be employed. An example
of a suitable vector is
pBR322 (derived from E. cola; see Bolivar et al., Gene, 2:95 (1977)) which
contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction enzyme and
dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector will
preferably include sequences which encode
for an antibiotic resistance gene, a trp promoter, a polyhis leader (including
the first six STII codons, polyhis
sequence, and enterokinase cleavage site), the SRT coding region, lambda
transcriptional terminator, and an
argU gene.
The ligation mixture is then used to transform a selected E. coli strain using
the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow
on LB plates and antibiotic resistant
colonies are then selected. Plasmid DNA can be isolated and confirmed by
restriction analysis and DNA
sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with
antibiotics. The overnight culture may subsequently be used to inoculate a
larger scale culture. The cells are
then grown to a desired optical density, during which the expression promoter
is turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell
pellet obtained by the centrifugation can be solubilized using various agents
known in the art, and the solubilized
SRT protein can then be purified using a metal chelating column under
conditions that allow tight binding of the
protein.
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SRT may be expressed in E. coli in a poly-His tagged form, using the following
procedure. The DNA
encoding SRT is initially amplified using selected PCR primers. The primers
will contain restriction enzyme
sites which correspond to the restriction enzyme sites on the selected
expression vector, and other useful
sequences providing for efficient and reliable translation initiation, rapid
purification on a metal ehelation
column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His
tagged sequences are then
ligated into an expression vector, which is used to transform an E. coli host
based on strain 52 (W3110
fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown
in LB containing 50 mg/ml
carbenicillin at 30°C with shaking until an O.D.600 of 3-5 is reached.
Cultures are then diluted 50-100 fold into
CRAP media (prepared by mixing 3.57 g (NH4)ZSO4, 0.71 g sodium citrate~2H20,
1.07 g KCI, 5.36 g Difco
yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM
MPOS, pH 7.3, 0.55 % (w/v)
glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30°C
with shaking. Samples are
removed to verify expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell
pellets are frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in
10 volumes (w/v) in 7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium
tetrathionate is added to make final
concentrations of O.1M and 0.02 M, respectively, and the solution is stirred
overnight at 4°C. This step results
in a denatured protein with all cysteine residues blocked by sulfitolization.
The solution is centrifuged at 40,000
rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-
5 volumes of metal chelate
column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22
micron filters to clarify. The
clarified extract is loaded onto a S ml Qiagen Ni-NTA metal chelate column
equilibrated in the metal chelate
column buffer. The column is washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol
grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractions containing the
desired protein are pooled and stored at 4°C. Protein concentration is
estimated by its absorbance at 280 nm
using the calculated extinction coefficient based on its amino acid sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared
refolding buffer consisting
of: 20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycine
and 1 mM EDTA.
Refolding volumes are chosen so that the final protein concentration is
between SO to 100 micrograms/ml. The
refolding solution is stirred gently at 4°C for 12-36 hours. The
refolding reaction is quenched by the addition
of TFA to a final concentration of 0.4 % (pH of approximately 3). Before
further purification of the protein, the
solution is filtered through a 0.22 micron filter and acetonitrile is added to
2-10% final concentration. The
refolded protein is chromatographed on a Poros R1/H reversed phase column
using a mobile buffer of 0.1
TFA with elution with a gradient of acetonitrile from 10 to 80% . Aliquots of
fractions with A280 absorbance
are analyzed on SDS polyacrylamide gels and fractions containing homogeneous
refolded protein are pooled.
Generally, the properly refolded species of most proteins are eluted at the
lowest concentrations of acetonitrile
since those species are the most compact with their hydrophobic interiors
shielded from interaction with the
reversed phase resin. Aggregated species are usually eluted at higher
aeetonitrile concentrations. In addition
to resolving misfolded forms of proteins from the desired form, the reversed
phase step also removes endotoxin
from the samples.
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Fractions containing the desired folded SRT polypeptide are pooled and the
acetonitrile removed using
a gentle stream of nitrogen directed at the solution. Proteins are formulated
into 20 mM Hepes, pH 6.8 with
0.14 M sodium chloride and 4 % mannitol by dialysis or by gel filtration using
G25 Superfine (Pharmacia) resins
equilibrated in the formulation buffer and sterile filtered.
EXAMPLE 5
Expression of SRT in mammalian cells
This example illustrates preparation of a potentially glycosylated form of SRT
by recombinant
expression in mammalian cells.
The vector, pRKS (see EP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the SRT DNA is ligated into pRKS with selected restriction enzymes
to allow insertion of the SRT
DNA using ligation methods such as described in Sambrook et al., su ra. The
resulting vector is called pRKS-
SRT.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells
(ATCC CCL 1573) are
grown to confluence in tissue culture plates in medium such as DMEM
supplemented with fetal calf serum and
optionally, nutrient components and/or antibiotics. About 10 pg pRKS-SRT DNA
is mixed with about 1 ~,g
DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and
dissolved in 500 ~.1 of 1 mM
Tris-HCI, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500
~,1 of 50 mM HEPES (pH
7.35), 280 mM NaCI, 1.5 mM NaP04, and a precipitate is allowed to form for 10
minutes at 25°C. The
precipitate is suspended and added to the 293 cells and allowed to settle for
about four hours at 37°C. The
culture medium is aspirated off and 2 ml of 20 % glycerol in PBS is added for
30 seconds. The 293 cells are
then washed with serum free medium, fresh medium is added and the cells are
incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed
and replaced with culture
medium (alone) or culture medium containing 200 ~Ci/ml 35S-cysteine and 200
~.Ci/ml 35S-methionine. After
a 12 hour incubation, the conditioned medium is collected, concentrated on a
spin filter, and loaded onto a 15 %
SDS gel. The processed gel may be dried and exposed to film for a selected
period of time to reveal the
presence of SRT polypeptide. The cultures containing transfected cells may
undergo further incubation (in serum
free medium) and the medium is tested in selected bioassays.
In an alternative technique, SRT may be introduced into 293 cells transiently
using the dextran sulfate
method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981).
293 cells are grown to
maximal density in a spinner flask and 700 ~g pRKS-SRT DNA is added. The cells
are first concentrated from
the spinner flask by centrifugation and washed with PBS. The DNA-dextran
precipitate is incubated on the cell
pellet for four hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture
medium, and re-introduced into the spinner flask containing tissue culture
medium, 5 ~g/ml bovine insulin and
0.1 ~,g/ml bovine transferrin. After about four days, the conditioned media is
centrifuged and filtered to remove
cells and debris. The sample containing expressed SRT can then be concentrated
and purified by any selected
method, such as dialysis and/or column chromatography.
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In another embodiment, SRT can be expressed in CHO cells. The pRKS-SRT can be
transfected into
CHO cells using known reagents such as CaP04 or DEAE-dextran. As described
above, the cell cultures can
be incubated, and the medium replaced with culture medium (alone) or medium
containing a radiolabel such as
35S-methionine. After determining the presence of SRT polypeptide, the culture
medium may be replaced with
serum free medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium
is harvested. The medium containing the expressed SRT can then be concentrated
and purified by any selected
method.
Epitope-tagged SRT may also be expressed in host CHO cells. The SRT may be
subcloned out of the
pRKS vector. The subclone insert can undergo PCR to fuse in frame with a
selected epitope tag such as a poly-
his tag into a Baculovirus expression vector. The poly-his tagged SRT insert
can then be subcloned into a SV40
driven vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the CHO cells
can be transfected (as described above) with the SV40 driven vector. Labeling
may be performed, as described
above, to verify expression. The culture medium containing the expressed poly-
His tagged SRT can then be
concentrated and purified by any selected method, such as by Niz+-chelate
affinity chromatography.
SRT may also be expressed in CHO and/or COS cells by a transient expression
procedure or in CHO
cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The
proteins are expressed
as an IgG construct (immunoadhesin), in which the coding sequences for the
soluble forms (e.g. extracellular
domains) of the respective proteins are fused to an IgGl constant region
sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using
standard techniques as described in Ausubel et al., Current Protocols of
Molecular Biolo~y, Unit 3.16, John
Wiley and Sons (1997). CHO expression vectors are constructed to have
compatible restriction sites 5' and 3'
of the DNA of interest to allow the convenient shuttling of cDNA's. The vector
used expression in CHO cells
is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and
uses the SV40 early
promoter/enhancer to drive expression of the cDNA of interest and
dihydrofolate reductase (DHFR). DHFR
expression permits selection for stable maintenance of the plasmid following
transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO cells
using commercially available transfection reagents Superfect (Quiagen),
Dosper° or Fugene' (Boehringer
Mannheim). The cells are grown as described in Lucas et al., supra.
Approximately 3 x 10'' cells are frozen
in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath
and mixed by
vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs
of media and centrifuged at 1000
rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended
in 10 mL of selective media (0.2
~m filtered PS20 with 5 % 0.2 ~cm diafiltered fetal bovine serum). The cells
are then aliquoted into a 100 mL
spinner containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner
filled with 150 mL selective growth medium and incubated at 37°C. After
another 2-3 days, 250 mL, 500 mL
and 2000 mL spinners are seeded with 3 x 105 cells/mL. The cell media is
exchanged with fresh media by
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centrifugation and resuspension in production medium. Although any suitable
CHO media may be employed,
a production medium described in U.S. Patent No. 5,122,469, issued June 16,
1992 may actually be used. A
3L production spinner is seeded at 1.2 x 106 cells/mL. On day 0, the cell
number pH ie determined. On day
1, the spinner is sampled and sparging with filtered air is commenced. On day
2, the spinner is sampled, the
temperature shifted to 33°C, and 30 mL of 500 g/L glucose and 0.6 mL of
10% antifoam (e.g., 35%
polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken.
Throughout the production,
the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or
until the viability dropped below
70 % , the cell culture is harvested by centrifugation and filtering through a
0.22 ~cm filter. The filtrate was either
stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA
column (Qiagen). Before
purification, imidazole is added to the conditioned media to a concentration
of S mM. The conditioned media
is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,
buffer containing 0.3 M NaCI
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading,
the column is washed with additional
equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly
purified protein is subsequently desalted into a storage buffer containing 10
mM Hepes, 0.14 M NaCI and 4
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -
80°C.
Inununoadhesin (Fc-containing) constructs are purified from the conditioned
media as follows. The
conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which
had been equilibrated in 20
mM Na phosphate buffer, pH 6.8. After loading, the column is washed
extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1
ml fractions into tubes containing 275 ~cL of 1 M Tris buffer, pH 9. The
highly purified protein is subsequently
desalted into storage buffer as described above for the poly-His tagged
proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation.
EXAMPLE 6
Expression of SRT in yeast
The following method describes recombinant expression of SRT in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of SRT from the
ADH2/GAPDH promoter. DNA encoding SRT and the promoter is inserted into
suitable restriction enzyme
sites in the selected plasmid to direct intracellular expression of SRT. For
secretion, DNA encoding SRT can
be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH
promoter, a native SRT
signal peptide or other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory
signal/leader sequence, and linker sequences (if needed) for expression of
SRT.
Yeast cells, such as yeast strain AB 110, can then be transformed with the
expression plasmids described
above and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by
precipitation with 10 % trichloroacetic acid and separation by SDS-PAGE,
followed by staining of the gels with
Coomassie Blue stain.
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Recombinant SRT can subsequently be isolated and purified by removing the
yeast cells from the
fermentation medium by centrifugation and then concentrating the medium using
selected cartridge filters. The
concentrate containing SRT may further be purified using selected column
chromatography resins.
EXAMPLE 7
Expression of SRT in baculovirus-infected insect cells
The following method describes recombinant expression of SRT in Baculovirus-
infected insect cells.
The sequence coding for SRT is fused upstream of an epitope tag contained
within a baculovirus
expression vector. Such epitope tags include poly-his tags and immunoglobulin
tags (like Fc regions of IgG).
A variety of plasmids may be employed, including plasmids derived from
commercially available plasmids such
as pVL1393 (Novagen). Briefly, the sequence encoding SRT or the desired
portion of the coding sequence of
SRT such as the sequence encoding the extracellular domain of a transmembrane
protein or the sequence
encoding the mature protein if the protein is extracellular is amplified by
PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product
is then digested with those selected restriction enzymes and subcloned into
the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoldT"' virus
DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711)
using lipofectin (commercially
available from GIBCO-BRL). After 4 - S days of incubation at 28°C, the
released viruses are harvested and used
for further amplifications. Viral infection and protein expression are
performed as described by O'Reilley et
al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
Expressed poly-his tagged SRT can then be purified, for example, by Niz+-
chelate affinity
chromatography as follows. Extracts are prepared from recombinant virus-
infected Sf9 cells as described by
Rupert et al. Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed,
resuspended in sonication buffer (25
mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10 % glycerol; 0.1 % NP-40; 0.4
M KCl), and sonicated
twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and
the supernatant is diluted 50-fold
in loading buffer (50 mM phosphate, 300 mM NaCI, 10 % glycerol, pH 7.8) and
filtered through a 0.45 ~m
filter. A Niz+-NTA agarose column (commercially available from Qiagen) is
prepared with a bed volume of 5
mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
The filtered cell extract is
loaded onto the column at 0.5 mL per minute. The column is washed to baseline
AZ$o with loading buffer, at
which point fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM
phosphate; 300 mM NaCI, 10 % glycerol, pH 6.0), which elutes nonspecifically
bound protein. After reaching
AZgo baseline again, the column is developed with a 0 to 500 mM Imidazole
gradient in the secondary wash
buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with
Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the
eluted Hislo tagged SRT are
pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) SRT can be
performed using known
chromatography techniques, including for instance, Protein A or protein G
column chromatography.
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EXAMPLE 8
Preparation of antibodies that bind SRT
This example illustrates preparation of monoclonal antibodies which can
specifically bind SRT.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for
instance, in Goding, supra. Immunogens that may be employed include purified
SRT, fusion proteins containing
SRT, and cells expressing recombinant SRT on the cell surface. Selection of
the immunogen can be made by
the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the SRT immunogen emulsified in
complete Freund's
adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-
100 micrograms. Alternatively,
the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
Hamilton, MT) and
injected into the animal's hind foot pads. The immunized mice are then boosted
10 to 12 days later with
additional immunogen emulsified in the selected adjuvant. Thereafter, for
several weeks, the mice may also be
boosted with additional immunization injections. Serum samples may be
periodically obtained from the mice
by retro-orbital bleeding for testing in ELISA assays to detect anti-SRT
antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected
with a final intravenous injection of SRT. Three to four days later, the mice
are sacrificed and the spleen cells
are harvested. The spleen cells are then fused (using 35 % polyethylene
glycol) to a selected murine myeloma
cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions
generate hybridoma cells
which can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and
thymidine) medium to inhibit proliferation of non-fused cells, myeloma
hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against SRT.
Determination of
"positive" hybridoma cells secreting the desired monoclonal antibodies against
SRT is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balb/c mice to produce
ascites containing the anti-SRT monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue
culture flasks or roller bottles. Purification of the monoclonal antibodies
produced in the ascites can be
accomplished using ammonium sulfate precipitation, followed by gel exclusion
chromatography. Alternatively,
affinity chromatography based upon binding of antibody to protein A or protein
G can be employed.
EXAMPLE 9
Purification of SRT nolyneytides using sQecific antibodies
Native or recombinant SRT polypeptides may be purified by a variety of
standard techniques in the art
of protein purification. For example, pro-SRT polypeptide, mature SRT
polypeptide, or pre-SRT polypeptide
is purified by immunoaffinity chromatography using antibodies specific for the
SRT polypeptide of interest. In
general, an immunoaffmity column is constructed by covalently coupling the
anti-SRT polypeptide antibody to
an activated chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium
sulfate or by purification on immobilized Protein A (Pharmacia LKB
Biotechnology, Piscataway, N.J.).
Likewise, monoclonal antibodies are prepared from mouse ascites fluid by
ammonium sulfate precipitation or
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chromatography on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a
chromatographic resin such as CnBr-activated SEPHAROSET"' (Pharmacia LKB
Biotechnology). The antibody
is coupled to the resin, the resin is blocked, and the derivative resin is
washed according to the manufacturer's
instructions.
Such an immunoaffmity column is utilized in the purification of SRT
polypeptide by preparing a fraction
from cells containing SRT polypeptide in a soluble form. This preparation is
derived by solubilization of the
whole cell or of a subcellular fraction obtained via differential
centrifugation by the addition of detergent or by
other methods well known in the art. Alternatively, soluble SRT polypeptide
containing a signal sequence may
be secreted in useful quantity into the medium in which the cells are grown.
A soluble SRT polypeptide-containing preparation is passed over the
immunoaffinity column, and the
column is washed under conditions that allow the preferential absorbance of
SRT polypeptide (e.g., high ionic
strength buffers in the presence of detergent). Then, the column is eluted
under conditions that disrupt
antibody/SRT polypeptide binding (e.g. , a low pH buffer such as approximately
pH 2-3, or a high concentration
of a chaotrope such as urea or thiocyanate ion), and SRT polypeptide is
collected.
EXAMPLE 10
Drug screenine
This invention is particularly useful for screening compounds by using SRT
polypeptides or binding
fragment thereof in any of a variety of drug screening techniques. The SRT
polypeptide or fragment employed
in such a test may either be free in solution, affixed to a solid support,
borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the SRT polypeptide or
fragment. Drugs are screened
against such transformed cells in competitive binding assays. Such cells,
either in viable or fixed form, can be
used for standard binding assays. One may measure, for example, the formation
of complexes between SRT
polypeptide or a fragment and the agent being tested. Alternatively, one can
examine the diminution in complex
formation between the SRT polypeptide and its target cell or target receptors
caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can
affect a SRT polypeptide-associated disease or disorder. These methods
comprise contacting such an agent with
an SRT polypeptide or fragment thereof and assaying (I) for the presence of a
complex between the agent and
the SRT polypeptide or fragment, or (ii) for the presence of a complex between
the SRT polypeptide or fragment
and the cell, by methods well known in the art. In such competitive binding
assays, the SRT polypeptide or
fragment is typically labeled. After suitable incubation, free SRT polypeptide
or fragment is separated from that
present in bound form, and the amount of free or uncomplexed label is a
measure of the ability of the particular
agent to bind to SRT polypeptide or to interfere with the SRT polypeptide/cell
complex.
Another technique for drug screening provides high throughput screening for
compounds having suitable
binding affinity to a polypeptide and is described in detail in WO 84/03564,
published on September 13, 1984.
Briefly stated, large numbers of different small peptide test compounds are
synthesized on a solid substrate, such
as plastic pins or some other surface. As applied to a SRT polypeptide, the
peptide test compounds are reacted
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with SRT polypeptide and washed. Bound SRT polypeptide is detected by methods
well known in the art.
Purified SRT polypeptide can also be coated directly onto plates for use in
the aforementioned drug screening
techniques. In addition, non-neutralizing antibodies can be used to capture
the peptide and immobilize it on the
solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing
antibodies capable of binding SRT polypeptide specifically compete with a test
compound for binding to SRT
polypeptide or fragments thereof. In this manner, the antibodies can be used
to detect the presence of any
peptide which shares one or more antigenic determinants with SRT polypeptide.
EXAMPLE 11
Rational drug desi,~n
The goal of rational drug design is to produce structural analogs of
biologically active polypeptide of
interest (i. e. , a SRT polypeptide) or of small molecules with which they
interact, e. g. , agonists, antagonists, or
inhibitors. Any of these examples can be used to fashion drugs which are more
active or stable forms of the
SRT polypeptide or which enhance or interfere with the function of the SRT
polypeptide in vivo (c.f., Hodgson,
Bio/Technology, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the SRT polypeptide, or of
an SRT
polypeptide-inhibitor complex, is determined by x-ray crystallography, by
computer modeling or, most typically,
by a combination of the two approaches. Both the shape and charges of the SRT
polypeptide must be ascertained
to elucidate the structure and to determine active sites) of the molecule.
Less often, useful information regarding
the structure of the SRT polypeptide may be gained by modeling based on the
structure of homologous proteins.
In both cases, relevant structural information is used to design analogous SRT
polypeptide-like molecules or to
identify efficient inhibitors. Useful examples of rational drug design may
include molecules which have improved
activity or stability as shown by Braxton and Wells, Biochemistry, 31:7796-
7801 (1992) or which act as
inhibitors, agonists, or antagonists of native peptides as shown by Athauda et
al., J. Biochem., 113:742-746
(1993).
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above,
and then to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein crystallography
altogether by generating anti-idiotypic
antibodies (anti-ids) to a functional, pharmacologically active antibody. As a
mirror image of a mirror image,
the binding site of the anti-ids would be expected to be an analog of the
original receptor. The anti-id could then
be used to identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated
peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the SRT polypeptide
may be made available
to perform such analytical studies as X-ray crystallography. In addition,
knowledge of the SRT polypeptide
amino acid sequence provided herein will provide guidance to those employing
computer modeling techniques
in place of or in addition to x-ray crystallography.
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The foregoing written specification is considered to be sufficient to enable
one skilled in the art to
practice the invention. The present invention is not to be limited in scope by
the construct deposited, since the
deposited embodiment is intended as a single illustration of certain aspects
of the invention and any constructs
that are functionally equivalent are within the scope of this invention. The
deposit of material herein does not
constitute an admission that the written description herein contained is
inadequate to enable the practice of any
aspect of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the
claims to the specific illustrations that it represents. Indeed, various
modifications of the invention in addition
to those shown and described herein will become apparent to those skilled in
the art from the foregoing
description and fall within the scope of the appended claims.
