Human prostatic and placental transglutaminases are identified and cloned. The human transglutaminases herein are useful for, inter alia, therapeutic wound repair, closure of skin grafts, stabilizing food preparations, and markers for identifying agents which act as agonists or antagonists of cellular apoptosis.

BACKGROUND OF THE INVENTION 
Transglutaminases are a group of calcium dependent enzymes that catalyze 
the crosslinking of proteins by promoting the formation of 
.epsilon.-(.gamma.-glutaminyl)lysine isopeptide bonds between 
protein-bound glutamine and lysine residues. These enzymes are believed to 
be widely distributed in nature, as the crosslinks are found in both 
prokaryotic and eukaryotic cells. Although different transglutaminases 
appear to be very similar in substrate specificity, several distinct forms 
of the enzymes have been identified. See generally, Folk, Ann. Rev. 
Biochem. 49:517-531 (1980). 
Transglutaminase-mediated protein crosslinking reactions have been 
implicated in both normal and pathological processes in mammalian cells 
and tissues. The crosslink may act to maintain some forms of protein 
structure, such as in the terminal differentiation of epidermal cell 
layers and in other cellular architecture. An intracellular 
transglutaminase known as epidermal or Type I transglutaminase has been 
isolated and cloned from rabbit epithelial cells (Floyd and Jetten, Mol. 
Cell. Biol. 9:4846-4851 (1989)), and a transglutaminase has been isolated 
and cloned from guinea pig liver cells (Ikura et al., Biochem. 27: 
2898-2905 (1988)). Other transglutaminase activities have been described 
including hair follicle transglutaminase, keratinocyte transglutaminase, 
and prostate transglutaminase (Wilson et al., Fed. Proc. 38:1809 (1979)). 
Lee et al., Prep. Biochem. 16:321-335 (1986) have described the 
purification of a transglutaminase from human erythrocytes. These 
transglutaminases have been shown to be distinct from a plasma 
transglutaminase, Factor XIII, an enzyme whose primary function appears to 
be stabilizing fibrin clots. Factor XIII has also been purified, cloned, 
and sequenced. (Ichinose, et al., Biochem. 25:6900-6906 (1986), Takahashi, 
et al., Proc. Natl. Acad. Sci. U.S.A. 83:8018-8023 (1986)). 
Transglutaminases have been employed for crosslinking purposes in a variety 
of fields. Certain microbial transglutaminases have found use in food 
technology to add texture to processed foods, particularly fish and 
cheese. Others have been used in enzyme-catalyzed fluorescent labeling of 
proteins, in the introduction of cleavable crosslinks, and in the 
solid-phase reversible removal of specific proteins from biological 
systems. Factor XIII preparations have been proposed for a variety of 
therapeutic uses, such as the treatment of subarachnoid hemorrhage and 
inflammatory bowel disease. 
Presently, a plasma derived Factor XIII is available as a fibrin sealant, 
but, as with most plasma-derived products, carries an inherent risk of 
viral contamination. Further, Factor XIII and certain other 
transglutaminases are zymogens, requiring some form of activation to 
become catalytically active. And, as each transglutaminase has a 
restricted range of substrates, their activity may be limited in certain 
applications. Accordingly, what is needed in the art are methods for 
producing by recombinant means human transglutaminases. The present 
invention fulfills these and other related needs. 
SUMMARY OF THE INVENTION 
The present invention provides the ability to produce human prostatic and 
placental transglutaminases and polypeptides or fragments thereof by 
recombinant means, preferably in cultured eukaryotic cells. The expressed 
transglutaminase may or may not have the biological activity of the native 
enzyme, depending on the intended use. Accordingly, isolated and purified 
polynucleotides are described which code for the transglutaminases and 
fragments thereof, where the polynucleotides may be in the form of DNA, 
such as cDNA or genomic DNA, or RNA. Based on these sequences probes may 
be designed for hybridization to identify these and related genes or 
transcription products thereof which encode human prostatic and placental 
transglutaminases. 
In related embodiments the invention concerns DNA constructs which comprise 
a transcriptional promoter, a DNA sequence which encodes the prostatic or 
placental transglutaminase or fragment thereof, and a transcriptional 
terminator, each operably linked for expression of the enzyme or enzyme 
fragment. The constructs are preferably used to transform or transfect 
host cells, preferably eukaryotic cells, more preferably yeast or 
mammalian cells. For large scale production the expressed prostatic or 
placental transglutaminase may be isolated from the cells by, for example, 
immunoaffinity purification. 
Nucleic acid sequences which encode the human prostatic or placental 
transglutaminases of the invention and the recombinant transglutaminases 
themselves can also be used to develop compounds which can alter 
transglutaminase-associated apoptosis of a eukaryotic cell. Compounds may 
be screened for agonistic or antagonistic effects on 
transglutaminase-mediated metabolism in the host cell. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The present invention provides isolated polynucleotide molecules encoding 
human prostatic and placental transglutaminases, thereby providing for the 
expression of human prostatic and placental transglutaminase polypeptides 
and fragments thereof. Isolated polynucleotide molecules are those that 
are separated from their natural environment and include cDNA and genomic 
clones. Isolated DNA molecules of the present invention are provided free 
of other genes with which they are naturally associated and may include 
naturally occurring 5' and 3' untranslated sequences that represent 
regulatory regions such as promoters and terminators. The identification 
of regulatory regions within the naturally occurring 5' and 3' 
untranslated regions will be evident to one of ordinary skill in the art 
(for review, see Dynan and Tijan, Nature 316: 774-778, 1985; Birnstiel et 
al., Cell 41: 349-359, 1985; Proudfoot, Trends in Biochem. Sci. 14: 
105-110, 1989; and Sambrook et al., Molecular Cloning: A Laboratory 
Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; which are 
incorporated herein by reference). 
As will be understood by one skilled in the art, the DNA molecules of the 
present invention encompass allelic variants and genetically engineered or 
synthetic variants of the transglutaminases that encode conservative amino 
acid substitutions and/or minor additions, or deletions of amino acids. 
Such variants also encompass DNA molecules containing degeneracies in the 
DNA code wherein host-preferred codons are substituted for the analogous 
codons in the human sequence. In addition, substantially similar DNA 
molecules of the present invention encompass those DNA molecules that are 
capable of hybridizing to the DNA sequences of the present invention under 
high or low stringency (see Sambrook et al., ibid.) and those sequences 
that are degenerate as a result of the genetic code to the amino acid 
sequences of the present invention. 
Recombinant DNA expression systems provide convenient means for obtaining 
large quantities of the human transglutaminases in relatively pure form. 
By human prostatic or placental transglutaminase polypeptides and 
fragments is meant to include sequences of amino acids from 9 to 20 amino 
acids up to entire proteins, which have at least about 85% homology, 
preferably at least 90%, and more preferably at least about 95% or more 
homology to the amino acid sequences of the human prostatic or placental 
transglutaminases of the invention. As will be appreciated by those 
skilled in the art, the invention also includes those polypeptides having 
slight variations in amino acid sequences or other properties. Such 
variations may arise naturally as allelic variations (e.g., due to genetic 
polymorphism) or may be produced by human intervention (e.g., by 
mutagenesis of cloned DNA sequences), such as induced point, deletion and 
insertion mutations. 
Nucleic acid molecules encoding the human transglutaminases as described 
herein can be cloned from a variety of human cell sources that express the 
enzymes. Preferred sources for human prostatic transglutaminase include 
human prostate or liver cells and tissues, and for human placental 
transglutaminase include, e.g., human placental tissue. Useful isolated 
nucleic acid sequences of the invention which encode the human 
transglutaminases include mRNA, genomic DNA and cDNA. For expression, 
cDNAs are generally preferred because they lack introns that may interfere 
with expression. 
To obtain human prostate and/or placental transglutaminase clones, a human 
prostate tissue cDNA library and/or human placental tissue cDNA library is 
amplified to obtain DNA molecules encoding transglutaminases using 
oligonucleotide primers in a polymerase chain reaction ("PCR"; U.S. Pat. 
Nos. 4,683,195, 4,683,202, incorporated herein by reference). The 
oligonucleotide primer sequences are designed by preparing a multiple 
sequence alignment of sequence information for a variety of 
transglutaminases and related proteins (e.g., rat keratinocyte 
transglutaminase, human keratinocyte transglutaminase, human 
transglutaminase K, human factor XIII, human endothelial cell 
transglutaminase, mouse macrophage transglutaminase, guinea pig 
transglutaminase, human erythrocyte membrane protein band 4.2, rabbit 
transglutaminase type I, and bovine factor XIII). The multiple alignment 
is subjected to analysis for least degenerate/most conserved regions from 
which primers, which are generally about 17-20 bases long, are designed. 
Primers were designed from three regions of multiple homology in Example I 
described below: one from the active site region, and two from other 
regions which seemed to have structural importance, based on, inter alia, 
the presence of hydrophobic residues and proline residues. Following 
amplification and enrichment for the desired DNA molecules, the molecules 
are identified and used to screen and isolate full length cDNA clones for 
the prostate and placental transglutaminases. 
cDNA libraries can be screened with, e.g., labeled probes from 
random-primed DNA molecules encoding human prostatic or placental 
transglutaminase, which probes preferably span the enzyme's active site 
and/or putative calcium binding site. To obtain the human placental 
transglutaminase clone, an oligo-d(T) primed cDNA library can be 
constructed from poly(A).sup.+ RNA purified from human placental tissues. 
Partial clones may be used as probes in additional screening until the 
complete coding sequence is obtained. 
In addition to the use of partial clones to obtain full length 
transglutaminase clones, PCR amplification may be used to obtain a 
complete cDNA. Synthetic oligonucleotide primers may be designed to 
hybridize to vector sequences near the cDNA insert boundary and to DNA 
sequences within the transglutaminase coding sequence. Polymerase chain 
amplification may be used in conjunction with such primers to obtain DNA 
segments encoding terminal DNA sequences for completing a partial cDNA 
clone. 
If necessary, partial clones are joined in the correct reading frame to 
construct the complete coding sequence. Joining is achieved by, for 
example, digesting clones with appropriate restriction endonucleases and 
joining the fragments enzymatically in the proper orientation. Depending 
on the fragments and the particular restriction endonucleases chosen, it 
may be necessary to remove unwanted DNA sequences through a "loop out" 
process of deletion mutagenesis or through a combination of restriction 
endonuclease cleavage and mutagenesis. It is preferred that the resultant 
sequence be in the form of a continuous open reading frame, that is, that 
it lack intervening sequences (introns). The sequence of one exemplary 
human prostate clone described herein is shown in SEQ. ID. NO.14. 
With the nucleotide and deduced amino acid sequences of human prostate 
transglutaminase provided herein, genomic or cDNA sequences encoding human 
prostatic transglutaminase may be obtained from libraries prepared from 
other cells and tissues according to known procedures. For instance, using 
oligonucleotide probes derived from human prostate transglutaminase 
sequences, generally of at least about fourteen nucleotides and up to 
twenty-five or more nucleotides in length, DNA sequences encoding 
transglutaminases of other cells or tissues may be obtained. If partial 
clones are obtained, it is necessary to join them in proper reading frame 
to produce a full length clone, using such techniques as endonuclease 
cleavage, ligation and loopout mutagenesis. 
For expression, a DNA sequence encoding human prostate or placental 
transglutaminase polypeptide is inserted into a suitable expression 
vector, which in turn is used to transform or transfect appropriate host 
cells for expression. Expression vectors for use in carrying out the 
present invention will generally comprise a promoter capable of directing 
the transcription of a cloned DNA and a transcriptional terminator, 
operably linked with the sequence encoding the prostate or placental 
transglutaminase polypeptide so as to produce a continuously transcribable 
gene sequence which produces sequences in reading frame and is 
continuously translated to produce a human prostate or placental 
transglutaminase polypeptide. The expression vectors of the present 
invention may further include enhancers and other elements such as 
secretory signal sequences to facilitate expression and/or secretion of 
the protein. One or more selectable markers may also be included. 
Secretory signal sequences, also called leader sequences, prepro sequences 
and/or pre sequences, are amino acid sequences that act to direct the 
secretion of mature polypeptides or proteins from a cell. Such sequences 
are characterized by a core of hydrophobic amino acids and are typically 
(but not exclusively) found at the amino termini of newly synthesized 
proteins. Very often the secretory peptide is cleaved from the mature 
protein during secretion. Such secretory peptides contain processing sites 
that allow cleavage of the secretory peptides from the mature proteins as 
they pass through the secretory pathway. A preferred processing site is a 
dibasic cleavage site, such as that recognized by the Saccharomyces 
cerevisiae KEX2 gene. A particularly preferred processing site is a 
Lys-Arg processing site. Processing sites may be encoded within the 
secretory peptide or may be added to the peptide by, for example, in vitro 
mutagenesis. 
The choice of a suitable secretory signal sequence is well within the level 
of ordinary skill in the art and will depend on the selected host system 
employed. Preferred secretory signals include the a factor signal sequence 
(prepro sequence: Kurjan and Herskowitz, Cell 30: 933-943, 1982; Kurjan et 
al., U.S. Pat. No. 4,546,082; Brake, U.S. Pat. No. 4,870,008), the PHO5 
signal sequence (Beck et al., WO 86/00637), the BAR1 secretory signal 
sequence (MacKay et al., U.S. Pat. No. 4,613,572; MacKay, WO 87/002670), 
the SUC2 signal sequence (Carlsen et al., Molecular and Cellular Biology 
3: 439-447, 1983), the .alpha.-1-antitrypsin signal sequence (Kurachi et 
al., Proc. Natl. Acad. Sci. U.S.A. 78: 6826-6830, 1981), and the .alpha.-2 
plasmin inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 
102:1033-1042, 1987). A particularly preferred signal sequence is the 
tissue plasminogen activator signal sequence (Pennica et al., Nature 301: 
214-221, 1983). Alternately, a secretory signal sequence may be 
synthesized according to the rules established, for example, by von Heinje 
(European Journal of Biochemistry 133: 17-21, 1983; Journal of Molecular 
Biology 184: 99-105, 1985; Nucleic Acids Research 14: 4683-4690, 1986). 
Secretory signal sequences may be used singly or may be combined. For 
example, a first secretory signal sequence may be used in combination with 
a sequence encoding the third domain of barrier (described in U.S. Pat. 
No. 5,037,243, which is incorporated by reference herein in its entirety). 
The third domain of barrier may be positioned in proper reading frame 3' 
of the DNA segment of interest or 5' to the DNA segment and in proper 
reading frame with both the secretory signal sequence and a DNA segment of 
interest. 
Host cells for use in practicing the present invention include mammalian, 
avian, plant, insect, bacterial and fungal cells, but preferably 
eukaryotic cells. Preferred eukaryotic cells include cultured mammalian 
cell lines (e.g., rodent or human cell lines) and fungal cells, including 
species of yeast (e.g., Saccharomyces spp., particularly S. cerevisiae, 
Schizosaccharomyces spp., or Kluyveromyces spp.) or filamentous fungi 
(e.g., Aspergillus spp., Neurospora spp.). Methods for producing 
recombinant proteins in a variety of prokaryotic and eukaryotic host cells 
are generally known in the art. 
Suitable yeast vectors for use in the present invention include YRp7 
(Struhl et al., Proc., Natl. Acad. Sci. U.S.A. 76: 1035-1039, 1978), YEp13 
(Broach et al., Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. 
Pat. No. 4,931,373, which is incorporated by reference herein), pJDB249 
and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof. 
Such vectors will generally include a selectable marker, which may be one 
of any number of genes that exhibit a dominant phenotype for which a 
phenotypic assay exists to enable transformants to be selected. Preferred 
selectable markers are those that complement host cell auxotrophy, provide 
antibiotic resistance or enable a cell to utilize specific carbon sources, 
and include LEU2 (Broach et al., ibid.), URA3 (Botstein et al., Gene 8: 
17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasaki et al., ibid.). 
Another suitable selectable marker is the CAT gene, which confers 
chloramphenicol resistance on yeast cells. 
Preferred promoters for use in yeast include promoters from yeast 
glycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; 
Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. 
Pat. No. 4,599,311) or alcohol dehydrogenase genes (Young et al., in 
Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., 
(eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101: 
192-201, 1983). In this regard, particularly preferred promoters are the 
TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311, 1986) and the 
ADH2-4.sup.c promoter (Russell et al., Nature 304: 652-654, 1983; Irani 
and Kilgore, U.S. patent application Ser. No. 07/631,763 and EP 284,044, 
which are incorporated herein by reference). The expression units may also 
include a transcriptional terminator. A preferred transcriptional 
terminator is the TPI1 terminator (Alber and Kawasaki, ibid.). 
Additional vectors, promoters and terminators for use in expressing the 
transglutaminases of the invention in yeast are well known in the art and 
are reviewed by, for example, Emr, Meth. Enzymol. 185:231-279, (1990), 
incorporated herein by reference. 
The transglutaminases of the invention may be expressed in Aspergillus spp. 
(McKnight and Upshall, described in U.S. Pat. No. 4,935,349, which is 
incorporated herein by reference). Useful promoters include those derived 
from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter 
(McKnight et al., EMBO J. 4:2093-2099, 1985) and the tpiA promoter. An 
example of a suitable terminator is the ADH3 terminator (McKnight et al., 
ibid.). 
Techniques for transforming fungi are well known in the literature, and 
have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. 
Natl. Acad. Sci. U.S.A. 75: 1929-1933, 1978), Yelton et al. (Proc. Natl. 
Acad. Sci. U.S.A. 81: 1740-1747, 1984), and Russell (Nature 301: 167-169, 
1983). The genotype of the host cell will generally contain a genetic 
defect that is complemented by the selectable marker present on the 
expression vector. Choice of a particular host and selectable marker is 
well within the level of ordinary skill in the art. 
In addition to fungal cells, cultured mammalian cells may be used as host 
cells within the present invention. Preferred cultured mammalian cells for 
use in the present invention include the COS-1 (ATCC CRL 1650) and BALB/c 
3T3 (ATCC CRL 163) cell lines. In addition, a number of other mammalian 
cell lines may be used within the present invention, including BHK (ATCC 
CRL 10314), 293 (ATCC CRL 1573), Rat Hep I (ATCC CRL 1600), Rat Hep II 
(ATCC CRL 1548), TCMK (ATCC CRL 139), Human lung (ATCC CCL 75.1), Human 
hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), Mouse liver (ATCC CCL 
29.1), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc. 
Natl. Acad. Sci U.S.A. 77: 4216-4220, 1980). 
Mammalian expression vectors for use in carrying out the present invention 
will include a promoter capable of directing the transcription of a cloned 
gene or cDNA. Preferred promoters include viral promoters and cellular 
promoters. Viral promoters include the immediate early cytomegalovirus 
promoter (Boshart et al., Cell 41: 521-530, 1985), the SV40 promoter 
(Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981), and the major late 
promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2: 
1304-1319, 1982). Cellular promoters include the mouse metallothionein-1 
promoter (Palmiter et al., U.S. Pat. No. 4,579,821), a mouse V.sub..kappa. 
promoter (Bergman et al., Proc. Natl. Acad. Sci. U.S.A. 81: 7041-7045, 
1983; Grant et al., Nuc. Acids Res. 15: 5496, 1987) and a mouse V.sub.H 
promoter (Loh et al., Cell 33: 85-93, 1983). Also contained in the 
expression vectors is a polyadenylation signal located downstream of the 
coding sequence of interest. Polyadenylation signals include the early or 
late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the 
polyadenylation signal from the Adenovirus 5 E1B region and the human 
growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9: 
3719-3730, 1981). Vectors can also include enhancer sequences, such as the 
SV40 enhancer and the mouse .mu. enhancer (Gillies, Cell 33: 717-728, 
1983). Expression vectors may also include sequences encoding the 
adenovirus VA RNAs. Vectors can be obtained from commercial sources (e.g., 
Stratagene, La Jolla, Calif.). 
Cloned DNA sequences may be introduced into cultured mammalian cells by, 
for example, calcium phosphate-mediated transfection (Wigler et al., Cell 
14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; 
Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann 
et al., EMBO J. 1: 841-845, 1982), DEAE-dextran mediated transfection 
(Ausubel et al., (ed.) Current Protocols in Molecular Biology, John Wiley 
and Sons, Inc., N.Y. (1987), incorporated herein by reference) or a 
commercially available transfection regent and method such as the 
Boehringer Mannheim Transfection-Reagent 
N-1-(2,3-Dioleoyloxy)propyl!-N,N,N-trimethyl ammoniummethylsulfate 
(Boehringer Mannheim, Indianapolis, Ind.). To identify cells that have 
stably integrated the cloned DNA, a selectable marker is generally 
introduced into the cells along with the gene or cDNA of interest. 
Preferred selectable markers for use in cultured mammalian cells include 
genes that confer resistance to drugs, such as neomycin, hygromycin, and 
methotrexate. The selectable marker may be an amplifiable selectable 
marker. Preferred amplifiable selectable markers are the DHFR gene and the 
neomycin resistance gene. Selectable markers are reviewed by Thilly 
(Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., which 
is incorporated herein by reference). The choice of selectable markers is 
well within the level of ordinary skill in the art. 
Selectable markers may be introduced into the cell on a separate vector at 
the same time as the transglutaminase sequence of interest, or they may be 
introduced on the same vector. If on the same vector, the selectable 
marker and the transglutaminase sequence of interest may be under the 
control of different promoters or the same promoter, the latter 
arrangement producing a dicistronic message. Constructs of this type are 
known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 
4,713,339). It may also be advantageous to add additional DNA, known as 
"carrier DNA" to the mixture which is introduced into the cells. 
Transfected mammalian cells are allowed to grow for a period of time, 
typically 1-2 days, to begin expressing the DNA sequence(s) of interest. 
Drug selection is then applied to select for growth of cells that are 
expressing the selectable marker in a stable fashion. For cells that have 
been transfected with an amplifiable selectable marker the drug 
concentration may be increased in a stepwise manner to select for 
increased copy number of the cloned sequences, thereby increasing 
expression levels. 
Promoters, terminators and methods for introducing expression vectors 
encoding transglutaminase into plant, avian and insect cells are well 
known in the art. The use of baculoviruses, for example, as vectors for 
expressing heterologous DNA sequences in insect cells has been reviewed by 
Atkinson et al. (Pestic. Sci. 28: 215-224,1990). The use of Agrobacterium 
rhizogenes as vectors for expressing genes in plant cells has been 
reviewed by Sinkar et al. (J. Biosci. (Banglaore) 11: 47-58, 1987). 
Host cells containing DNA constructs of the present invention are then 
cultured to produce the transglutaminase. The cells are cultured according 
to standard methods in a culture medium containing nutrients required for 
growth of the chosen host cells. A variety of suitable media are known in 
the art and generally include a carbon source, a nitrogen source, 
essential amino acids, vitamins and minerals, as well as other components, 
e.g., growth factors or serum, that may be required by the particular host 
cells. The growth medium will generally select for cells containing the 
DNA construct by, for example, drug selection or deficiency in an 
essential nutrient which is complemented by the selectable marker on the 
DNA construct or co-transfected with the DNA construct. 
Yeast cells, for example, are preferably cultured in a medium which 
comprises a nitrogen source (e.g., yeast extract), inorganic salts, 
vitamins and trace elements. The pH of the medium is preferably maintained 
at a pH greater than 2 and less than 8, preferably at pH 5-6. Methods for 
maintaining a stable pH include buffering and constant pH control, 
preferably through the addition of sodium hydroxide. Preferred buffering 
agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, 
Mo.). Cultured mammalian cells are generally cultured in commercially 
available serum-containing or serum-free media. Selection of a medium 
appropriate for the particular cell line used is within the level of 
ordinary skill in the art. 
In a preferred embodiment, the human prostate and placental 
transglutaminases are expressed in yeast as intracellular products. The 
yeast host can be a diploid strain homozygous for pep4, a mutation that 
reduces vacuolar protease levels, as described in Jones et al., Genetics 
85:23-33 (1977), incorporated herein by reference. The strain is also 
homozygous for disruption of the endogenous TPI (triose phosphate 
isomerase) gene, thereby allowing the S. pombe POT1 gene to be used as a 
selectable marker. The vector includes the POT1 marker, a leu2-d marker 
and the ADH2-4.sup.c promoter. The POT1 marker in the TPI.sup.- host 
allows for selection by growth in glucose. The host strain is grown in 
glucose-containing synthetic media with a glucose feed. An ethanol feed is 
then substituted for glucose to de-repress the promoter. The pH is 
maintained with NaOH. Other preferred means for expression are generally 
described in, e.g., EPO publication EP 268,772, incorporated herein by 
reference. 
In another preferred embodiment, the human prostate and placental 
transglutaminases are expressed in cultured mammalian cells. Preferably, 
the cultured mammalian cells are BHK 570 cells (deposited with the 
American Type Culture Collection under accession number 10314). 
The human prostate and placental transglutaminases produced according to 
the present invention may be purified by affinity chromatography on an 
antibody column using antibodies directed against the transglutaminases. 
Additional purification may be achieved by conventional chemical 
purification means, such as liquid chromatography, gradient 
centrifugation, and gel electrophoresis, among others. Methods of protein 
purification are known in the art (see generally, Scopes, R., Protein 
Purification, Springer-Verlag, NY (1982), which is incorporated herein by 
reference) and may be applied to the purification of the recombinant 
transglutaminase described herein. Antibodies prepared against the novel 
transglutaminases may be either polyclonal or monoclonal, and can be used 
to isolate and substantially purify the recombinant or native 
transglutaminases of the invention. Substantially pure recombinant human 
prostatic or placental transglutaminase of at least about 50% is 
preferred, at least about 70-80% more preferred, and 95-99% or more 
homogeneity most preferred, particularly for pharmaceutical uses. Once 
purified, partially or to homogeneity, as desired, the recombinant or 
native human prostatic and placental transglutaminases described herein 
may be used as desired. 
The human prostatic and placental transglutaminases produced according to 
the present invention find a variety of uses. These transglutaminases can 
be used therapeutically in humans or other mammals. For example, human 
transglutaminase may be used in the repair of wounds, ulcerated lesions, 
skin grafts, etc. As the human transglutaminases are relatively stable, 
active extracellularly, and bind avidly to collagen, they can be used to 
stabilize basement membrane structures. An appropriate endogenous 
substrate for transglutaminase is fibronectin, which thus serves as a 
basis for crosslinking and stabilizing collagen/fibronectin complexes. 
Pharmaceutical compositions of the invention comprise therapeutically 
effective amounts of human prostatic and/or placental transglutaminase and 
an appropriate physiologically acceptable carrier. The pharmaceutical 
compositions are intended primarily for topical or local administration, 
for use in methods of wound closure, as tissue adhesives, and the like. 
Typically the transglutaminase will be administered concurrently with or 
prior to compositions of thrombin to the wound site to increase 
effectiveness. 
A variety of aqueous carriers may be used in the compositions, e.g., water, 
buffered water, saline, 0.3% glycine and the like, including glycoproteins 
for enhanced stability, such as albumin, lipoprotein, fibronectin and/or 
globulin. The compositions may be sterilized by well known sterilization 
techniques, and the solutions packaged for use or lyophilized. Other 
components of the pharmaceutical compositions of the invention can include 
pharmaceutically acceptable auxiliary substances as required to 
approximate physiological conditions, such as pH adjusting and buffering 
agents, tonicity adjusting agents and the like, for example, sodium 
acetate, sodium lactate, sodium chloride, potassium chloride, calcium 
chloride, etc. 
Other components may also be added to the transglutaminase compositions to 
enhance their effectiveness, such as calcium ions, protease inhibitors 
(e.g., aprotinin), fibrinogen, etc. Admixtures of prostaglandins, 
coagulation factors, antihistamines, vasopressins, growth factors, 
vitamins, antibiotics (e.g., aminoglycosides, penicillins, carbapenems, 
sulfonamides, tetracyclines) and the like may also be provided. The 
formulation of various wound tissue adhesives is discussed in detail in 
U.S. Pat. Nos. 4,427,650, 4,442,655, and 4,655,211, each of which is 
incorporated herein by reference. 
The concentration of human prostatic and/or placental transglutaminase in 
the pharmaceutical formulations can vary widely, i.e., from about 20 
.mu.g/ml to 20 mg/ml or more, usually at least about 50 .mu.g to 1 mg/ml, 
preferably from about 100 .mu.g to 500 .mu.g/ml and will be selected 
primarily by volumes, viscosities, strength of the resulting complex, 
etc., in accordance with the particular use intended, the severity of the 
wound, the mode of administration selected, etc. Amounts effective for 
these uses will depend on the severity of the wound, injury or disease and 
the general state of the patient, but generally range from about 100 .mu.g 
to about 500 mg or more of transglutaminase per site, with dosages of from 
about 500 .mu.g to about 50 mg of transglutaminase per site being more 
commonly used. It must be kept in mind that the materials of the present 
invention may generally be employed in serious disease or injury states, 
that is, life-threatening or potentially life threatening situations. In 
such cases, in view of the minimization of extraneous substances, 
decreased immunogenicity and the prolonged half-life and stability of the 
human prostatic and placental transglutaminases made feasible by this 
invention, it is possible and may be felt desirable by the treating 
physician to administer substantial excesses of these transglutaminase 
compositions. 
The transglutaminases described herein can also be used in the preparation 
of food material, such as paste food or cheese, and can be added to 
dehydrated fish to prevent deterioration caused by protozoans, e.g., 
myxamoeba. The transglutaminases can also be used in the preparation of 
ground meat of okiomi (Euphasia superba), by adding to dehydrated meat 
parts from 0.1 to 100 units, preferably about 1-40 U per gram of protein 
to improve meat texture and quality. Frozen granular meats can be improved 
by combining meat material with transglutaminase of the invention at 1-500 
U per gram protein, at 30-60.degree. C. for 10-120 min. to promote 
crosslinking between glutamine groups and lysine contained in meat 
preparations. 
Other uses of the human prostatic and placental transglutaminases described 
herein include use in the enzyme-catalyzed labeling of proteins and cell 
membranes (Iwanij, Eur. J. Biochem. 80:359-368 (1977), incorporated herein 
by reference), in the introduction of cleavable crosslinks, and in the 
solid phase reversible removal of specific proteins from biological 
systems. 
Transglutaminase expression can be used as a marker for screening for 
agonists and antagonists of cellular apoptosis. Identifying agents which 
inhibit the expression of transglutaminase by a cell provides a means to 
prevent or delay atrophic changes characteristic of many degenerative 
changes, particularly degenerative nerve diseases, such as Parkinson's 
disease and Alzheimer's disease. Inhibition of apoptosis may also enhance 
blood cell counts in chemotherapy patients. The human prostatic and 
placental transglutaminase or the nucleic acids which encode the 
transglutaminases of the invention can also be used to identify agents 
which induce apoptotic activity by a cell, for the control of, e.g., 
hyperproliferative disorders. The growth of cells such as adipocytes can 
be regulated with agents identified using the transglutaminases provided 
herein as a marker, providing a means for controlling fat deposits in 
certain forms of obesity without the necessity for surgical intervention. 
Polynucleotide molecules which encode the prostatic and placental 
transglutaminases may be directly detected in cells with labeled synthetic 
oligonucleotide probes in a hybridization procedure similar to the 
Southern or dot blot. Also, PCR (including Saiki et al., Science 239:487 
(1988)) may be used to amplify DNA sequences, which are subsequently 
detected by their characteristic size on agarose gels, Southern blots of 
the gels using transglutaminase sequences or oligonucleotide probes, or a 
dot blot using similar probes. The probes of the present invention are at 
least 85% homologous to a corresponding DNA sequence of a human prostate 
transglutaminase sequence of Sequence ID No. 14 or its complement or a 
human placental transglutaminase sequence of Sequence ID No. 22 or its 
complement. For use as probes, the molecules may comprise from about 14 
nucleotides to about 25 or more nucleotides, sometimes 40 to 60 
nucleotides, and in some instances a substantial portion or even the 
entire cDNA of a transglutaminase gene of the invention may be used. The 
probes are labeled to provide a detectable signal, such as an enzyme, 
biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic 
particle, etc. 
The following examples are provided by way of illustration, not limitation.

EXAMPLE I 
Cloning of Human Prostatic Transglutaminase 
This Example describes the construction of oligonucleotide primers for 
amplification via PCR of sequences encoding human prostate 
transglutaminase, the cloning of the human prostate transglutaminase gene, 
and its nucleotide sequencing. 
A series of synthetic degenerate oligonucleotide primers were generated to 
encode three regions of conserved amino acid sequences identified from a 
multiple alignment of known transglutaminase sequences, human erythrocyte 
membrane protein band 4.2 and the rat dorsal protein-1 (Ho et al., Prog. 
Clin. Biol. Res. 239: 125-153, (1987)). The multiple alignment employed 
sequences of rat keratinocyte transglutaminase, human keratinocyte 
transglutaminase, human transglutaminase K, human factor XIII, human 
endothelial cell transglutaminase, mouse macrophage transglutaminase, 
guinea pig transglutaminase, human erythrocyte membrane protein band 4.2, 
rabbit transglutaminase type I, and bovine factor XIII. The multiple 
alignment was subjected to analysis of subsequence for least 
degenerate/most conserved regions to design primers of 17-20 bases in 
length. The amino acid sequences across three regions of multiple homology 
were chosen as the basis from which to design degenerate primers: One 
region corresponding to the active site region of factor XIII, and two 
other regions which seemed to have structural importance, based on, inter 
alia, the presence of hydrophobic residues and proline residues. 
Degenerate oligonucleotides ZC4109, ZC4110, ZC4111 and ZC4112 (Sequence ID 
Nos. 1, 2, 3 and 4) were designed to provide DNA segments corresponding to 
the conserved amino acid coding sequences. Degenerate oligonucleotides 
ZC4120, ZC4121, ZC4122, ZC4127, ZC4128, and ZC4129 (Sequence ID Nos. 5, 6, 
7, 8, 9 and 10; Table 1) were designed such that each primer contained a 
5' prime sequence to facilitate cloning into prime vectors described by 
Hagen (U.S. Pat. No. 5,075,227, incorporated herein by reference) in 
addition to a DNA segment corresponding to the conserved amino acid coding 
sequence. The prime sequences shown in Table 1 are underlined. 
TABLE 1 
______________________________________ 
Degenerate Oligonucleotide Primers (5' to 3') 
______________________________________ 
ZC4120 (Sequence ID Number 5) 
CATCCACGGA CTACGACGAR TAYSTNCTNA MYGA 
ZC4121 (Sequence ID Number 6) 
CATCCACGGA CTACGACGAR TAYSTNCTNA MRGA 
ZC4122 (Sequence ID Number 7) 
CATCCACGGA CTACGACGAR TAYSTNCTNA MNCA 
ZC4127 (Sequence ID Number 8) 
CATCCACGGA CTACGACTAY GGNCARTGY TGGGTNTT 
ZC4128 (Sequence ID Number 9) 
ACTCTCCGGT ACGACAGAAN ACCCARCAYT GNCC 
ZC4128 (Sequence ID Number 10) 
ACTCTCCGGT ACGACAGCCY TCNKGRWRYT TRTA 
______________________________________ 
The oligonucleotide primers were paired as shown in Table 2, and each pair 
was used in a PCR reaction using a .lambda.gt11 human prostate tissue cDNA 
library obtained from Clontech Laboratories, Inc., Palo Alto, Calif. as a 
template. Fifty microliter reactions were set up with each reaction 
containing 0.2 mM each of dCTP, dGTP, dATP and dTTP, 2 pmol of each 
primer, 1 .mu.l of the cDNA library, 3 units of Taq polymerase (Promega 
Corp., Madison, Wis.) and 5 .mu.l of 10.times. Promega PCR buffer (Promega 
Corp., Madison, Wis.). The reactions were each overlaid with mineral oil 
and amplified with two cycles (90 seconds at 94.degree. C., 90 seconds at 
40.degree. C., 2 minutes at 72.degree. C.), thirty-eight cycles (45 
seconds at 94.degree. C., 45 seconds at 45.degree. C., two minutes at 
72.degree. C.) and an incubation at 72.degree. C. for seven minutes. 
TABLE 2 
______________________________________ 
Oligonucleotide Primer Combinations And Expected 
Fragment Sizes (Base Pairs) 
EXPECTED 
RXN SENSE OLIGO ANTISENSE OLIGO 
FRAGMENT SIZE 
______________________________________ 
1. ZC4110 ZC4112 344 
2. ZC4110 ZC4111 851 
3. ZC4109 ZC4111 527 
4. ZC4127 ZC4129 561 
5. ZC4120 ZC4128 378 
6. ZC4121 ZC4128 378 
7. ZC4122 ZC4128 378 
8. ZC4120 ZC4129 885 
9. ZC4121 ZC4129 885 
10. ZC4122 ZC4129 885 
______________________________________ 
Aliquots of the reaction mixtures were electrophoresed on an agarose gel. 
Reactions 3, 4 and 6 exhibited bands of expected size (527 bp, 561 bp and 
378 bp, respectively). The PCR reaction products were isolated by agarose 
gel electrophoresis, and the DNA fragments were extracted with a Bio-Rad 
PREP-A-GENE kit (Bio-Rad, Richmond, Calif.) using the manufacturer's 
directions. The purified fragments were ligated into pCR1000 (Invitrogen, 
San Diego, Calif.) from the TA Cloning Kit (Invitrogen) and transformed 
into E. coli strain INVaF' (Invitrogen) using the manufacturer's protocol 
(Invitrogen TA Cloning Instruction Manual K2000-1). Two clones from PCR 
reaction 4, designated PTG561/1 and PTG561/2, were selected for subsequent 
analysis. Sequence analysis of the PCR-generated cDNA inserts in plasmids 
PTG561/1 and PTG561/2 showed that PTG561/2 (SEQ. ID. NO. 13) contained a 
unique sequence. 
To generate a full-length prostate transglutaminase cDNA clone, sense and 
antisense oligonucleotide primers were designed to specific sequences in 
the PTG561/2 clone. Oligonucleotides ZC4248 and ZC4249 (Sequence ID Nos. 
11 and 12) were used to amplify a 468 base pair fragment from clone 
PTG561/2 that was used to probe the prostate cDNA library. A fifty 
microliter reaction mixture was set up containing 2 pmols each of ZC4248 
and ZC4249, 0.025 mM dGTP, 0.025 mM dTTP, 6.6.times.10.sup.-3 mM 
.alpha.-.sup.32 P dCTP, 6.6.times.10.sup.-3 mM .alpha.-.sup.32 P dATP, 
1.times.Promega PCR buffer, 1 .mu.l of purified plasmid diluted 1:100 and 
0.5 .mu.l Taq polymerase. The reaction mixture was layered with mineral 
oil, and the mixture was preheated for approximately three minutes at 
87.degree. C. The reaction was amplified for six cycles (one minute at 
94.degree. C., one minute at 45.degree. C., one minute at 72.degree. C.) 
and one incubation at 72.degree. C. for five minutes. A 45 .mu.l aliquot 
of the amplified reaction mixture was isopropanol precipitated, and the 
radiolabeled PCR product was used to probe the .lambda.gt11 human prostate 
cDNA library (Clontech). Six positive clones were selected for further 
analysis. 
The six clones were subjected to PCR amplification using oligonucleotides 
ZC4362 and ZC4363 (Sequence ID Nos. 19 and 20, respectively), which were 
designed to anneal to sequences in the .lambda.gt11 vector, to 
characterize the cDNA inserts. Six 50 .mu.l reaction mixtures were 
prepared, each of which contained 1 .mu.l of a plate lysate of one of the 
selected clones, 1.times.Promega PCR buffer, 0.2 mM of each dNTP, 2 pmole 
each of ZC4362 and ZC4363 (Sequence ID Nos. 19 and 20, respectively), and 
3 units of Taq polymerase. The reactions were overlaid with mineral oil, 
and the mixtures were pre-heated to 94.degree. C. for two minutes to 
disrupt the phage. The reactions were amplified through 30 cycles (1 
minute at 94.degree. C., 1 minute at 50.degree. C., three minutes at 
72.degree. C.) followed by one cycle at 72.degree. C. for 7 minutes. The 
reaction products were isolated by agarose gel electrophoresis, and each 
reaction product was subcloned into PCR1000 (Invitrogen, San Diego, 
Calif.) and transformed into E. coli strain INV.alpha.F' (Invitrogen) 
using the TA cloning kit (Invitrogen). 
One of the six lambda clones, 8c2, was selected for sequence analysis. 
Lambda DNA was prepared from the 8c2 clone, and the cDNA insert was 
isolated as an Eco RI fragment and subcloned into Eco RI-linearized pUC18 
to obtain plasmid pDT43. The 8c2 cDNA insert was subjected to DNA sequence 
analysis. Based on homology with a published rat prostate protein sequence 
(Ho et al., J. Biol. Chem. 267: 12660-12667, 1992), it was determined that 
the prostate transglutaminase clone 8c2 lacked the 5' coding sequence. 
To confirm the presence of additional 5' sequences, the original six lambda 
clones were used as templates for PCR reactions using oligonucleotides 
ZC5509 (Sequence ID No. 21) and ZC4048 (Sequence ID No. 18). Each reaction 
mixture contained 1.times.PCR buffer, 1.25 MM MgCl.sub.2, 0.2 mM of each 
dNTP, 20 .mu.M ZC5509, 17.5 .mu.l of 20 .mu.M ZC4048, 1.5 units of Taq 
polymerase. The reaction mixture was divided into 24 .mu.l aliquots. Each 
aliquot received 1 .mu.l of template, and the reaction mixtures were 
amplified for thirty cycles (94.degree. C. for one minute, 42.degree. C. 
for one minute, 72.degree. C. for two minutes) followed by a seven minute 
incubation at 72.degree. C. The reaction mixtures were subjected to 
agarose gel electrophoresis. Analysis of the PCR products showed that 
clones 11A2 and 11A3 generated the largest PCR products relative to clone 
8c2, suggesting that these two clones contained additional 5' prostate 
transglutaminase coding sequences. 
The 5' human prostate coding sequence was obtained by amplification from 
the two lambda clones (11A2 and 11A3) described above. Synthetic 
oligonucleotide ZC4048 (Sequence ID No. 18) was designed to hybridize to 
the antisense lambda sequences near the Eco RI site of the .lambda.gt11 
vector. Synthetic oligonucleotide ZC5509 (Sequence ID No. 21) was designed 
to hybridize to the sense sequences in the 5' coding sequence of the 
PTG561/2 cDNA (Sequence ID No. 13). 
Two 50 .mu.l reaction mixture were prepared containing 9.3 .mu.l of either 
11A2 or 11A3 phage from plate lysates, 5 .mu.l 10.times.Promega PCR 
buffer, 5 .mu.l of a solution containing 0.2 mM of each dNTP, 2.5 .mu.l 
each of 20 pMol/.mu.l ZC4048 and 20 pMol/.mu.l ZC5509 (Sequence ID Nos. 18 
and 21, respectively), 25.1 .mu.l of water and 0.6 .mu.l of Taq 
polymerase. The reactions were incubated at 94.degree. C. for two minutes 
to disrupt the phage followed by thirty cycles (45 seconds at 94.degree. 
C., 45 seconds at 42.degree. C., 90 seconds at 72.degree. C.). After the 
final amplification cycle, the reactions were incubated at 72.degree. C. 
for five minutes. The reactions were subjected to agarose gel 
electrophoresis, and an approximately 530 bp band was isolated from each 
reaction. The PCR-generated fragments were subcloned into pCRII 
(Invitrogen, San Diego, Calif.) using the manufacturer's supplied 
instructions. Sequence analysis of several clones showed identical 
sequences spanning the .lambda.gt11 Eco RI cloning site and sequences 
present in the 8c2 clone. One clone, pDT46-1 was selected for further 
manipulation. 
The 5' transglutaminase coding sequence present in pDT46-1 but missing from 
the 8c2 clone was obtained by digesting pDT46-1 with Spe I and Ava I to 
isolate the 351 bp fragment. The 3' transglutaminase coding sequence was 
obtained by digesting plasmid pDT43 with Ava I and Xba I and isolating the 
fragment containing the transglutaminase and vector sequences. The Spe I 
and Xba I digestion produce complementary adhesive ends. The Spe I-Ava I 
fragment from pDT46-1 and the Ava I-Xba I fragment from pDT43 were ligated 
to obtain plasmid pDT47-15, which contained the prostate transglutaminase 
coding sequence of Sequence ID No. 14. 
EXAMPLE II 
Expression of Human Prostate Transglutaminase This Example describes the 
expression of a human prostate transglutaminase from cultured mammalian 
cells. 
The prostate transglutaminase cDNA insert present in plasmid pDT47-15 was 
subcloned into the mammalian expression vector Zem229R. Plasmid Zem229 is 
a pUC18-based expression vector containing a unique Bam HI site for 
insertion of cloned DNA between the mouse metallothionein-1 promoter and 
SV40 transcription terminator and an expression unit containing the SV40 
early promoter, mouse dihydrofolate reductase gene, and SV40 terminator. 
Zem229 was modified to delete the two Eco RI sites by partial digestion 
with Eco RI, blunting with DNA polymerase I (Klenow fragment) and dNTPs, 
and re-ligation. Digestion of the resulting plasmid with Bam HI followed 
by ligation of the linearized plasmid with Bam HI-Eco RI adapters resulted 
in a unique Eco RI cloning site. The resultant plasmid was designated 
Zem229R. 
Plasmid pDT47-15 was digested with Hind III to isolate the approximately 3 
kb Hind III fragment containing the prostate transglutaminase cDNA. 
Synthetic oligonucleotides ZC1157 and ZC1158 (Sequence ID Nos. 16 and 17, 
respectively) were kinased and annealed to form Eco RI-Hind III adapters. 
The kinased, annealed oligonucleotides and the 3 kb Hind III fragment were 
ligated to Eco RI-linearized Zem229R. The ligation mixture was transformed 
into E. coli strain DH10B cells, and transformants were selected for 
growth in the presence of ampicillin. Plasmid DNA prepared from selected 
transformants was subjected to restriction endonuclease analysis. A 
plasmid clone, pPTG/229R, suspected of having the insert in the correct 
orientation relative to the promoter, was selected for DNA sequence 
analysis to confirm the orientation of the insert. DNA sequence analysis 
confirmed the orientation of the insert and also disclosed the presence of 
polylinker sequences between the promoter sequence of Zem229R and the 
beginning of the prostate transglutaminase coding sequence. These 
sequences appeared to be remnants from the initial cloning procedure. 
The polylinker sequences between the promoter sequence of Zem229R and the 
prostate transglutaminase coding sequence were removed by first digesting 
plasmid pPTG/229R with Eco RI to completion. The approximately 236 base 
pair fragment containing the 5'-most transglutaminase coding sequence and 
the approximately 2.7 kb fragment containing the remainder of the 
transglutaminase coding sequence were isolated by agarose gel 
electrophoresis. The two Eco RI fragments were ligated with Eco 
RI-linearized Zem229R that had been treated with calf alkaline phosphatase 
to prevent recircularization. The ligation mixture was transformed into E. 
coli strain DH10B cells, and transformants were selected in the presence 
of ampicillin. Plasmid DNA prepared from selected transformants was 
analyzed by restriction enzyme analysis. A plasmid containing the prostate 
transglutaminase cDNA insert in the proper orientation relative to the 
promoter in Zem229R was designated pPTGR/229R. 
Both plasmids pPTG/229R and pPTGR/229R were transfected into BHK 570 cells 
(deposited with the American Type Culture Collection under accession 
number 10314) using Boehringer Mannheim Transfection-Reagent 
N-1-(2,3-Dioleoyloxy)propyl!-N,N,N-trimethyl ammoniummethylsulfate using 
the manufacturer-supplied directions. The cells were cultured under 
non-selective conditions for two days. After two days the pPTG/229R 
transfectants were selected in media containing 1 .mu.M methotrexate, and 
the pPTGR/229R transfectants were selected in media containing either 1 
.mu.M or 10 .mu.M methotrexate. 
Transfectant colonies were overlaid with a nitrocellulose filter, and the 
colonies were incubated for 3 hours. After incubation, the filter was 
lifted and probed with rabbit anti-rat prostate transglutaminase 
antiserum, obtained from Dr. V. Gentile (University of Texas-Medical 
School, Houston, Tex.). The filters were incubated with a 
peroxidase-conjugated goat anti-rabbit IgG, and colonies bound by the 
rabbit anti-rat prostate transglutaminase antibodies were visualized using 
the chemiluminescent ECL REAGENT (Amersham Corp., Arlington Heights, Ill.) 
using the manufacturer's instructions. Six positive pPTG/229R transfectant 
colonies were each picked into a well of a 24-well plate. Of the 
pPTGR/229R transfectant colonies, 12 positive colonies were picked from 
those colonies selected in the presence of 1 .mu.M methotrexate, and 12 
positive colonies were picked from those colonies selected in the presence 
of 10 .mu.M methotrexate. 
The colonies are subjected to in vivo labeling followed by 
radioimmunoprecipitation of the protein with the rabbit anti-rat prostate 
transglutaminase antiserum. Briefly, the medium in each well is replaced 
with 1 ml of serum-free medium (DMEM-Lys-Met, 1 mM sodium pyruvate, 2 mM 
L-glutamine, 5 mg/l insulin, 2 .mu.g/l selenium, 10 mg/l fetuin, 10 mg/l 
transferrin and 25 mM pH 7.2 HEPES buffer) containing 20 .mu.Ci of .sup.35 
S-EXPRESS (Du Pont-NEN Research Products, Boston, Mass.), and the cells 
are incubated overnight at 37.degree. C. After incubation, 1 ml of each 
supernatant is harvested, and the cells are rinsed with PBS. Cell extracts 
from each culture are obtained by incubating the cells with 1 ml RIPa 
buffer (10 mM Tris, pH 7.4, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 5 
mM EDTA, 0.7 M NaCl). The labeled proteins are incubated with a 1:300 
dilution of rabbit anti-rat prostate transglutaminase antiserum on ice for 
one hour. After incubation, 10 .mu.l of PANSORBIN (S. aureus cells, 
Calbiochem, San Diego, Calif.) is added to each reaction, and the mixtures 
are incubated on ice for one hour. The reactions are centrifuged, and the 
pellets are resuspended in 1 ml of RIP wash buffer (0.1% SDS, 5 mM EDTA, 
0.7 M NaCl). The reactions are centrifuged, the pellets are each 
resuspended in 20 .mu.l of loading buffer and the samples are applied to a 
10/20 gradient polyacrylamide gel (Daichi). The gel is fixed for thirty 
minutes in 40% methanol, 10% acetic acid, following which the gel is 
incubated in AMPLIFY (Amersham) for 30 minutes. The gel is dried and 
exposed to film at -80.degree. C. The autoradiograph is examined to 
identify the presence of prostate transglutaminase. 
EXAMPLE III 
Cloning of Human Placental Transalutaminase 
This Example describes the cloning of a human placental transglutaminase 
from human placental cDNA and identification of a confirmatory human 
prostatic transglutaminase clone from human liver cDNA. 
Two other cDNA sources were used in conjunction with selected degenerate 
oligonucleotide primers described in Example I to obtain unique 
transglutaminase cDNAs. QUICK-CLONE human liver cDNA (Clontech) and 
QUICK-CLONE human placenta cDNA (Clontech) were used as templates with 
oligonucleotide primers paired as shown in Table 3. Fifty microliter 
reactions were set up with each reaction containing 0.2 mM each of dCTP, 
dGTP, DATP and dTTP, 2 pmol of each primer, 1 .mu.g of the cDNA library, 3 
units of Taq polymerase (Promega Corp., Madison, Wis.) and 5 .mu.l of 
10.times.Promega PCR buffer (Promega Corp., Madison, Wis.). The reactions 
were layered with mineral oil and amplified with two cycles (90 seconds at 
94.degree. C., 90 seconds at 50.degree. C., 2 minutes at 72.degree. C.), 
twenty-five cycles (45 seconds at 94.degree. C., 45 seconds at 55.degree. 
C., one minute at 72.degree. C.) and one incubation at 72.degree. C. for 
seven minutes. 
TABLE 3 
______________________________________ 
Oligonucleotide Primer Combinations And Expected 
Fragment Sizes (Base Pairs) 
SENSE ANTISENSE 
RXN TEMPLATE OLIGO OLIGO EXP. FRAG. SIZE 
______________________________________ 
1. LIVER ZC4127 ZC4129 561 
2. ZC4120 ZC4128 378 
3. ZC4121 ZC4128 378 
4. ZC4122 ZC4128 378 
5. ZC4120 ZC4129 885 
6. ZC4121 ZC4129 885 
7. ZC4122 ZC4129 885 
14. PLACENTA ZC4127 ZC4129 561 
15. ZC4120 ZC4128 378 
16. ZC4121 ZC4128 378 
17. ZC4122 ZC4128 378 
18. ZC4120 ZC4129 885 
19. ZC4121 ZC4129 885 
20. ZC4122 ZC4129 885 
______________________________________ 
Aliquots of the amplified DNA were electrophoresed on agarose gels. 
Reactions 1, 3, 14, 15, 16, and 17 yielded fragments of expected size 
(Table 3). The PCR-generated cDNA fragments were electrophoresed on 
agarose gels, and the fragments were extracted with a Bio-Rad PREP-A-GENE 
Kit (Bio-Rad, Richmond, Calif.) using the manufacturer's directions. The 
purified fragments were ligated into pCR1000 (Invitrogen, San Diego, 
Calif.) and transformed into E. coli strain INVaF' (Invitrogen) according 
to the TA Cloning Kit (Invitrogen) using the manufacturer's protocol 
(Invitrogen TA Cloning Instruction Manual K2000-1). Clones from reactions 
1 and 14 were selected for subsequent analysis. Sequence analysis of a 
clone arising from reaction 1 revealed the same human prostatic 
transglutaminase sequence as found in PTG561/2. Sequence analysis of a 
clone arising from reaction 14 PCR cDNA, designated p1TG561/5, revealed a 
novel transglutaminase sequence. The nucleotide sequence of p1TG561/5 is 
shown in Sequence ID No. 22. 
It is evident from the above results that compositions are provided which 
encode novel prostatic and placental human transglutaminases. 
Pharmaceutical preparations of these transglutaminases are particularly 
useful as wound tissue adhesives, in view of the minimization of 
extraneous substances when produced by recombinant means, decreased 
immunogenicity in humans and prolonged half-life and stability. The 
efficacy, convenience of administration, and reduced cost are among the 
advantages conferred by the compositions of the invention. 
The transglutaminases described herein can also be used, inter alia, in the 
preparation of food material, in the enzyme-catalyzed labeling of proteins 
and cell membranes, as markers for screening for agonists and antagonists 
of cellular apoptosis, and for the detection or monitoring of expression 
in cells with labeled synthetic oligonucleotide probes or other convenient 
assays. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 22 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4109 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
# 20 TNTT 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4110 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 17 A 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4111 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
# 17 A 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4112 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 17 C 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4120 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 34 CGAR TAYSTNCTNA MYGA 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4121 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 34 CGAR TAYSTNCTNA MRGA 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4122 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 34 CGAR TAYSTNCTNA MNCA 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 37 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4127 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
# 37 CTAY GGNCARTGYT GGGTNTT 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4128 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 34 GAAN ACCCARCAYT GNCC 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4129 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
# 34 GCCY TCNKGRWRYT TRTA 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 36 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4248 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 36 CGCC TGTCTTGGCC CACTGC 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 36 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4249 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
# 36 TGCT GAGAGCGTTG GGCATC 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 521 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: PTG562 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- TATGGACAGT GCTGGGTATT TGCTGGGATC CTGACTACAG TGCTGAGAGC GT - #TGGGCATC 
60 
- CCAGCACGCA GTGTGACAGG CTTCGATTCA GCTCACGACA CAGAAAGGAA CC - #TCACGGTG 
120 
- GACACCTATG TGAATGAGAA TGGCGAGAAA ATCACCAGTA TGACCCACGA CT - #CTGTCTGG 
180 
- AATTTCCATG TGTGGACGGA TGCCTGGATG AAGCGACCCT ACGACGGCTG GC - #AGGCTGTG 
240 
- GACGCAACGC CGCAGGAGCG AAGCCAGGGT GTCTTCTGCT GTGGGCCATC AC - #CACTGACC 
300 
- GCCATCCGCA AAGGTGACAT CTTTATTGTC TATGACACCA GATTCGTCTT CT - #CAGAAGTG 
360 
- AATGGTGACA GGCTCATCTG GTTGGTGAAG ATGGTGAATG GGCAGGAGGA GT - #TACACGTA 
420 
- ATTTCAATGG AGACCACAAG CATCGGGAAA AACATCAGCA CCAAGGCAGT GG - #GCCAAGAC 
480 
# 521 CCTC TGAGTACAAG CTCCCCGAAG G 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 3064 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 147..2186 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- AATTCTAAAA ATGCTTTTGC AAGCTTGCAT GCCTGCAGGT GCAGCGGCCG CC - #AGTGTGAT 
60 
- GGATATCTGC AGAATTCGGC TTGCGCTCAG CTGGAATTCC GCAGAGATAG AG - #TCTTCCCT 
120 
#AAA GAG CTG CAA 173GAAGGG ATG ATG GAT GCA TCA 
#Ser Lys Glu Leu GlnAsp Ala 
# 5 1 
- GTT CTC CAC ATT GAC TTC TTG AAT CAG GAC AA - #C GCC GTT TCT CAC CAC 
221 
Val Leu His Ile Asp Phe Leu Asn Gln Asp As - #n Ala Val Ser His His 
# 25 
- ACA TGG GAG TTC CAA ACG AGC AGT CCT GTG TT - #C CGG CGA GGA CAG GTG 
269 
Thr Trp Glu Phe Gln Thr Ser Ser Pro Val Ph - #e Arg Arg Gly Gln Val 
# 40 
- TTT CAC CTG CGG CTG GTG CTG AAC CAG CCC CT - #A CAA TCC TAC CAC CAA 
317 
Phe His Leu Arg Leu Val Leu Asn Gln Pro Le - #u Gln Ser Tyr His Gln 
# 55 
- CTG AAA CTG GAA TTC AGC ACA GGG CCG AAT CC - #T AGC ATC GCC AAA CAC 
365 
Leu Lys Leu Glu Phe Ser Thr Gly Pro Asn Pr - #o Ser Ile Ala Lys His 
# 70 
- ACC CTG GTG GTG CTC GAC CCG AGG ACG CCC TC - #A GAC CAC TAC AAC TGG 
413 
Thr Leu Val Val Leu Asp Pro Arg Thr Pro Se - #r Asp His Tyr Asn Trp 
# 85 
- CAG GCA ACC CTT CAA AAT GAG TCT GGC AAA GA - #G GTC ACA GTG GCT GTC 
461 
Gln Ala Thr Leu Gln Asn Glu Ser Gly Lys Gl - #u Val Thr Val Ala Val 
#105 
- ACC AGT TCC CCC AAT GCC ATC CTG GGC AAG TA - #C CAA CTA AAC GTG AAA 
509 
Thr Ser Ser Pro Asn Ala Ile Leu Gly Lys Ty - #r Gln Leu Asn Val Lys 
# 120 
- ACT GGA AAC CAC ATC CTT AAG TCT GAA GAA AA - #C ATC CTA TAC CTT CTC 
557 
Thr Gly Asn His Ile Leu Lys Ser Glu Glu As - #n Ile Leu Tyr Leu Leu 
# 135 
- TTC AAC CCA TGG TGT AAA GAG GAC ATG GTT TT - #C ATG CCT GAT GAG GAC 
605 
Phe Asn Pro Trp Cys Lys Glu Asp Met Val Ph - #e Met Pro Asp Glu Asp 
# 150 
- GAG CGC AAA GAG TAC ATC CTC AAT GAC ACG GG - #C TGC CAT TAC GTG GGG 
653 
Glu Arg Lys Glu Tyr Ile Leu Asn Asp Thr Gl - #y Cys His Tyr Val Gly 
# 165 
- GCT GCC AGA AGT ATC AAA TGC AAA CCC TGG AA - #C TTT GGT CAG TTT GAG 
701 
Ala Ala Arg Ser Ile Lys Cys Lys Pro Trp As - #n Phe Gly Gln Phe Glu 
170 1 - #75 1 - #80 1 - 
#85 
- AAA AAT GTC CTG GAC TGC TGC ATT TCC CTG CT - #G ACT GAG AGC TCC CTC 
749 
Lys Asn Val Leu Asp Cys Cys Ile Ser Leu Le - #u Thr Glu Ser Ser Leu 
# 200 
- AAG CCC ACA GAT AGG AGG GAC CCC GTG CTG GT - #G TGC AGG GCC ATG TGT 
797 
Lys Pro Thr Asp Arg Arg Asp Pro Val Leu Va - #l Cys Arg Ala Met Cys 
# 215 
- GCT ATG ATG AGC TTT GAG AAA GGC CAG GGC GT - #G CTC ATT GGG AAT TGG 
845 
Ala Met Met Ser Phe Glu Lys Gly Gln Gly Va - #l Leu Ile Gly Asn Trp 
# 230 
- ACT GGG GAC TAC GAA GGT GGC ACA GCC CCA TA - #C AAG TGG ACA GGC AGT 
893 
Thr Gly Asp Tyr Glu Gly Gly Thr Ala Pro Ty - #r Lys Trp Thr Gly Ser 
# 245 
- GCC CCG ATC CTG CAG CAG TAC TAC AAC ACG AA - #G CAG GCT GTG TGC TTT 
941 
Ala Pro Ile Leu Gln Gln Tyr Tyr Asn Thr Ly - #s Gln Ala Val Cys Phe 
250 2 - #55 2 - #60 2 - 
#65 
- GGC CAG TGC TGG GTG TTT GCT GGG ATC CTG AC - #T ACA GTG CTG AGA GCG 
989 
Gly Gln Cys Trp Val Phe Ala Gly Ile Leu Th - #r Thr Val Leu Arg Ala 
# 280 
- TTG GGC ATC CCA GCA CGC AGT GTG ACA GGC TT - #C GAT TCA GCT CAC GAC 
1037 
Leu Gly Ile Pro Ala Arg Ser Val Thr Gly Ph - #e Asp Ser Ala His Asp 
# 295 
- ACA GAA AGG AAC CTC ACG GTG GAC ACC TAT GT - #G AAT GAG AAT GGC GAG 
1085 
Thr Glu Arg Asn Leu Thr Val Asp Thr Tyr Va - #l Asn Glu Asn Gly Glu 
# 310 
- AAA ATC ACC AGT ATG ACC CAC GAC TCT GTC TG - #G AAT TTC CAT GTG TGG 
1133 
Lys Ile Thr Ser Met Thr His Asp Ser Val Tr - #p Asn Phe His Val Trp 
# 325 
- ACG GAT GCC TGG ATG AAG CGA CCC TAC GAC GG - #C TGG CAG GCT GTG GAC 
1181 
Thr Asp Ala Trp Met Lys Arg Pro Tyr Asp Gl - #y Trp Gln Ala Val Asp 
330 3 - #35 3 - #40 3 - 
#45 
- GCA ACG CCG CAG GAG CGA AGC CAG GGT GTC TT - #C TGC TGT GGG CCA TCA 
1229 
Ala Thr Pro Gln Glu Arg Ser Gln Gly Val Ph - #e Cys Cys Gly Pro Ser 
# 360 
- CCA CTG ACC GCC ATC CGC AAA GGT GAC ATC TT - #T ATT GTC TAT GAC ACC 
1277 
Pro Leu Thr Ala Ile Arg Lys Gly Asp Ile Ph - #e Ile Val Tyr Asp Thr 
# 375 
- AGA TTC GTC TTC TCA GAA GTG AAT GGT GAC AG - #G CTC ATC TGG TTG GTG 
1325 
Arg Phe Val Phe Ser Glu Val Asn Gly Asp Ar - #g Leu Ile Trp Leu Val 
# 390 
- AAG ATG GTG AAT GGG CAG GAG GAG TTA CAC GT - #A ATT TCA ATG GAG ACC 
1373 
Lys Met Val Asn Gly Gln Glu Glu Leu His Va - #l Ile Ser Met Glu Thr 
# 405 
- ACA AGC ATC GGG AAA AAC ATC AGC ACC AAG GC - #A GTG GGC CAA GAC AGG 
1421 
Thr Ser Ile Gly Lys Asn Ile Ser Thr Lys Al - #a Val Gly Gln Asp Arg 
410 4 - #15 4 - #20 4 - 
#25 
- CGG AGA GAT ATC ACC TAT GAG TAC AAG TAT CC - #A GAA GGC TCC TCT GAG 
1469 
Arg Arg Asp Ile Thr Tyr Glu Tyr Lys Tyr Pr - #o Glu Gly Ser Ser Glu 
# 440 
- GAG AGG CAG GTC ATG GAT CAT GCC TTC CTC CT - #T CTC AGT TCT GAG AGG 
1517 
Glu Arg Gln Val Met Asp His Ala Phe Leu Le - #u Leu Ser Ser Glu Arg 
# 455 
- GAG CAC AGA CAG CCT GTA AAA GAG AAC TTT CT - #T CAC ATG TCG GTA CAA 
1565 
Glu His Arg Gln Pro Val Lys Glu Asn Phe Le - #u His Met Ser Val Gln 
# 470 
- TCA GAT GAT GTG CTG CTG GGA AAC TCT GTT AA - #T TTC ACC GTG ATT CTT 
1613 
Ser Asp Asp Val Leu Leu Gly Asn Ser Val As - #n Phe Thr Val Ile Leu 
# 485 
- AAA AGG AAG ACC GCT GCC CTA CAG AAT GTC AA - #C ATC TTG GGC TCC TTT 
1661 
Lys Arg Lys Thr Ala Ala Leu Gln Asn Val As - #n Ile Leu Gly Ser Phe 
490 4 - #95 5 - #00 5 - 
#05 
- GAA CTA CAG TTG TAC ACT GGC AAG AAG ATG GC - #A AAA CTG TGT GAC CTC 
1709 
Glu Leu Gln Leu Tyr Thr Gly Lys Lys Met Al - #a Lys Leu Cys Asp Leu 
# 520 
- AAT AAG ACC TCG CAG ATC CAA GGT CAA GTA TC - #A GAA GTG ACT CTG ACC 
1757 
Asn Lys Thr Ser Gln Ile Gln Gly Gln Val Se - #r Glu Val Thr Leu Thr 
# 535 
- TTG GAC TCC AAG ACC TAC ATC AAC AGC CTG GC - #T ATA TTA GAT GAT GAG 
1805 
Leu Asp Ser Lys Thr Tyr Ile Asn Ser Leu Al - #a Ile Leu Asp Asp Glu 
# 550 
- CCA GTT ATC AGA GGT TTC ATC ATT GCG GAA AT - #T GTG GAG TCT AAG GAA 
1853 
Pro Val Ile Arg Gly Phe Ile Ile Ala Glu Il - #e Val Glu Ser Lys Glu 
# 565 
- ATC ATG GCC TCT GAA GTA TTC ACG TCA AAC CA - #G TAC CCT GAG TTC TCT 
1901 
Ile Met Ala Ser Glu Val Phe Thr Ser Asn Gl - #n Tyr Pro Glu Phe Ser 
570 5 - #75 5 - #80 5 - 
#85 
- ATA GAG TTG CCT AAC ACA GGC AGA ATT GGC CA - #G CTA CTT GTC TGC AAT 
1949 
Ile Glu Leu Pro Asn Thr Gly Arg Ile Gly Gl - #n Leu Leu Val Cys Asn 
# 600 
- TGT ATC TTC AAG AAT ACC CTG GCC ATC CCT TT - #G ACT GAC GTC AAG TTC 
1997 
Cys Ile Phe Lys Asn Thr Leu Ala Ile Pro Le - #u Thr Asp Val Lys Phe 
# 615 
- TCT TTG GAA AGC CTG GGC ATC TCC TCA CTA CA - #G ACC TCT GAC CAT GGG 
2045 
Ser Leu Glu Ser Leu Gly Ile Ser Ser Leu Gl - #n Thr Ser Asp His Gly 
# 630 
- ACG GTG CAG CCT GGT GAG ACC ATC CAA TCC CA - #A ATA AAA TGC ACC CCA 
2093 
Thr Val Gln Pro Gly Glu Thr Ile Gln Ser Gl - #n Ile Lys Cys Thr Pro 
# 645 
- ATA AAA ACT GGA CCC AAG AAA TTT ATC GTC AA - #G TTA AGT TCC AAA CAA 
2141 
Ile Lys Thr Gly Pro Lys Lys Phe Ile Val Ly - #s Leu Ser Ser Lys Gln 
650 6 - #55 6 - #60 6 - 
#65 
- GTG AAA GAG ATT AAT GCT CAG AAG ATT GTT CT - #C ATC ACC AAG TAGCCTTGTC 
2193 
Val Lys Glu Ile Asn Ala Gln Lys Ile Val Le - #u Ile Thr Lys 
# 680 
- TGATGCTGTG GAGCCTTAGT TGAGATTTCA GCATTTCCTA CCTTGTGCTT AG - #CTTTCAGA 
2253 
- TTATGGATGA TTAAATTTGA TGACTTATAT GAGGGCAGAT TCAAGAGCCA GC - #AGGTCAAA 
2313 
- AAGGCCAACA CAACCATAAG CAGCCAGACC CACAAGGCCA GGTCCTGTGC TA - #TCACAGGG 
2373 
- TCACCTCTTT TACAGTTAGA AACACCAGCC GAGGCCACAG AATCCCATCC CT - #TTCCTGAG 
2433 
- TCATGGCCTC AAAAATCAGG GCCACCATTG TCTCAATTCA AATCCATAGA TT - #TCGAAGCC 
2493 
- ACAGAGCTCT TCCCTGGAGC AGCAGACTAT GGGCAGCCCA GTGCTGCCAC CT - #GCTGACGA 
2553 
- CCCTTGAGAA GCTGCCATAT CTTCAGGCCA TGGGTTCACC AGCCCTGAAG GC - #ACCTGTCA 
2613 
- ACTGGAGTGC TCTCTCAGCA CTGGGATGGG CCTGATAGAA GTGCATTCTC CT - #CCTATTGC 
2673 
- CTCCATTCTC CTCTCTCTAT CCCTGAAATC CAGGAAGTCC CTCTCCTGGT GC - #TCCAAGCA 
2733 
- GTTTGAAGCC CAATCTGCAA GGACATTTCT CAAGGGCCAT GTGGTTTTGC AG - #ACAACCCT 
2793 
- GTCCTCAGGC CTGAACTCAC CATAGAGACC CATGTCAGCA AACGGTGACC AG - #CAAATCCT 
2853 
- CTTCCCTTAT TCTAAAGCTG CCCCTTGGGA GACTCCAGGG AGAAGGCATT GC - #TTCCTCCC 
2913 
- TGGTGTGAAC TCTTTCTTTG GTATTCCATC CACTATCCTG GCAACTCAAG GC - #TGCTTCTG 
2973 
- TTAACTGAAG CCTGCTCCTT CTTGTTCTGC CCTCCAGAGA TTTGCTCAAA TG - #ATCAATAA 
3033 
# 3064 GAAT CCGCGGAATT C 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 679 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- Met Met Asp Ala Ser Lys Glu Leu Gln Val Le - #u His Ile Asp Phe Leu 
# 15 
- Asn Gln Asp Asn Ala Val Ser His His Thr Tr - #p Glu Phe Gln Thr Ser 
# 30 
- Ser Pro Val Phe Arg Arg Gly Gln Val Phe Hi - #s Leu Arg Leu Val Leu 
# 45 
- Asn Gln Pro Leu Gln Ser Tyr His Gln Leu Ly - #s Leu Glu Phe Ser Thr 
# 60 
- Gly Pro Asn Pro Ser Ile Ala Lys His Thr Le - #u Val Val Leu Asp Pro 
# 80 
- Arg Thr Pro Ser Asp His Tyr Asn Trp Gln Al - #a Thr Leu Gln Asn Glu 
# 95 
- Ser Gly Lys Glu Val Thr Val Ala Val Thr Se - #r Ser Pro Asn Ala Ile 
# 110 
- Leu Gly Lys Tyr Gln Leu Asn Val Lys Thr Gl - #y Asn His Ile Leu Lys 
# 125 
- Ser Glu Glu Asn Ile Leu Tyr Leu Leu Phe As - #n Pro Trp Cys Lys Glu 
# 140 
- Asp Met Val Phe Met Pro Asp Glu Asp Glu Ar - #g Lys Glu Tyr Ile Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Asn Asp Thr Gly Cys His Tyr Val Gly Ala Al - #a Arg Ser Ile Lys Cys 
# 175 
- Lys Pro Trp Asn Phe Gly Gln Phe Glu Lys As - #n Val Leu Asp Cys Cys 
# 190 
- Ile Ser Leu Leu Thr Glu Ser Ser Leu Lys Pr - #o Thr Asp Arg Arg Asp 
# 205 
- Pro Val Leu Val Cys Arg Ala Met Cys Ala Me - #t Met Ser Phe Glu Lys 
# 220 
- Gly Gln Gly Val Leu Ile Gly Asn Trp Thr Gl - #y Asp Tyr Glu Gly Gly 
225 2 - #30 2 - #35 2 - 
#40 
- Thr Ala Pro Tyr Lys Trp Thr Gly Ser Ala Pr - #o Ile Leu Gln Gln Tyr 
# 255 
- Tyr Asn Thr Lys Gln Ala Val Cys Phe Gly Gl - #n Cys Trp Val Phe Ala 
# 270 
- Gly Ile Leu Thr Thr Val Leu Arg Ala Leu Gl - #y Ile Pro Ala Arg Ser 
# 285 
- Val Thr Gly Phe Asp Ser Ala His Asp Thr Gl - #u Arg Asn Leu Thr Val 
# 300 
- Asp Thr Tyr Val Asn Glu Asn Gly Glu Lys Il - #e Thr Ser Met Thr His 
305 3 - #10 3 - #15 3 - 
#20 
- Asp Ser Val Trp Asn Phe His Val Trp Thr As - #p Ala Trp Met Lys Arg 
# 335 
- Pro Tyr Asp Gly Trp Gln Ala Val Asp Ala Th - #r Pro Gln Glu Arg Ser 
# 350 
- Gln Gly Val Phe Cys Cys Gly Pro Ser Pro Le - #u Thr Ala Ile Arg Lys 
# 365 
- Gly Asp Ile Phe Ile Val Tyr Asp Thr Arg Ph - #e Val Phe Ser Glu Val 
# 380 
- Asn Gly Asp Arg Leu Ile Trp Leu Val Lys Me - #t Val Asn Gly Gln Glu 
385 3 - #90 3 - #95 4 - 
#00 
- Glu Leu His Val Ile Ser Met Glu Thr Thr Se - #r Ile Gly Lys Asn Ile 
# 415 
- Ser Thr Lys Ala Val Gly Gln Asp Arg Arg Ar - #g Asp Ile Thr Tyr Glu 
# 430 
- Tyr Lys Tyr Pro Glu Gly Ser Ser Glu Glu Ar - #g Gln Val Met Asp His 
# 445 
- Ala Phe Leu Leu Leu Ser Ser Glu Arg Glu Hi - #s Arg Gln Pro Val Lys 
# 460 
- Glu Asn Phe Leu His Met Ser Val Gln Ser As - #p Asp Val Leu Leu Gly 
465 4 - #70 4 - #75 4 - 
#80 
- Asn Ser Val Asn Phe Thr Val Ile Leu Lys Ar - #g Lys Thr Ala Ala Leu 
# 495 
- Gln Asn Val Asn Ile Leu Gly Ser Phe Glu Le - #u Gln Leu Tyr Thr Gly 
# 510 
- Lys Lys Met Ala Lys Leu Cys Asp Leu Asn Ly - #s Thr Ser Gln Ile Gln 
# 525 
- Gly Gln Val Ser Glu Val Thr Leu Thr Leu As - #p Ser Lys Thr Tyr Ile 
# 540 
- Asn Ser Leu Ala Ile Leu Asp Asp Glu Pro Va - #l Ile Arg Gly Phe Ile 
545 5 - #50 5 - #55 5 - 
#60 
- Ile Ala Glu Ile Val Glu Ser Lys Glu Ile Me - #t Ala Ser Glu Val Phe 
# 575 
- Thr Ser Asn Gln Tyr Pro Glu Phe Ser Ile Gl - #u Leu Pro Asn Thr Gly 
# 590 
- Arg Ile Gly Gln Leu Leu Val Cys Asn Cys Il - #e Phe Lys Asn Thr Leu 
# 605 
- Ala Ile Pro Leu Thr Asp Val Lys Phe Ser Le - #u Glu Ser Leu Gly Ile 
# 620 
- Ser Ser Leu Gln Thr Ser Asp His Gly Thr Va - #l Gln Pro Gly Glu Thr 
625 6 - #30 6 - #35 6 - 
#40 
- Ile Gln Ser Gln Ile Lys Cys Thr Pro Ile Ly - #s Thr Gly Pro Lys Lys 
# 655 
- Phe Ile Val Lys Leu Ser Ser Lys Gln Val Ly - #s Glu Ile Asn Ala Gln 
# 670 
- Lys Ile Val Leu Ile Thr Lys 
675 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC1157 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
#21 TTGC A 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC1158 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
#21 TTTA G 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 15 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4048 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
# 15 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 24 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4362 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
# 24CCAT ATGG 
- (2) INFORMATION FOR SEQ ID NO:20: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 24 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC4363 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
# 24CCAA CTGG 
- (2) INFORMATION FOR SEQ ID NO:21: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: ZC5509 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
#21 ACCA T 
- (2) INFORMATION FOR SEQ ID NO:22: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 527 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: plTG 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- TATGGTCAGT GTTGGGTTTT TGCTGGGACC CTCAACACAG CGCTGCGGTC TT - #TGGGGATT 
60 
- CCTTCCCGGG TGATCACCAA CTTCAACTCA GCTCATGACA CAGACCGAAA TC - #TCAGTGTG 
120 
- GATGTGTACT ACGACCCCAT GGGAAACCCC CTGGACAAGG GTAGTGATAG CG - #TATGGAAT 
180 
- TTCCATGTCT GGAATGAAGG CTGGTTTGTG AGGTCTGACC TGGGCCCCTC GT - #ACGGTGGA 
240 
- TGGCAGGTGT TGGATGCTAC CCCGCAGGAA AGAAGCCAAG GGGTGTTCCA GT - #GCGGCCCC 
300 
- GCTTCGGTCA TTGGTGTTCG AGAGGGTGAT GTGCAGCTGA ACTTCGACAT GC - #CCTTTATC 
360 
- TTCGCGGAGG TTAATGCCGA CCGCATCACC TGGCTGTACG ACAACACCAC TG - #GCAAACAG 
420 
- TGGAAGAATT CCGTGAACAG TCACACCATT GGCAGGTACA TCAGCACCAA GG - #CGGTGGGC 
480 
# 527ACGT CACGGACAAG TACAAGCTCC ACGAGGG 
__________________________________________________________________________