Purified coprinus laccases and nucleic acids encoding the same

The present invention relates to polypeptides having laccase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to polypeptides having laccase activity and 
isolated nucleic acid sequences encoding the polypeptides. The invention 
also relates to nucleic acid constructs, vectors, and host cells 
comprising the nucleic acid sequences as well as methods for producing the 
polypeptides. 
2. Description of the Related Art 
Laccases (benzenediol:oxygen oxidoreductases) are multi-copper containing 
enzymes that catalyze the oxidation of phenolics. Laccase-mediated 
oxidations result in the production of aryloxy-radical intermediates from 
suitable phenolic substrate; the ultimate coupling of the intermediates so 
produced provides a combination of dimeric, oligomeric, and polymeric 
reaction products. Such reactions are important in nature in biosynthetic 
pathways which lead to the formation of melanin, alkaloids, toxins, 
lignins, and humic acids. Laccases are produced by a wide variety of 
fungi, including ascomycetes such as Aspergillus, Neurospora, and 
Podospora, the deuteromycete Botrytis, and basidiomycetes such as 
Collybia, Fomes, Lentinus, Pleurotus, Trametes, and perfect forms of 
Rhizoctonia. Laccase exhibits a wide range of substrate specificity, and 
each different fungal laccase usually differs only quantitatively from 
others in its ability to oxidize phenolic substrates. Because of the 
substrate diversity, laccases generally have found many potential 
industrial applications. Among these are lignin modification, paper 
strengthening, dye transfer inhibition in detergents, phenol 
polymerization, juice manufacture, phenol resin production, and waste 
water treatment. 
Although the catalytic capabilities are similar, laccases made by different 
fungal species do have different temperature and pH optima. A number of 
these fungal laccases have been isolated, and the genes for several of 
these have been cloned. For example, Choi et al. (1992, Mol. Plant-Microbe 
Interactions 5: 119-128) describe the molecular characterization and 
cloning of the gene encoding the laccase of the chestnut blight fungus 
Cryphonectria parasitica. Kojima et al. (1990, Journal of Biological 
Chemistry 265: 15224-15230; JP 2-238885) provide a description of two 
allelic forms of the laccase of the white-rot basidiomycete Coriolus 
hirsutus. Germann and Lerch (1985, Experientia 41: 801; 1986, Proceedings 
of the National Academy of Sciences USA 83: 8854-8858) have reported the 
cloning and partial sequencing of the Neurospora crassa laccase gene. 
Saloheimo et al. (1985, Journal of General Microbiology 137:1537-1544; WO 
92/01046) have disclosed a structural analysis of the laccase gene from 
the fungus Phlebia radiata. 
It is an object of the present invention to provide polypeptides having 
laccase activity and nucleic acid constructs encoding these polypeptides. 
SUMMARY OF THE INVENTION 
The present invention relates to isolated polypeptides having laccase 
activity, obtained from a Coprinus strain. The present invention further 
relates to isolated polypeptides having laccase activity which have: (a) a 
pH optimum in the range of about 5 to about 9 at 20.degree. C. using 
syringaldazine as a substrate; and (b) an isoelectric point in the range 
of about 3.7 to about 4.0. The present invention also relates to isolated 
polypeptides which have an amino acid sequence which has at least 65% 
identity with the amino acid sequence set forth in SEQ ID NO:27, SEQ ID 
NO:29, or SEQ ID NO:33. 
The present invention further relates to isolated nucleic acid sequences 
encoding the polypeptides and to nucleic acid constructs, vectors, and 
host cells comprising the nucleic acid sequences as well as methods for 
producing the polypeptides.

DETAILED DESCRIPTION OF THE INVENTION 
Polypeptides Having Laccase Activity 
The present invention relates to isolated polypeptides having laccase 
activity (hereinafter "polypeptides"), obtained from a Coprinus strain. 
The present invention further relates to isolated polypeptides having 
laccase activity which have: 
(a) a pH optimum in the range of about 5 to about 9 at 20.degree. C. using 
syringaldazine as a substrate; and 
(b) an isoelectric point in the range of about 3.7 to about 4.0. 
The polypeptides preferably have a molecular weight of about 63 kDa (using 
SDS-PAGE). In another embodiment, the polypeptides are obtained from a 
strain of the family Coprinaceae, preferably a Coprinus strain, and more 
preferably a Coprinus cinereus strain, e.g., Coprinus cinereus IFO 8371 or 
a mutant strain thereof. In a most preferred embodiment, the polypeptide 
has the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29, or 
SEQ ID NO:33. 
The present invention also relates to polypeptides obtained from 
microorganisms which are synonyms of Coprinus as defined by, for example, 
Webster, 1980, In Introduction to the Fungi, Second Edition, Cambridge 
University Press, New York. Strains of Coprinus are readily accessible to 
the public in a number of culture collections, such as the American Type 
Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und 
Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and 
Agricultural Research Service Patent Culture Collection, Northern Regional 
Research Center (NRRL), e.g., from the American Type Culture Collection, 
ATCC 12890, 36519, 38628, 42727, 48566 (Coprinus cinereus); 15744 
(Coprinus clastophyllus); 12640, 22314 (Coprinus comatus); 46457, 46972 
(Coprinus congregatus); 48096 (Coprinus cothurnatus); 48098 (Coprinus 
curtus); 46973 (Coprinus disseminatus); 26829 (Coprinus domesticus); 48100 
(Coprinus ephemeroides); 36567 (Coprinus fimentarius); 48097 (Coprinus 
gonophyllus); 20122 (Coprinus micaceus); from the Institute for 
Fermentation (IFO, Osaka, Japan), IFO 8371, 30116 (Coprinus cinereus); 
from Centraalbureau voor Schimmelcultures (CBS; Netherlands) CBS 147.39, 
148.39, 175.51 (Coprinus angulatus), 147.29 (Coprinus astramentarius); 
143,39 (Coprinus auricomus); 185.52 (Coprinus callinus); 159.39, 338.69 
(Coprinus cinereus); 631.95 (Coprinus comatus); 629.95 (Coprinus friesii); 
627.95 (Coprinus plicatilis); 628.95 (Psathyrella condolleana); 630.95 
(Panaeolus papilionaceus) from Deutsche Sammlung von Mikroorganismenn und 
Zellkulturen (DSM; Germany) DSM 888 (Coprinus radians); 4916 (Csprinus 
xanthothrix); 3341 (Coprinus sterquilinius). The invention also embraces 
polypeptides having laccase activity of other fungi and other members of 
the family Coprinaceae, for example, laccases from the genera Podaxis, 
Montagnea, Macrometrula, Psathyrella, Panaeolina, Panaeolus, Copelandia, 
Anellaria, Limnoperdon, Panaelopsis, and Polyplocium. 
For purposes of the present invention, the term "obtained from" as used 
herein in connection with a given source shall mean that the polypeptide 
is produced by the source or by a cell in which a gene from the source has 
been inserted. 
The present invention also relates to polypeptides which are encoded by 
nucleic acid sequences which are capable of hybridizing under standard 
conditions with an oligonucleotide probe which hybridizes under the same 
conditions with the nucleic acid sequence set forth in SEQ ID NO:26, SEQ 
ID NO:28, SEQ ID NO:32 as well as a complementary strand thereof or a 
subsequence thereof (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, 
Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, 
N.Y.). Hybridization indicates that the analogous nucleic acid sequence 
hybridizes to the oligonucleotide probe corresponding to the polypeptide 
encoding part of the nucleic acid sequence shown in SEQ ID NO:26, SEQ ID 
NO:28, SEQ ID NO:32, or a subsequence thereof, under medium to high 
stringency conditions (for example, prehybridization and hybridization at 
42.degree. C. in 5.times. SSPE, 0.3% SDS, 200 .mu.g/ml sheared and 
denatured salmon sperm DNA, and either 35 or 50% formamide for medium and 
high stringencies, respectively), following standard Southern blotting 
procedures. 
The nucleic acid sequences set forth in SEQ ID NO:26, SEQ ID NO:28, SEQ ID 
NO:32, or subsequences thereof may be used to identify and clone DNA 
encoding laccases from other strains of different genera or species 
according to methods well known in the art. Thus, a genomic or cDNA 
library prepared from such other organisms may be screened for DNA which 
hybridizes with SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, or subsequences 
thereof. Genomic or other DNA from such other organisms may be separated 
by agarose or polyacrylamide gel electrophoresis, or other separation 
techniques. DNA from the libraries or the separated DNA may be transferred 
to and immobilized on nitrocellulose or other suitable carrier material. 
In order to identify clones or DNA which are homologous with SEQ ID NO:26, 
SEQ ID NO:28, SEQ ID NO:32, or subsequences thereof, the carrier material 
is used in a Southern blot in which the carrier material is finally washed 
three times for 30 minutes each using 0.2.times. SSC, 0.1% SDS at 
40.degree. C., more preferably not higher than 45.degree. C., more 
preferably not higher than 50.degree. C., more preferably not higher than 
55.degree. C., even more preferably not higher than 60.degree. C., 
especially not higher than 65.degree. C. Molecules to which the 
oligonucleotide probe hybridizes under these conditions are detected using 
a X-ray film. 
The present invention also relates to polypeptides which have an amino acid 
sequence which has a degree of identity to the amino acid sequence set 
forth in SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33 of at least about 
65%, preferably about 70%, preferably about 75%, preferably about 80%, 
preferably about 85%, more preferably about 90%, even more preferably 
about 95%, and most preferably about 97%, which qualitatively retain the 
activity of the polypeptides (hereinafter "homologous polypeptides"). In a 
preferred embodiment, the homologous polypeptides have an amino acid 
sequence which differs by five amino acids, preferably by four amino 
acids, more preferably by three amino acids, even more preferably by two 
amino acids, and most preferably by one amino acid from the amino acid 
sequence set forth SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. The degree 
of identity between two or more amino acid sequences may be determined by 
means of computer programs known in the art such as GAP provided in the 
GCG program package (Needleman and Wunsch, 1970, Journal of Molecular 
Biology 48:443-453). For purposes of determining the degree of identity 
between two amino acid sequences for the present invention, the Clustal 
method (DNASTAR, Inc., Madison, Wis.) is used with an identity table, a 
gap penalty of 10, and a gap length of 10. 
The amino acid sequences of the homologous polypeptides differ from the 
amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29, or SEQ ID 
NO:33 by an insertion or deletion of one or more amino acid residues 
and/or the substitution of one or more amino acid residues by different 
amino acid residues. Preferably, amino acid changes are of a minor nature, 
that is conservative amino acid substitutions that do not significantly 
affect the folding and/or activity of the protein; small deletions, 
typically of one to about 30 amino acids; small amino- or 
carboxyl-terminal extensions, such as an amino-terminal methionine 
residue; a small linker peptide of up to about 20-25 residues; or a small 
extension that facilitates purification by changing net charge or another 
function, such as a poly-histidine tract, an antigenic epitope or a 
binding domain. 
Examples of conservative substitutions are within the group of basic amino 
acids (such as arginine, lysine and histidine), acidic amino acids (such 
as glutamic acid and aspartic acid), polar amino acids (such as glutamine 
and asparagine), hydrophobic amino acids (such as leucine, isoleucine and 
valine), aromatic amino acids (such as phenylalanine, tryptophan and 
tyrosine) and small amino acids (such as glycine, alanine, serine, 
threonine and methionine). Amino acid substitutions which do not generally 
alter the specific activity are known in the art and are described, e.g., 
by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New 
York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, 
Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, 
Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these 
in reverse. 
The present invention also relates to polypeptides having immunochemical 
identity or partial immunochemical identity to the polypeptides having 
laccase activity which are native to Coprinus cinereus IFO 8371. A 
polypeptide having immunochemical identity to the polypeptide native to 
Coprinus cinereus IFO 8371 means that an antiserum containing antibodies 
against the antigens of the native polypeptide reacts with the antigens of 
the other polypeptide in an identical fashion such as total fusion of 
precipitates, identical precipitate morphology, and/or identical 
electrophoretic mobility using a specific immunochemical technique. A 
further explanation of immunochemical identity is described by Axelsen, 
Bock, and Kroll, In N. H. Axelsen, J. Kr.o slashed.ll, and B. Weeks, 
editors, A Manual of Quantitative Immunoelectrophoresis, Blackwell 
Scientific Publications, 1973, Chapter 10. Partial immunochemical identity 
means that an antiserum containing antibodies against the antigens of the 
native polypeptide reacts with the antigens of the other polypeptide in an 
partially identical fashion such as partial fusion of precipitates, 
partially identical precipitate morphology, and/or partially identical 
electrophoretic mobility using a specific immunochemical technique. A 
further explanation of partial immunochemical identity is described by 
Bock and Axelsen, In N. H. Axelsen, J. Kr.o slashed.ll, and B. Weeks, 
editors, A Manual of Quantitative Immunoelectrophoresis, Blackwell 
Scientific Publications, 1973, Chapter 11. The immunochemical properties 
are determined by immunological cross-reaction identity tests by the 
well-known Ouchterlony double immunodiffusion procedure. Specifically, an 
antiserum against the polypeptide of the invention is raised by immunizing 
rabbits (or other rodents) according to the procedure described by Harboe 
and Ingild, In N. H. Axelsen, J. Kr.o slashed.ll, and B. Weeks, editors, A 
Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific 
Publications, 1973, Chapter 23, or Johnstone and Thorpe, Immunochemistry 
in Practice, Blackwell Scientific Publications, 1982 (more specifically 
pages 27-31). Monoclonal antibodies may be prepared, e.g., according to 
the methods of E. Harlow and D. Lane, editors, 1988, Antibodies, A 
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 
Purified immunoglobulins may be obtained from the antiserum, e.g., by 
ammonium sulfate precipitation, followed by dialysis and ion exchange 
chromatography (e.g., DEAE-Sephadex). 
Homologous polypeptides and polypeptides having identical or partially 
identical immunological properties may be obtained from microorganisms of 
any genus, preferably from a bacterial or fungal source. Sources for 
homologous genes are strains of the family Coprinaceae, preferably of the 
genus Coprinus and species thereof available in public depositories. 
Furthermore, homologous genes may be identified and obtained from other 
sources including microorganisms isolated from nature (e.g., soil, 
composts, water, etc.) using the above-mentioned probes. Techniques for 
isolating microorganisms from natural habitats are well known in the art. 
The nucleic acid sequence may then be derived by similarly screening a 
cDNA library of another microorganism. Once a nucleic acid sequence 
encoding a polypeptide has been detected with the probe(s), the sequence 
may be isolated or cloned by utilizing techniques which are known to those 
of ordinary skill in the art (see, e.g., Sambrook et al., supra). 
As defined herein, an "isolated" polypeptide is a polypeptide which is 
essentially free of other non-laccase polypeptides, e.g., at least about 
20% pure, preferably at least about 40% pure, more preferably about 60% 
pure, even more preferably about 80% pure, most preferably about 90% pure, 
and even most preferably about 95% pure, as determined by SDS-PAGE. 
Nucleic Acid Sequences 
The present invention also relates to isolated nucleic acid sequences 
obtained from a Coprinus strain, which encode a polypeptide of the present 
invention. In a preferred embodiment, the nucleic acid sequence encodes a 
polypeptide obtained from Coprinus cinereus and in a more preferred 
embodiment, the nucleic acid sequence is obtained from Coprinus cinereus 
IFO 8371, e.g., the nucleic acid sequence set forth in SEQ ID NO:26, SEQ 
ID NO:28, or SEQ ID NO:32. The present invention also encompasses nucleic 
acid sequences which encode a polypeptide having the amino acid sequence 
set forth in SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33, which differ 
from SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:32, respectively, by virtue 
of the degeneracy of the genetic code. 
As described above, the nucleic acid sequences may be obtained from 
microorganisms which are synonyms of Coprinus as defined by Webster, 1980, 
supra. 
The techniques used to isolate or clone a nucleic acid sequence encoding a 
polypeptide are known in the art and include isolation from genomic DNA, 
preparation from cDNA, or a combination thereof. The cloning of the 
nucleic sequences of the present invention from such genomic DNA can be 
effected, e.g., by using the well known polymerase chain reaction (PCR). 
See, e.g., Innis et al., 1990, A Guide to Methods and Application, 
Academic Press, New York. The nucleic acid sequence may be cloned from a 
strain of the Coprinus producing the polypeptide, or another or related 
organism and thus, for example, may be an allelic or species variant of 
the polypeptide encoding region of the nucleic acid sequence. 
The term "isolated nucleic acid sequence" as used herein refers to a 
nucleic acid sequence encoding a polypeptide of the present invention 
which is isolated by standard cloning procedures used in genetic 
engineering to relocate the nucleic acid sequence from its natural 
location to a different site where it will be reproduced. The cloning 
procedures may involve excision and isolation of a desired nucleic acid 
fragment comprising the nucleic acid sequence encoding the polypeptide, 
insertion of the fragment into a vector molecule, and incorporation of the 
recombinant vector into a host cell where multiple copies or clones of the 
nucleic acid sequence will be replicated. The nucleic acid sequence may be 
of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any 
combinations thereof. 
The present invention also relates to nucleic acid sequences which have a 
nucleic acid sequence which has a degree of identity to the nucleic acid 
sequence set forth in SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, or 
subsequences thereof of at least about 65%, preferably about 70%, 
preferably about 75%, preferably about 80%, preferably about 85%, more 
preferably about 90%, even more preferably about 95%, and most preferably 
about 97%, which encode an active polypeptide. The degree of identity 
between two nucleic acid sequences may be determined by means of computer 
programs known in the art such as GAP provided in the GCG program package 
(Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453). For 
purposes of determining the degree of identity between two nucleic acid 
sequences for the present invention, the Clustal method (DNASTAR, Inc., 
Madison, Wis.) is used with an identity table, a gap penalty of 10, and a 
gap length of 10. 
Modification of the nucleic acid sequence encoding the polypeptide may be 
necessary for the synthesis of polypeptides substantially similar to the 
polypeptide. The term "substantially similar" to the polypeptide refers to 
non-naturally occurring forms of the polypeptide. These polypeptides may 
differ in some engineered way from the polypeptide isolated from its 
native source. For example, it may be of interest to synthesize variants 
of the polypeptide where the variants differ in specific activity, 
thermostability, pH optimum, or the like using, e.g., site-directed 
mutagenesis. The analogous sequence may be constructed on the basis of the 
nucleic acid sequence presented as the polypeptide encoding part of SEQ ID 
NO:26, SEQ ID NO:28, or SEQ ID NO:32, e.g., a sub-sequence thereof, and/or 
by introduction of nucleotide substitutions which do not give rise to 
another amino acid sequence of the polypeptide encoded by the nucleic acid 
sequence, but which corresponds to the codon usage of the host organism 
intended for production of the enzyme, or by introduction of nucleotide 
substitutions which may give rise to a different amino acid sequence. For 
a general description of nucleotide substitution, see, e.g., Ford et al., 
1991, Protein Expression and Purification 2:95-107. 
It will be apparent to those skilled in the art that such substitutions can 
be made outside the regions critical to the function of the molecule and 
still result in an active polypeptide. Amino acid residues essential to 
the activity of the polypeptide encoded by the isolated nucleic acid 
sequence of the invention, and therefore preferably not subject to 
substitution, may be identified according to procedures known in the art, 
such as site-directed mutagenesis or alanine-scanning mutagenesis (see, 
e.g., Cunningham and Wells, 1989, Science 244:1081-1085). In the latter 
technique mutations are introduced at every residue in the molecule, and 
the resultant mutant molecules are tested for laccase activity to identify 
amino acid residues that are critical to the activity of the molecule. 
Sites of substrate-enzyme interaction can also be determined by analysis 
of crystal structure as determined by such techniques as nuclear magnetic 
resonance analysis, crystallography or photoaffinity labelling (see, e.g., 
de Vos et al., 1992, Science 255, 306-312; Smith et al., 1992, Journal of 
Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Letters 309, 
59-64). 
Polypeptides of the present invention also include fused polypeptides in 
which another polypeptide is fused at the N-terminus or the C-terminus of 
the polypeptide or fragment thereof. A fused polypeptide is produced by 
fusing a nucleic acid sequence (or a portion thereof) encoding another 
polypeptide to a nucleic acid sequence (or a portion thereof) of the 
present invention. Techniques for producing fusion polypeptides are known 
in the art, and include, ligating the coding sequences encoding the 
polypeptides so that they are in frame and that expression of the fused 
polypeptide is under control of the same promoter(s) and terminator. 
The present invention also relates to nucleic acid sequences which are 
capable of hybridizing under standard conditions with an oligonucleotide 
probe which hybridizes under the same conditions with the nucleic acid 
sequence set forth in SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, a 
subsequence thereof, or its complementary strand (Sambrook et al., supra). 
Hybridization indicates that the analogous nucleic acid sequence 
hybridizes to the oligonucleotide probe corresponding to the polypeptide 
encoding part of the nucleic acid sequence shown in SEQ ID NO:26, SEQ ID 
NO:28, or SEQ ID NO:32 under standard conditions. 
The amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29, or SEQ ID 
NO:33 or a partial amino acid sequence thereof may be used to design an 
oligonucleotide probe, or a gene encoding a polypeptide of the present 
invention or a subsequence thereof can also be used as a probe, to isolate 
homologous genes of any genus or species. In particular, such probes can 
be used for hybridization with the genomic or cDNA of the genus or species 
of interest, following standard Southern blotting procedures, in order to 
identify and isolate the corresponding gene therein. Such probes can be 
considerably shorter than the entire sequence, but should be at least 15, 
preferably at least 25, and more preferably at least 40 nucleotides in 
length. Longer probes, preferably no more than 1200 nucleotides in length, 
can also be used. Both DNA and RNA probes can be used. The probes are 
typically labeled for detecting the corresponding gene (for example, with 
.sup.32 P, .sup.1 H, biotin, or avidin). A PCR reaction using the 
degenerate probes mentioned herein and genomic DNA or first-strand cDNA 
from a Coprinus cinereus can also yield a Coprinus cinereus 
laccase-specific product which can then be used as a probe to clone the 
corresponding genomic or cDNA. 
Nucleic Acid Constructs 
The present invention also relates to nucleic acid constructs comprising a 
nucleic acid sequence of the present invention operably linked to one or 
more control sequences capable of directing the expression of the coding 
sequence in a suitable host cell under conditions compatible with the 
control sequences. 
"Nucleic acid construct" is defined herein as a nucleic acid molecule, 
either single- or double-stranded, which is isolated from a naturally 
occurring gene or which has been modified to contain segments of nucleic 
acid which are combined and juxtaposed in a manner which would not 
otherwise exist in nature. The term nucleic acid construct may be 
synonymous with the term expression cassette when the nucleic acid 
construct contains all the control sequences required for expression of a 
coding sequence of the present invention. The term "coding sequence" as 
defined herein is a sequence which is transcribed into mRNA and translated 
into a polypeptide of the present invention when placed under the control 
of the above mentioned control sequences. The boundaries of the coding 
sequence are generally determined by a translation start codon ATG at the 
5'-terminus and a translation stop codon at the 3'-terminus. A coding 
sequence can include, but is not limited to, DNA, cDNA, and recombinant 
nucleic acid sequences. 
An isolated nucleic acid sequence encoding a polypeptide of the present 
invention may be manipulated in a variety of ways to provide for 
expression of the polypeptide. Manipulation of the nucleic acid sequence 
encoding a polypeptide prior to its insertion into a vector may be 
desirable or necessary depending on the expression vector. The techniques 
for modifying nucleic acid sequences utilizing cloning methods are well 
known in the art. 
The term "control sequences" is defined herein to include all components 
which are necessary or advantageous for expression of the coding sequence 
of the nucleic acid sequence. Each control sequence may be native or 
foreign to the nucleic acid sequence encoding the polypeptide. Such 
control sequences include, but are not limited to, a leader, a 
polyadenylation sequence, a propeptide sequence, a promoter, a signal 
sequence, and a transcription terminator. At a minimum, the control 
sequences include a promoter, and transcriptional and translational stop 
signals. The control sequences may be provided with linkers for the 
purpose of introducing specific restriction sites facilitating ligation of 
the control sequences with the coding region of the nucleic acid sequence 
encoding a polypeptide. 
The control sequence may be an appropriate promoter sequence, a nucleic 
acid sequence which is recognized by a host cell for expression of the 
nucleic acid sequence. The promoter sequence contains transcription and 
translation control sequences which mediate the expression of the 
polypeptide. The promoter may be any nucleic acid sequence which shows 
transcriptional activity in the host cell of choice and may be obtained 
from genes encoding extracellular or intracellular polypeptides either 
homologous or heterologous to the host cell. 
Examples of suitable promoters for directing the transcription of the 
nucleic acid constructs of the present invention, especially in a 
bacterial host cell, are the promoters obtained from the E. coli lac 
operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus 
subtilis levansucrase gene (sacB), the Bacillus licheniformis 
alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic 
amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene 
(amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus 
subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene 
(Villa-Kamaroff et al., 1978, Proceedings of the National Academy of 
Sciences USA 75:3727-3731), as well as the tac gene (DeBoer et al., 1983, 
Proceedings of the National Academy of Sciences USA 80:21-25). Further 
promoters are described in "Useful proteins from recombinant bacteria" in 
Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra. 
Examples of suitable promoters for directing the transcription of the 
nucleic acid constructs of the present invention in a filamentous fungal 
host cell are promoters obtained from the genes encoding Aspergillus 
oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus 
niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, 
Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor 
miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae 
triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium 
oxysporum trypsin-like protease (as described in U.S. Pat. No. 4,288,627, 
which is incorporated herein by reference), and hybrids thereof. 
Particularly preferred promoters for use in filamentous fungal host cells 
are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes 
encoding Aspergillus niger neutral .alpha.-amylase and Aspergillus oryzae 
triose phosphate isomerase), and glaA promoters. 
In a yeast host, useful promoters are obtained from the Saccharomyces 
cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae 
galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol 
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), 
and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other 
useful promoters for yeast host cells are described by Romanos et al., 
1992, Yeast 8:423-488. In a mammalian host cell, useful promoters include 
viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma 
virus (RSV), adenovirus, and bovine papilloma virus (BPV). 
The control sequence may also be a suitable transcription terminator 
sequence, a sequence recognized by a host cell to terminate transcription. 
The terminator sequence is operably linked to the 3' terminus of the 
nucleic acid sequence encoding the polypeptide. Any terminator which is 
functional in the host cell of choice may be used in the present 
invention. 
Preferred terminators for filamentous fungal host cells are obtained from 
the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger 
glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus 
niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. 
Preferred terminators for yeast host cells are obtained from the genes 
encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae 
cytochrome C (CYC1), or Saccharomyces cerevisiae 
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for 
yeast host cells are described by Romanos et al., 1992, supra. Terminator 
sequences are well known in the art for mammalian host cells. 
The control sequence may also be a suitable leader sequence, a 
nontranslated region of a mRNA which is important for translation by the 
host cell. The leader sequence is operably linked to the 5' terminus of 
the nucleic acid sequence encoding the polypeptide. Any leader sequence 
which is functional in the host cell of choice may be used in the present 
invention. 
Preferred leaders for filamentous fungal host cells are obtained from the 
genes encoding Aspergillus oryzae TAKA amylase and Aspergillus oryzae 
triose phosphate isomerase. 
Suitable leaders for yeast host cells are obtained from the Saccharomyces 
cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae 
3-phosphoglycerate kinase gene, the Saccharomyces cerevisiae alpha-factor, 
and the Saccharomyces cerevisiae alcohol 
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP). 
The control sequence may also be a polyadenylation sequence, a sequence 
which is operably linked to the 3' terminus of the nucleic acid sequence 
and which, when transcribed, is recognized by the host cell as a signal to 
add polyadenosine residues to transcribed mRNA. Any polyadenylation 
sequence which is functional in the host cell of choice may be used in the 
present invention. 
Preferred polyadenylation sequences for filamentous fungal host cells are 
obtained from the genes encoding Aspergillus oryzae TAKA amylase, 
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate 
synthase, and Aspergillus niger alpha-glucosidase. 
Useful polyadenylation sequences for yeast host cells are described by Guo 
and Sherman, 1995, Molecular Cellular Biology 15:5983-5990. 
Polyadenylation sequences are well known in the art for mammalian host 
cells. 
The control sequence may also be a signal peptide coding region, which 
codes for an amino acid sequence linked to the amino terminus of the 
polypeptide which can direct the expressed polypeptide into the cell's 
secretory pathway. The 5' end of the coding sequence of the nucleic acid 
sequence may inherently contain a signal peptide coding region naturally 
linked in translation reading frame with the segment of the coding region 
which encodes the secreted polypeptide. Alternatively, the 5' end of the 
coding sequence may contain a signal peptide coding region which is 
foreign to that portion of the coding sequence which encodes the secreted 
polypeptide. The foreign signal peptide coding region may be required 
where the coding sequence does not normally contain a signal peptide 
coding region. Alternatively, the foreign signal peptide coding region may 
simply replace the natural signal peptide coding region in order to obtain 
enhanced secretion of the laccase relative to the natural signal peptide 
coding region normally associated with the coding sequence. The signal 
peptide coding region may be obtained from a glucoamylase or an amylase 
gene from an Aspergillus species, a lipase or proteinase gene from a 
Rhizomucor species, the gene for the .alpha.-factor from Saccharomyces 
cerevisiae, an amylase or a protease gene from a Bacillus species, or the 
calf preprochymosin gene. However, any signal peptide coding region 
capable of directing the expressed laccase into the secretory pathway of a 
host cell of choice may be used in the present invention. 
An effective signal peptide coding region for bacterial host cells is the 
signal peptide coding region obtained from the maltogenic amylase gene 
from Bacillus NCEB 11837, the Bacillus stearothermophilus alpha-amylase 
gene, the Bacillus licheniformis subtilisin gene, the Bacillus 
licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral 
proteases genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA gene. 
Further signal peptides are described by Simonen and Palva, 1993, 
Microbiological Reviews 57:109-137. 
An effective signal peptide coding region for filamentous fungal host cells 
is the signal peptide coding region obtained from Aspergillus oryzae TAKA 
amylase gene, Aspergillus niger neutral amylase gene, the Rhizomucor 
miehei aspartic proteinase gene, the Humicola lanuginosa cellulase gene, 
or the Rhizomucor miehei lipase gene. 
Useful signal peptides for yeast host cells are obtained from the genes for 
Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae 
invertase. Other useful signal peptide coding regions are described by 
Romanos et al., 1992, supra. 
The control sequence may also be a propeptide coding region, which codes 
for an amino acid sequence positioned at the amino terminus of a 
polypeptide. The resultant polypeptide is known as a proenzyme or 
propolypeptide (or a zymogen in some cases). A propolypeptide is generally 
inactive and can be converted to a mature active polypeptide by catalytic 
or autocatalytic cleavage of the propeptide from the propolypeptide. The 
propeptide coding region may be obtained from the Bacillus subtilis 
alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene 
(nprT), the Saccharomyces cerevisiae alpha-factor gene, or the 
Myceliophthora thermophilum laccase gene (WO 95/33836). 
The nucleic acid constructs of the present invention may also comprise one 
or more nucleic acid sequences which encode one or more factors that are 
advantageous in the expression of the polypeptide, e.g., an activator 
(e.g., a trans-acting factor), a chaperone, and a processing protease. Any 
factor that is functional in the host cell of choice may be used in the 
present invention. The nucleic acids encoding one or more of these factors 
are not necessarily in tandem with the nucleic acid sequence encoding the 
polypeptide. 
An activator is a protein which activates transcription of a nucleic acid 
sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal 
9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244; 
Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding an 
activator may be obtained from the genes encoding Bacillus 
stearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activator 
protein 1 (hap1), Saccharomyces cerevisiae galactose metabolizing protein 
4 (gal4), and Aspergillus nidulans ammonia regulation protein (areA). For 
further (examples, see Verdier, 1990, supra and MacKenzie et al., 1993, 
Journal of General Microbiology 139:2295-2307. 
A chaperone is a protein which assists another polypeptide in folding 
properly (Hartl et al., 1994, TIBS 19:20-25; Bergeron et al., 1994. TIBS 
19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189; 
Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature 
355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 
269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; 
Robinson et al., 1994, Bio/Technology 1:381-384). The nucleic acid 
sequence encoding a chaperone may be obtained from the genes encoding 
Bacillus subtilis GroE proteins, Aspergillus oryzae protein disulphide 
isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae 
BiP/GRP78, and Saccharomyces cerevisiae Hsp70. For further examples, see 
Gething and Sambrook, 1992, supra, and Hartl et al., 1994, supra. 
A processing protease is a protease that cleaves a propeptide to generate a 
mature biochemically active polypeptide (Enderlin and Ogrydziak, 1994, 
Yeast 10:67-79; Fuller et al., 1989, Proceedings of the National Academy 
of Sciences USA 86:1434-1438; Julius et al., 1984, Cell 37:1075-1089; 
Julius et al., 1983, Cell 32:839-852). The nucleic acid sequence encoding 
a processing protease may be obtained from the genes encoding 
Saccharomyces cerevisiae dipeptidylarninopeptidase, Saccharomyces 
cerevisiae Kex2, and Yarrowia lipolytica dibasic processing endoprotease 
(xpr6). 
It may also be desirable to add regulatory sequences which allow the 
regulation of the expression of the polypeptide relative to the growth of 
the host cell. Examples of regulatory systems are those which cause the 
expression of the gene to be turned on or off in response to a chemical or 
physical stimulus, including the presence of a regulatory compound. 
Regulatory systems in prokaryotic systems would include the lac, tac, and 
trp operator systems. In yeast, the ADH2 system or GALl system may be 
used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus 
niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase 
promoter may be used as regulatory sequences. Other examples of regulatory 
sequences are those which allow for gene amplification. In eukaryotic 
systems, these include the dihydrofolate reductase gene which is amplified 
in the presence of methotrexate, and the metallothionein genes which are 
amplified with heavy metals. In these cases, the nucleic acid sequence 
encoding the polypeptide would be placed in tandem with the regulatory 
sequence. 
Expression Vectors 
The present invention also relates to recombinant expression vectors 
comprising a nucleic acid sequence of the present invention, a promoter, 
and transcriptional and translational stop signals. The various nucleic 
acid and control sequences described above may be joined together to 
produce a recombinant expression vector which may include one or more 
convenient restriction sites to allow for insertion or substitution of the 
nucleic acid sequence encoding the polypeptide at such sites. 
Alternatively, the nucleic acid sequence of the present invention may be 
expressed by inserting the nucleic acid sequence or a nucleic acid 
construct comprising the sequence into an appropriate vector for 
expression. In creating the expression vector, the coding sequence is 
located in the vector so that the coding sequence is operably linked with 
the appropriate control sequences for expression, and possibly secretion. 
The recombinant expression vector may be any vector which can be 
conveniently subjected to recombinant DNA procedures and can bring about 
the expression of the nucleic acid sequence. The choice of the vector will 
typically depend on the compatibility of the vector with the host cell 
into which the vector is to be introduced. The vectors may be linear or 
closed circular plasmids. The vector may be an autonomously replicating 
vector, i.e., a vector which exists as an extrachromosomal entity, the 
replication of which is independent of chromosomal replication, e.g., a 
plasmid, an extrachromosomal element, a minichromosome, or an artificial 
chromosome. The vector may contain any means for assuring 
self-replication. Alternatively, the vector may be one which, when 
introduced into the host cell, is integrated into the genome and 
replicated together with the chromosome(s) into which it has been 
integrated. The vector system may be a single vector or plasmid or two or 
more vectors or plasmids which together contain the total DNA to be 
introduced into the genome of the host cell, or a transposon. 
The vectors of the present invention preferably contain one or more 
selectable markers which permit easy selection of transformed cells. A 
selectable marker is a gene the product of which provides for biocide or 
viral resistance, resistance to heavy metals, prototrophy to auxotrophs, 
and the like. Examples of bacterial selectable markers are the dal genes 
from Bacillus subtilis or Bacillus licheniformis, or markers which confer 
antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or 
tetracycline resistance. A frequently used mammalian marker is the 
dihydrofolate reductase gene. Suitable markers for yeast host cells are 
ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use 
in a filamentous fungal host cell may be selected from the group 
including, but not limited to, amdS (acetamidase), argB (ornithine 
carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB 
(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG 
(orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), 
trpC (anthranilate synthase), and glufosinate resistance markers, as well 
as equivalents from other species. Preferred for use in an Aspergillus 
cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus 
oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, 
selection may be accomplished by co-transformation, e.g., as described in 
WO 91/17243, where the selectable marker is on a separate vector. 
The vectors of the present invention preferably contain an element(s) that 
permits stable integration of the vector into the host cell genome or 
autonomous replication of the vector in the cell independent of the genome 
of the cell. 
The vectors of the present invention may be integrated into the host cell 
genome when introduced into a host cell. For integration, the vector may 
rely on the nucleic acid sequence encoding the polypeptide or any other 
element of the vector for stable integration of the vector into the genome 
by homologous or nonhomologous recombination. Alternatively, the vector 
may contain additional nucleic acid sequences for directing integration by 
homologous recombination into the genome of the host cell. The additional 
nucleic acid sequences enable the vector to be integrated into the host 
cell genome at a precise location(s) in the chromosome(s). To increase the 
likelihood of integration at a precise location, the integrational 
elements should preferably contain a sufficient number of nucleic acids, 
such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and 
most preferably 800 to 1,500 base pairs, which are highly homologous with 
the corresponding target sequence to enhance the probability of homologous 
recombination. The integrational elements may be any sequence that is 
homologous with the target sequence in the genome of the host cell. 
Furthermore, the integrational elements may be non-encoding or encoding 
nucleic acid sequences. On the other hand, the vector may be integrated 
into the genome of the host cell by non-homologous recombination. These 
nucleic acid sequences may be any sequence that is homologous with a 
target sequence in the genome of the host cell, and, furthermore, may be 
non-encoding or encoding sequences. 
For autonomous replication, the vector may further comprise an origin of 
replication enabling the vector to replicate autonomously in the host cell 
in question. Examples of bacterial origins of replication are the origins 
of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB10, 
pE194, pTA1060, and pAM.beta.1. Examples of origin of replications for use 
in a yeast host cell are the 2 micron origin of replication, the 
combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The 
origin of replication may be one having a mutation which makes its 
functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 
1978, Proceedings of the National Academy of Sciences USA 75:1433). 
More than one copy of a nucleic acid sequence encoding a polypeptide of the 
present invention may be inserted into the host cell to amplify expression 
of the nucleic acid sequence. Stable amplification of the nucleic acid 
sequence can be obtained by integrating at least one additional copy of 
the sequence into the host cell genome using methods well known in the art 
and selecting for transformants. 
The procedures used to ligate the elements described above to construct the 
recombinant expression vectors of the present invention are well known to 
one skilled in the art (see, e.g., Sambrook et al., 1989, supra). 
Host Cells 
The present invention also relates to recombinant host cells, comprising a 
nucleic acid sequence of the invention, which are advantageously used in 
the recombinant production of the polypeptides. The cell is preferably 
transformed with a vector comprising a nucleic acid sequence of the 
invention followed by integration of the vector into the host chromosome. 
"Transformation" means introducing a vector comprising a nucleic acid 
sequence of the present invention into a host cell so that the vector is 
maintained as a chromosomal integrant or as a self-replicating 
extra-chromosomal vector. Integration is generally considered to be an 
advantage as the nucleic acid sequence is more likely to be stably 
maintained in the cell. Integration of the vector into the host chromosome 
may occur by homologous or non-homologous recombination as described 
above. 
The choice of a host cell will to a large extent depend upon the gene 
encoding the polypeptide and its source. The host cell may be a 
unicellular microorganism or a non-unicellular microorganism. Useful 
unicellular cells are bacterial cells such as gram positive bacteria 
including, but not limited to, a Bacillus cell, e.g., Bacillus subtilis, 
Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus 
stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, 
Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus 
megaterium, and Bacillus thuringiensis; or a Streptomyces cell, e.g., 
Streptomyces lividans or Streptomyces murinus, or gram negative bacteria 
such as E. coli and Pseudomonas sp. In a preferred embodiment, the 
bacterial host cell is a Bacillus lentus, a Bacillus licheniformis, a 
Bacillus subtilis, or a Bacillus stearothermophilus cell. The 
transformation of a bacterial host cell may, for instance, be effected by 
protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular 
General Genetics 168:111-115), by using competent cells (see, e.g., Young 
and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar and 
Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by 
electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 
6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, 
Journal of Bacteriology 169:5771-5278). 
The host cell may be a eukaryote, such as a mammalian cell, an insect cell, 
a plant cell or, preferably, a fungal cell. Useful mammalian cells include 
Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) 
cells, COS cells, or any number of other immortalized cell lines 
available, e.g., from the American Type Culture Collection. The fungal 
host cell may be a yeast cell or a filamentous fungal cell. 
"Yeast" as used herein includes ascosporogenous yeast (Endomycetales), 
basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti 
(Blastomycetes). The ascosporogenous yeasts are divided into the families 
Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four 
subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), 
Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, 
Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include 
the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and 
Filobasidiella. Yeast belonging to the Fungi Imperfecti are divided into 
two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) 
and Cryptococcaceae (e.g., genus Candida). Since the classification of 
yeast may change in the future, for the purposes of this invention, yeast 
shall be defined as described in Biology and Activities of Yeast (Skinner, 
F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. 
Symposium Series No. 9, 1980. The biology of yeast and manipulation of 
yeast genetics are well known in the art (see, e.g., Biochemistry and 
Genetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M., 
editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J. S., 
editors, 2nd edition, 1987; and The Molecular Biology of the Yeast 
Saccharomyces, Strathern et al., editors, 1981). 
"Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, 
Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, 
Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB 
International, University Press, Cambridge, UK) as well as the Oomycota 
(as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic 
fungi (Hawksworth et al., 1995, supra). Representative groups of 
Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium), 
Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts 
listed above. Examples of Basidiomycota include mushrooms, rusts, and 
smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, 
Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of 
Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) 
such as Achlya. Examples of mitosporic fungi include Aspergillus, 
Penicillium, Candida, and Alternaria. Representative groups of Zygomycota 
include, e.g., Rhizopus and Mucor. 
"Filamentous fungi" include all filamentous forms of the subdivision 
Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The 
filamentous fungi are characterized by a vegetative mycelium composed of 
chitin, cellulose, glucan, chitosan, mannan, and other complex 
polysaccharides. Vegetative growth is by hyphal elongation and carbon 
catabolism is obligately aerobic. In contrast, vegetative growth by yeasts 
such as Saccharomyces cerevisiae is by budding of a unicellular thallus 
and carbon catabolism may be fermentative. 
In a preferred embodiment, the fungal host cell is a yeast cell. In a more 
preferred embodiment, the yeast host cell is a cell of a species of 
Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, or 
Yarrowia. In a most preferred embodiment, the yeast host cell is a 
Saccharomyces cerevisiae, a Saccharomyces carlsbergensis, a Saccharomyces 
diastaticus, a Saccharomyces douglasii, a Saccharomyces kluyveri, a 
Saccharomyces norbensis, or a Saccharomyces oviformis cell. In another 
most preferred embodiment, the yeast host cell is a Kluyveromyces lactis 
cell. In another most preferred embodiment, the yeast host cell is a 
Yarrowia lipolytica cell. 
In another preferred embodiment, the fungal host cell is a filamentous 
fungal cell. In a more preferred embodiment, the filamentous fungal host 
cell is a cell of a species of, but not limited to, Acremonium, 
Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, 
Penicillium, Thielavia, Tolypocladium, and Trichoderma. In an even more 
preferred embodiment, the filamentous fungal host cell is an Aspergillus 
cell. In another even more preferred embodiment, the filametitous fungal 
host cell is a Fusariun cell. In a most preferred embodiment, the 
filamentous fungal host cell is an Aspergillus oryzae, an Aspergillus 
niger, an Aspergillus foetidus, Lan or an Aspergillus japonicus cell. In 
another most preferred embodiment, the filamentous fungal host cell is a 
Fusarium oxysporum or a Fusarium graminearum cell. 
Fungal cells may be transformed by a process involving protoplast 
formation, transformation of the protoplasts, and regeneration of the cell 
wall in a manner known per se. Suitable procedures for transformation of 
Aspergillus host cells are described in EP 238 023 and Yelton et al., 
1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A 
suitable method of transforming Fusarium species is described by Malardier 
et al., 1989, Gene 78:147-156 or in copending U.S. Ser. No. 08/269,449. 
Yeast may be transformed using the procedures described by Becker and 
Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast 
Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 
182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of 
Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National 
Academy of Sciences USA 75:1920. Mammalian cells may be transformed by 
direct uptake using the calcium phosphate precipitation method of Graham 
and Van der Eb (1978, Virology 52:546). 
Methods of Production 
The present invention also relates to methods for producing a polypeptide 
of the present invention comprising (a) cultivating a Coprinus strain to 
produce a supernatant comprising the polypeptide; and (b) recovering the 
polypeptide. 
The present invention also relates to methods for producing a polypeptide 
of the present invention comprising (a) cultivating a host cell under 
conditions conducive to expression of the polypeptide; and (b) recovering 
the polypeptide. 
In both methods, the cells are cultivated in a nutrient medium suitable for 
production of the polypeptide using methods known in the art. For example, 
the cell may be cultivated by shake flask cultivation, small-scale or 
large-scale fermentation (including continuous, batch, fed-batch, or solid 
state fermentations) in laboratory or industrial fermentors performed in a 
suitable medium and under conditions allowing the polypeptide to be 
expressed and/or isolated. The cultivation takes place in a suitable 
nutrient medium comprising carbon and nitrogen sources and inorganic 
salts, using procedures known in the art (see, e.g., references for 
bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene 
Manipulations in Fungi, Academic Press, Calif., 1991). Suitable media are 
available from commercial suppliers or may be prepared according to 
published compositions (e.g., in catalogues of the American Type Culture 
Collection). If the polypeptide is secreted into the nutrient medium, the 
polypeptide can be recovered directly from the medium. If the polypeptide 
is not secreted, it is recovered from cell lysates. 
The polypeptides may be detected using methods known in the art that are 
specific for the polypeptides. These detection methods may include use of 
specific antibodies, formation of an enzyme product, or disappearance of 
an enzyme substrate. For example, an enzyme assay may be used to determine 
the activity of the polypeptide. Procedures for determining laccase 
activity are known in the art and include, e.g., the oxidation of 
2,2'-azinobis-(3-ethybenzthiazoline-6-sulfonic acid (ABTS) (Childs et al., 
1975, Biochemical Journal 145:93-103) or syringaldazine (Bauer et al., 
1971, Analytical Chemistry 43: 421-425) as substrate. 
The resulting polypeptide may be recovered by methods known in the art. For 
example, the polypeptide may be recovered from the nutrient medium by 
conventional procedures including, but not limited to, centrifugation, 
filtration, extraction, spray-drying, evaporation, or precipitation. The 
recovered polypeptide may then be further purified by a variety of 
chromatographic procedures, e.g., ion exchange chromatography, gel 
filtration chromatography, affinity chromatography, or the like. 
The polypeptides of the present invention may be purified by a variety of 
procedures known in the art including, but not limited to, chromatography 
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size 
exclusion), electrophoretic procedures (e.g., preparative isoelectric 
focusing (IEF), differential solubility (e.g., ammonium sulfate 
precipitation), or extraction (see, e.g., Protein Purification, J. -C. 
Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). 
Uses 
The polypeptides of the present invention may be used in a number of 
different industrial processes. These processes include polymerization of 
lignin, both Kraft and lignosulfates, in solution, in order to produce a 
lignin with a higher molecular weight. A neutral/alkaline laccase is a 
particular advantage in that Kraft lignin is more soluble at higher pHs. 
Such methods are described in, for example, Jin et al., 1991, 
Holzforschung 45: 467-468; U.S. Pat. No. 4,432,921; EP 0 275 544; 
PCT/DK93/00217, 1993. Laccase is also useful in the copolymerization of 
lignin with low molecular weight compounds, such as is described by 
Milstein et al., 1994, Appl. Microbiol. Biotechnol. 40: 760-767. 
The laccase of the present invention can also be used for in-situ 
depolymerization of lignin in Kraft pulp, thereby producing a pulp with 
lower lignin content. This use of laccase is an improvement over the 
current use of chlorine for depolymerization of lignin, which leads to the 
production of chlorinated aromatic compounds, which are an environmentally 
undesirable by-product of paper mills. Such uses are described in, for 
example, Current Opinion in Biotechnology 3: 261-266, 1992; Journal of 
Biotechnology 25: 333-339, 1992; Hiroi et al., 1976, Svensk Papperstidning 
5:162-166, 1976. Since the environment in a paper mill is typically 
alkaline, the present laccase is more useful for this purpose than other 
known laccases, which function best under acidic conditions. 
Oxidation of dyes or dye precursors and other chromophoric compounds leads 
to decolorization of the compounds. Laccase can be used for this purpose, 
which can be particularly advantageous in a situation in which a dye 
transfer between fabrics is undesirable, e.g., in the textile industry and 
in the detergent industry. Methods for dye transfer inhibition and dye 
oxidation can be found in WO 92/01406; WO 92/18683; WO 92/18687; WO 
91/05839; EP 0495836; Calvo, 1991, Mededelingen van de Faculteit 
Landbouw-wetenschappen/Rijiksuniversitet Gent. 56: 1565-1567; Tsujino et 
al., 1991, J. Soc. Chem. 42: 273-282. Laccases of the present invention 
are particularly useful in oxidation at high pH, i.e., over pH 7, as 
disclosed in DK 0982/94, the contents of which are incorporated herein by 
reference. Use of laccase in oxidation of dye precursors for hair dyeing 
is disclosed in U.S. Pat. No. 3,251,742, the contents of which are 
incorporated herein by reference. 
The present laccase can also be used for the polymerization or oxidation of 
phenolic compounds present in liquids. An example of such utility is the 
treatment of juices, such as apple juice, so that the laccase will 
accelerate a precipitation of the phenolic compounds present in the juice, 
thereby producing a more stable juice. Such applications have been 
described by Stutz, Fruit processing 7/93, 248-252, 1993; Maier et al., 
1990, Dt. Lebensmittel-rindschau 86: 137-142; Dietrich et al., 1990, 
Fluss. Obst. 57: 67-73. 
Laccases of the present invention are also useful in soil detoxification 
(Nannipieri et al., 1991, J. Environ. Qual. 20: 510-517; Dec and Bollag, 
1990, Arch. Environ. Contam. Toxicol. 19: 543-550). 
The present invention is further described by the following examples which 
should not be construed as limiting the scope of the invention. 
EXAMPLES 
Materials and strains 
Chemicals used as buffers and substrates are commercial products of at 
least reagent grade. Strains used are Coprinus cinereus A3387 (IFO 8371), 
E. coli Y1090(ZL) (GIBCO BRL, Gaithersburg, Md.), E. coli DH10B(ZL) (GIBCO 
BRL), E. coli DH5.alpha. (Stratagene, La Jolla, Calif.), Aspergillus 
oryzae HowB712, Aspergillus oryzae JeRS317, and Aspergillus oryzae 
JeRS316. 
Example 1 
Purification and characterization of Coprinus cinereus laccase 
The laccase is initially isolated from Coprinus cinereus strain A3387 
culture broth by filtration (Propex 23+HSC) and concentration (Filtron 
2.times.10K). The cationic flocculent Magnifloc.RTM. 521C (American 
Cyanamid, Wallingford, Conn.) is added to the resulting preparation, mixed 
for 30 minutes, and then centrifuged. This step removes colored substances 
from the preparation. The supernate is then precipitated with ammonium 
sulfate (55% saturation) and resuspended twice in ammonium sulfate (40% 
saturation), which also results in color removal. The resuspension is 
further concentrated to reduce the volume, and filtered, but not washed 
out. The concentrate in ammonium sulfate (40% saturation) is then 
subjected to Butyl ToyoPearl hydrophobic chromatography (Tosoh Corp., 
Tokyo, Japan) and eluted with an ammonium sulfate gradient from 40% to 0% 
saturation. Buffer exchange to 20 mM MES pH 6.0 and concentration with an 
Amicon cell equipped with a membrane of 20,000 molecular weight cut-off is 
then conducted. The resulting soluti.on is then subjected to Q-Sepharose 
(Pharmacia, Uppsala, Sweden) anion exchange chromatography (150 ml) in 20 
mM MES pH 6.0 with a linear gradient from 0 to 0.4 M NaCl. The sample is 
finally rechromatographed by HPQ-Sepharose (Pharmacia, Upsala, Sweden) 
chromatography (50 ml) in 20 mM MES pH 6.0 with a linear gradient from 0 
to 0.4 M NaCl. The laccase elutes at 0.25-0.30 M NaCl. 
The purified laccase is about 95% pure as determined by SDS-PAGE which 
shows the laccase as a band of M.sub.W =63,000. Isoelectric focusing shows 
two dominating bands with pIs of 3.7 and 4.0. 
The N-terminal amino acid residue of the purified laccase is blocked. The 
laccase is therefore reduced, S-carboxymethylated, and digested with 
Endoproteinase Lys-C (Boehringer Mannheim, Indianapolis, Ind.) and with 
chymotrypsin. The resulting peptides are purified by reversed phase HPLC 
using a Vydac C18 column (Vydac, Inc., Hesperia, Calif.) eluted with a 
linear gradient of either acetonitrile or 2-propanol in 0.1% aqueous 
trifluoroacetic acid. The purified peptides are sequenced on an Applied 
Biosystems 473A Protein Sequencer according to the manufacture's 
instructions. 
Several distinct peptides which result from the protease digestion are 
listed below. In the following sequences, Xaa represents an indeterminable 
residue. Peptide 3 apparently encompasses peptide 2. In peptides 4 and 9, 
residues designated Xaa/Yaa indicate both residues are found at that 
position. Residues in parentheses are uncertain. Peptide 9 is included in 
peptide 13. 
__________________________________________________________________________ 
Peptide 1 (SEQ ID NO: 1): 
Glu-Val-Asp-Gly-Gln-Leu-Thr-Glu-Pro-His-Thr-Val-Asp-Arg-Leu-Gln-Ile-Phe- 
Thr-Gly-Gln- 
Arg-Tyr-Ser-Phe-Val-Leu-Asp-Ala-Asn-Gln-Pro-Val-Asp-Asn-Tyr-Trp-Ile-Arg-A 
la 
Peptide 2 (SEQ ID NO: 2): 
Xaa-Xaa-Asp-Asn-Pro-Gly-Pro 
Peptide 3 (SEQ ID NO: 3): 
Phe-Val-Thr-Asp-Asn-Pro-Gly-Pro 
Peptides 2 and 3 combined (SEQ ID NO: 4): 
Phe-Val-Thr-Asp-Asn-Pro-Gly-Pro-Trp 
Peptide 4 (SEQ ID NO: 5): 
Ile/Leu-Asp-Pro-Ala-Xaa-Pro-Gly-Ile-Pro-Thr-Pro-Gly-Ala-(Ala)-Asp-Val 
Peptide 5 (SEQ ID NO: 6): 
Gly-Val-Leu-Gly-Asn-Pro-Gly-Ile 
Peptide 6 (SEQ ID NO: 7): 
Xaa-Phe-Asp-Asn-Leu-Thr-Asn 
Peptide 7 (SEQ ID NO: 8): 
Tyr-Arg-Xaa-Arg-Leu-Ile-Ser-Leu-Ser-Cys-Asn-Pro-Asp-(Trp)-Gln-Phe 
Peptide 8 (SEQ ID NO: 9): 
Ala-Asp-Trp-Tyr 
Peptide 9 (SEQ ID NO: 10): 
Ile-Pro-Ala/Asp-Pro-Ser-Ile-Gln 
Peptide 10 (SEQ ID NO: 11): 
Glue-Ser-Pro-Ser-Val-Pro-Thr-Leu-Ile-Arg-Phe 
Peptide 11 (SEQ ID NO: 12): 
Ala-Gly-Thr-Phe 
Peptide 12 (SEQ ID NO: 13): 
Ser-Gly-Ala-Gln-Ser-Ala-Asn-Asp-Leu-Leu-Pro-Ala-Gly 
Peptide 13 (SEQ ID NO: 14): 
Ile-Pro-Ala-Pro-Ser-Ile-Gln-Gly-Ala-Ala-Gln-Pro-Asx-Ala-Thr 
__________________________________________________________________________ 
Most of the peptides show considerable homology with portions of the amino 
acid sequence of a Polyporus pinsitus laccase (Yaver et al., 1995, Applied 
and Environmental Microbiology 62: 834-841). 
Example 2 
RNA isolation 
Coprinus cinereus strain A3387 is cultivated at 26.degree. C. in FG4 medium 
comprised of 30 g of soybean meal, 15 g of maltodextrin, 5 g of Bacto 
peptone, and 0.2 g of pluronic acid per liter. The mycelia are harvested 
after six days of growth, frozen in liquid N.sub.2, and stored at 
-80.degree. C. Total RNA is prepared from the frozen, powdered mycelium of 
Coprinus cinereus A3387 by extraction with guanidinium thiocyanate 
followed by ultracentrifugation through a 5.7 M cesium chloride cushion 
(Chirgwin et al., 1979, Biochemistry 18: 5294-5299). Poly(A)+ RNA is 
isolated by oligo(dT)-cellulose affinity chromatography according to Aviv 
and Leder (1972, Proceedings of the National Academy of Sciences USA 69: 
1408-1412). 
Example 3 
Construction of a cDNA library 
Double-stranded cDNA is synthesized from 5 .mu.g of Coprinus cinereus 
poly(A)+ RNA of Example 2 as described by Gubler and Hoffman (1983, Gene 
25: 263-269) and Sambrook et al. (1989, Molecular Cloning: A Laboratory 
Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), except that 
an oligo(dT)-NotI anchor primer, instead of an oligo(dT)12-18 primer, is 
used in the first strand reaction. After synthesis, the cDNA is treated 
with Mung bean nuclease (Life Technologies, Gaithersburg, Md.), 
blunt-ended with T4 DNA polymerase (Boehringer Mannheim, Indianapolis, 
Ind.), and ligated to non-palindromic BstXI adaptors (Invitrogen, San 
Diego, Calif.), using about 50-fold molar excess of the adaptors. The 
adapted cDNA is digested with NotI, size-fractionated for 1.2-3.0 kb cDNAs 
by agarose gel electrophoresis, and ligated into BstXI/NotI cleaved 
pYES2.0 vector (Invitrogen, San Diego, Calif.). The ligation mixture is 
transformed into electrocompetent E. coli DH10B cells (Life Technologies, 
Gaithersburg, Md.) according to the manufacturer's instructions. The 
library consisting of 1.times.10.sup.6 independent clones is stored as 
individual pools (25,000-30,000 colony forming units/pool) in 20% glycerol 
at -80.degree. C., and as double stranded cDNA and ligation mixture at 
-20.degree. C. 
Example 4 
Generation of a cDNA probe from a Coprinus cinereus cDNA using PCR 
Three oligonucleotides (sense s1 and s2 and antisense as1) with low codon 
degeneracy are designed based on two conserved motifs in laccases from 
Rhizoctonia, Phlebia, Polyporus, and Coriolus. The oligos have the 
following sequences: 
s1: 5'-ATI CAc/t TGG CAc/t GGI c/tTI c/tTI-3' (SEQ ID NO:15) 
s2: 5'-ATI CAc/t TGG CAc/t GGI TTc/t Ttc/t-3' (SEQ ID NO:16) 
as1: 3'-GGI ACC AAa/g a/gAI GTa/g ACa/g GTa/g TAI CT-5' (SEQ ID NO:17) 
One .mu.g of plasmid DNA from the Coprinus cinereus library pool described 
in Example 3 is PCR amplified in a thermal cycler according to Frohman et 
al., 1988, Proceedings of the National Academy of Sciences USA 85: 
8998-9002 using 500 pmol of each laccase sense primer in two combinations 
(s1 and as1, s2 and as1) with 500 pmol of the laccase antisense primer, 
and 2.5 units of Taq polymerase (Perkin Elmer Cetus, Branchburg, N.J.). 
Thirty cycles of PCR are performed using a cycle profile of denaturation 
at 94.degree. C. for 1 minute, annealing at 55.degree. C. for two minutes, 
and extension at 72.degree. C. for 3 minutes. Analysis of the PCR products 
reveals a 1.2 kb major PCR product with one primer pair, s1 and as1, 
whereas the other pair does not amplify any major products. The PCR 
fragment of interest is subcloned into a pUC18 vector and sequenced 
according to Siggaard-Andersen et al., 1991, Proceedings of the National 
Academy of Sciences USA 88: 4114-4118. Sequencing of the ends of two PCR 
subclones in pUC18 reveals a cDNA sequence coding for a laccase 
polypeptide. In addition to the primer encoding residues, the deduced 
amino acid sequence aligns with two peptide sequences obtained from the 
purified wild-type laccase, indicating that PCR has specifically amplified 
the desired region of a Coprinus cinereus laccase cDNA. 
Example 5 
Subcloning and sequencing of partial cDNAs 
The PCR product described in Example 4 is ligated into pCRII using a TA 
Cloning Kit (Invitrogen, San Diego, Calif.) according to the 
manufacturer's instructions. Seven subclones are prepared and sequenced 
using both the M13 universal -21mer oligonucleotide and the M13 -48 
reverse oligonucleotide. Nucleotide sequences are determined on both 
strands by primer walking using Taq polymerase cycle-sequencing with 
fluorescent-labeled nucleotides, and reactions are electrophoresed on an 
Applied Biosystems Automatic DNA Sequencer (Model 373A, version 2.0.1). 
The seven clones based on deduced amino acid sequence and percent 
identities between them appear to encode for 3 laccases (Table 1). Clones 
CCLACC4, 8 and 7 are designated as partial cDNAs of Coprinus cinereus lcc1 
(SEQ ID NOS:18 and 19). Clones CCLACC 1, 3 and 11 (pDSY71) are designated 
as partial cDNAs of Coprinus cinereus lcc2 (SEQ ID NOS:20 and 21). Clone 
CCLACC 15 (pDSY72) is designated as a partial cDNA of Coprinus cinereus 
lcc3 (SEQ ID NOS:22 and 23). The deduced amino acid sequences of the 
partial cDNAs of lcc1, lcc2, and lcc3 (SEQ ID NOS:19, 21, and 23) are 
compared to the peptide sequences determined above, and the closest match 
is found between lcc1 and the peptide sequences. In order to obtain a 
full-length clone for heterologous expression of lcc1 in Aspergillus 
oryzae, a genomic library of Coprinus cinereus A3387 is constructed in 
.lambda.ZipLox. 
TABLE 1 
______________________________________ 
Percent identities between Coprinus cinereus cDNAs 
1 2 3 4 5 6 7 
______________________________________ 
1 CCLACC4 98 100 65 65 65 62 
2 CCLACC7 98 65 64 65 63 
3 CCLACC8 65 65 65 62 
4 CCLACC1 100 99 81 
5 CCLACC3 99 81 
6 CCLACC11 81 
7 CCLACC15 
______________________________________ 
Example 6 
Genomic DNA isolation 
A culture of Coprinus cinereus A3387 is grown at room temperature for 4 
days with shaking at 200 rpm in YEG medium comprised of 0.5% yeast extract 
and 2% dextrose. Mycelia are harvested through Miracloth (Calbiochem, La 
Jolla, Calif.), washed twice with 10 mM Tris-0.1 mM EDTA pH 7.4 buffer 
(TE) and frozen quickly in liquid nitrogen. DNA is isolated as described 
by Timberlake and Barnard, 1981, Cell 26: 29-37. 
Example 7 
Preparation of Coprinus cinereus genomic library 
A genomic library of Coprinus cinereus A3387 is constructed using a 
.lambda.ZipLox Kit (Life Technologies, Gaithersburg, Md.) according to the 
manufacturer's instructions. Genomic DNA (.about.30 .mu.g) is digested 
with Tsp509I (New England Biolabs, Beverly, Mass.) at 65.degree. C. in a 
total volume of 150 .mu.l in the buffer provided by the supplier. Samples 
of 30 .mu.l are taken at 3, 5, 7, 8, and 9 minutes and electrophoresed on 
a 1% agarose preparative gel. Bands of 3 to 8 kb in size are excised from 
the gel. The DNA is then isolated from the gel slices using a Qiaex Kit 
(Qiagen, San Diego, Calif.). The size-fractionated DNA is ligated 
overnight at room temperature to .lambda.ZipLox EcoRI arms following the 
protocols provided with the kit. The ligations are packaged into phage 
using a Giga Pak Gold Packaging Kit (Stratagene, La Jolla, Calif.), and 
the packaging reactions are titered using E. coli Y1090 cells. A total of 
6.times.10.sup.5 pfu are obtained. The packaging extract is plated to 
amplify the library, and the titer of the library is determined to be 
1.times.10.sup.11 pfu/ml. Twenty individual plaques are picked, and the 
plasmids are excised from the plaques by passage through E. coli DH10B. 
Plasmid DNA is isolated from the cultures and is digested with PstI/NotI 
to determine the percent of molecules in the library which have inserts. 
Eight of the twenty, or 40% of those tested, have inserts which range in 
size from 3 to 6 kb. 
Example 8 
Probe preparation for library screening 
A DIG-labeled probe for nonradioactive screening of the library is prepared 
by PCR using the Coprinus cinereus partial lcc1 cDNA described in Example 
5 as a template. The primers used in the reaction are shown below: 
5' ACTGCGATGGTCTCCGTGGTC 3' (SEQ ID NO:24) 
5' GGGGCCTGGGTTATCGGTGAC 3' (SEQ ID NO:25) 
The PCR conditions are 1 cycle at 95.degree. C. for 5 minutes, 50.degree. 
C. for 1 minute, and 72.degree. C. for 1.5 minutes; 29 cycles each at 
95.degree. C. for 1 minute, 50.degree. C. for 1 minute, and 72.degree. C. 
for 1.5 minutes; and 1 cycle at 95.degree. C. for 30 seconds, 50.degree. 
C. for 1 minute, and 72.degree. C. for 3 minutes. The reaction contains 
0.1 .mu.g of the Coprinus cinereus partial lcc1 cDNA, 10 .mu.l 10.times. 
PCR Buffer (Perkin Elmer, Branchburg, N.J.), 5 .mu.l 10.times. DIG 
labeling mix (Boehringer Mannheim, Indianapolis, Ind.), 75 pmol of each 
primer, and 0.5 unit of Taq DNA polymerase (Perkin-Elmer Corp., 
Branchburg, N.J.). A probe concentration of 250 ng/.mu.l is determined 
after PCR following protocols provided with the Genius Kit (Boehringer 
Mannheim, Indianapolis, Ind.). 
.sup.32 P-labeled probes of Coprinus cinereus lcc2 and lcc3 partial cDNAs 
are prepared using a RadPrime Kit (Life Technologies, Gaithersburg, Md.) 
according to the manufacturer's instructions. 
Example 9 
Genomic library screening 
Appropriate dilutions of the .lambda.ZipLox Coprinus cinereus genomic 
library are plated with E. coli Y1090 cells on NZY plates comprised of 
0.5% NaCl, 0.2% MgSO.sub.4, 0.5% yeast extract, and 1% NZ amine pH 7.5 per 
liter with 0.7% top agarose. The plaques are lifted to Hybond N+ filters 
(Amersham Co., Amersham, UK) using standard procedures (Sambrook et al. 
1989, supra). The filters are hybridized in Engler Blue hybridization 
buffer at 65.degree. C. for 1 hour. After prehybridization, the DIG 
labeled probe of Example 8 is added at a final concentration of 3 ng/ml 
and allowed to hybridize overnight at 65.degree. C. The filters are washed 
at 65.degree. C. twice for 5 minutes in 2.times. SSC, 0.1% SDS, twice for 
15 minutes in 0.5.times. SSC, 0.1%SDS, and then are processed to detect 
the hybridized DIG-label using the Genius Kit and Lumi-Phos 530 substrate 
according to the manufacturer's instructions. Following the detection 
protocol, film is placed on top of the filters for 2 hours. 
For screening of the library using the .sup.32 P-labeled probes described 
in Example 8, filter lifts are prepared as described above, and 
prehybridized at 65.degree. C. in 2.times. SSPE, 1% SDS, 0.5% nonfat dry 
milk and 200 .mu.g denatured salmon sperm DNA. After 1 hour 
prehybridization, the .sup.32 P-labeled probes are added to a final 
concentration of 10.sup.6 cpm/ml and hybridizations are continued 
overnight at 65.degree. C. The filters are washed twice at 65.degree. C. 
for 15 minutes in 0.2.times. SSC, 1% SDS, and 0.1% sodium pyrophosphate. 
The genomic library is probed with the DIG-labeled fragment of lcc1. 
Approximately 200,000 plaques are screened using the conditions described 
above, and 9 positive clones are obtained. The plasmids are excised from 
the clones by passage through E. coli DH10B(ZL), and then are 
characterized by digestion with PstI/NotI. All 9 clones contain inserts. 
Based on the nucleotide sequence of the partial lcc1 cDNA, the genomic 
clones which may be lcc1 genomic clones are determined. All 8 unique 
clones are digested with BamHI/PstI and PstI/BsmI for which fragments of 
205 bp and 382 bp, respectively, are expected (neither lcc2 nor lcc3 
partial cDNAs contain these fragments). Four of the 8 unique clones 
contain both predicted fragments. DNA sequencing reactions on all four 
clones using universal sequencing primers are performed as described in 
Example 5 to determine which clones are full-length. 
The nucleotide sequence of clone 4-19 (pDSY73) is determined completely on 
both strands and shown to contain the full length lcc1 gene (FIG. 1, SEQ 
ID NO:26). The deduced amino acid sequence (FIG. 1, SEQ ID NO:27) of the 
genomic lcc1 matches 100% with the determined N-terminal sequence (see 
Example 14) although the predicted signal peptide cleavage site is between 
A18 and Q19 while the peptide sequence begins 4 residues downstream at 
S23. The lcc1 gene contains 7 introns ranging in size from 54 to 77 bp. 
The deduced protein contains 3 potential N-glycosylation sites 
(AsnXaaThr/Ser), and the predicted mature protein after removal of the 
signal peptide is 521 amino acids in length. The percent identities of the 
lcc1 protein to other fungal laccases is shown in Table 2. The highest 
percent identity, 57.8%, is found when compared to the laccase from the 
unidentified basidiomycete PM1 (Coll et al., 1993, supra). When alignments 
of Lccl and other basidiomycete laccases are performed, it appears that 
Lcc1 may have either a C-terminal extension or a C-terminal peptide that 
is removed by processing. 
The genomic library is also screened with the .sup.32 P-labeled probes for 
the Coprinus cinereus lcc2 and lcc3 partial cDNAs. For screening with the 
lcc2 probe, approximately 50,000 plaques are hybridized with the probe, 
and 4 positive clones are obtained. For screening of the library with the 
lcc3 probe, approximately 35,000 plaques are probed, and 2 positive clones 
are obtained. After passage through E. coli and isolation of the plasmid 
DNA, the nucleotide sequence of one of the lcc3 clones (pDSY100) is 
determined by primer walking as described in Example 5 (FIG. 2, SEQ ID 
NO:28). The lcc3 gene contains 13 introns (as indicated by lowercase in 
FIG. 2). The positions of introns 4 through 10 are confirmed from the 
partial cDNA while the positions of the other 6 introns are deduced based 
on the consensus sequences found at the 5' and 3' splice sites of fungal 
introns and by homology of the deduced amino acid sequence (FIG. 2, SEQ ID 
NO:29) to other laccases. The lcc3 gene encodes for a precursor protein of 
517 amino acids. There is one potential N-glycosylation site, and the 
mature protein after the predicted signal peptide cleavage (indicated by 
an arrow) is 501 amino acids in length. 
From the nucleotide sequences of the 4 positive lcc2 clones, it is observed 
that none of the clones are full-length. The clone with the largest insert 
(CCLACC1-4) is missing the sequence coding for the last approximately 100 
amino acids based on homology to other fungal laccases. 
TABLE 2 
__________________________________________________________________________ 
Percent identities of the Coprinus cinereus lcc1 to other fungal* 
laccases 
Cc Cc Cc Tv Tv Tv Tv Tv 
lcc1 lcc2 lcc3 lcc1 lcc2 lcc3 lcc4 lcc5 Ch Pr PMI Ab Nc 
__________________________________________________________________________ 
Cclcc1 
Cclcc2 59.3 
Cclcc3 57.5 79.6 
Tvlcc1 55.5 61.3 59.5 
Tvlcc2 55.7 60.9 59.5 79.6 
Tvlcc3 57.0 61.0 58.2 62.8 84.6 
Tvlcc4 55.5 59.2 58.8 70.3 67.1 61.4 
Tvlcc5 54.4 59.3 57.9 71.1 69.1 64.6 76.5 
Ch 55.5 61.5 59.3 91.4 81.4 63.0 70.1 71.3 
Pr 50.3 59.1 57.5 63.3 61.5 62.2 63.9 63.9 6.41 
PMI 57.8 62.8 59.4 79.6 73.7 62.2 69.1 70.1 80.2 65.7 
Ab 40.3 41.7 41.9 43.7 43.1 43.6 44.6 43.1 44.1 42.5 44.4 
Nc 25.3 25.3 24.0 25.1 23.8 24.8 21.9 24.2 25.1 23.0 24.4 25.5 
__________________________________________________________________________ 
*Cc = Coprinus cinereus; Tv = Trametes villosa; Ch = Coriolus hirsutus; 
PM1 = unidentified basidiomycete; Pr = Phlebia radiata; Nc = Neurospora 
crassa; Ab = Agaricus bisporus; 1cc = laccase gene. 
Example 10 
Probe preparation for library screening to obtain the full length lcc2 gene 
A DIG-labeled probe for nonradioactive screening of the library is prepared 
by PCR using the Coprinus cinereus lcc2 partial genomic clone as template 
in order to obtain a full-length clone of lcc2. The primers used in the 
reaction are shown below: 
AGCTCGATGACTTTGTTACGG (1868R CCLCC2) (SEQ ID NO:30) 
CAGCGCTACTCGTTFCGTTCTC (1460 CCLCC2) (SEQ ID NO:31) 
The PCR conditions are 1 cycle at 95.degree. C. for 1 minute; and 30 cycles 
each at 94.degree. C. for 1 minute, 55.degree. C. for 1 minute, and 
72.degree. C. for 2 minutes. The reaction contains 0.1 .mu.g of Coprinus 
cinereus lcc2 partial genomic clone (CCLACC1-4), 10 .mu.l of 10.times. PCR 
Buffer (Perkin Elmer, Branchburg, N.J.), 5 .mu.l of 10.times. DIG labeling 
mix (Boehringer Mannheim, Indianapolis, Ind.), 75 pmol of each primer, and 
0.5 Unit of Taq DNA polymerase. The concentration of the DIG-labeled probe 
is determined using the Genius Kit according to the manufacturer's 
instructions. 
Example 11 
Genomic library screening to obtain the full length lcc2 gene 
Appropriate dilutions of the .lambda.ZipLox Coprinus cinereus genomic 
library prepared as described in Example 7 are plated with E. coli Y1090 
cells on NZY plates (0.5% NaCl, 0.2% MgSO.sub.4, 0.5% yeast extract, and 
1% NZ amine pH 7.5) with 0.7% top agarose. The plaques are lifted to 
Hybond N+ filters using standard procedures (Sambrook et al., 1989, 
supra). 
Filters are prehybridized in Easy Hyb hybridization buffer (Boehringer 
Mannheim, Indianapolis, Ind.) at 42.degree. C. for 1 hour, and after 
prehybridization the DIG labeled probe mentioned above is added at a final 
concentration of 1 ng/ml. The filters and probe are allowed to hybridize 
overnight at 42.degree. C. The filters are then washed twice at room 
temperature for 5 minutes in 2.times. SSC-0.1% SDS and twice at 68.degree. 
C. for 15 minutes in 0.1.times. SSC-0.1% SDS. The filters are next 
processed to detect the hybridized DIG-label using the Genius Kit and CSPD 
Ready-To-Use (Boehringer Mannheim, Indianapolis, Ind.) as substrate 
according to the manufacturer's instructions. Following the detection 
protocol, film is placed on the filters for 20 minutes to 2 hours. 
In order to obtain a full-length clone, the genomic library is screened 
(.about.42,000 plaques) using a DIG-labeled fragment containing the 3' 
most 400 bp of the CCLACC1-4 insert. Five positive clones are isolated and 
purified. Plasmid DNA is excised from all five clones by passage through 
E. coli DH10B. Using a specific primer to the 3' end of the CCLACC1-4 
insert in sequencing reactions as described in Example 5, it is determined 
that only one of the clones (LCC2-5B-1) contains the 3' missing portion of 
lcc2 gene. However, further sequencing demonstrates that (LCC2-5B-1) does 
not contain the whole gene but is missing part of the 5' end. Overlapping 
the sequences of CCLACC1-4 and CCLACC2-5B-1 yields the sequence of the 
entire gene (FIG. 3, SEQ ID NO:32). 
A plasmid pDSY105 containing the full-length lcc2 genomic clone is 
constructed by ligating together fragments from the LCC2-5B-1 and 
CCLACC1-4 clones. Clone LCC2-5B-1 is digested with EagI and BglII and 
electrophoresed on a 1% agarose gel. The gel slice containing the 1.3 kb 
EagI/BglII fragment is excised, and the DNA is isolated using a Spin Bind 
column (FMC). A PCR reaction is performed to obtain an EcoRI/BglII 
fragment containing the N-terminal half of lcc2. The PCR reaction mixture 
contains 0.1 mg of CCLACC1-4 DNA, 50 pmol each of oligonucleotides 96-0545 
and 96-0546, 0.01 mM each of dATP, dCTP, dGTP, and dTTP, and 0.5 U Taq DNA 
polymerase. PCR conditions are 1 cycle at 95.degree. C. for 5 minutes, 
55.degree. C. for 1 minute, and 72.degree. C. for 1 minute; and 30 cycles 
each at 95.degree. C. for 30 seconds, 55.degree. C. for 1 minute, and 
72.degree. C. for 1 minute. The primers used in the reaction are: 
96-0545: AGAATTGACTCCACCGACGAA (SEQ ID NO:34) 
96-0546: GAATTCTGGCATTCCTGACCTTTGTTC (SEQ ID NO:35) 
The desired product of 1.6 kb is subcloned into pCRII using the TA Cloning 
Kit (Invitrogen, San Diego, Calif.). Partial nucleotide sequences of the 
subclones are determined using M13-20 universal and M13 -48 reverse 
universal primers. The fmal plasmid is constructed by digesting 
pBluescript SK- with EcoRI/EagI and ligating with the EagI/BglII fragment 
from LCC2-5B-1 and the BglII/EcoRI fragment from the pCRII subclone. The 
resulting subclones are screened by restriction digests, and the desired 
product is designated pDSY105. 
The lcc2 gene contains 13 introns (indicated by lowercase in FIG. 3). The 
positions of introns 4 through 10 are confirmed from the partial cDNA 
while the positions of the other 6 introns are deduced based on the 
consensus sequences found at the 5' and 3' splice sites of fungal introns 
and by homology of the deduced amino acid sequence (FIG. 3, SEQ ID NO:33) 
to other laccases. The lcc2 gene encodes for a precursor protein of 517 
amino acids in length. There is one potential N-glycosylation site, and 
the mature protein after the predicted signal peptide cleavage is 499 
amino acids in length. 
From the alignment of the Lcc1, Lcc2 and Lcc3 predicted mature proteins, it 
appears that unlike Lcc1 neither Lcc2 nor Lcc3 contains the 23 amino acid 
extension present on Lcc1. Lcc1 shares 59.3% and 57.5% identity with Lcc2 
and Lcc3, respectively (Table 2). When compared to other fungal laccases, 
Lcc2 and Lcc3 have the highest identity (79.6%) with one another. The 
percent identities shared with other fungal laccases range from a high of 
62.8% for Lcc2 and the basidiomycete PM1 laccase to a low of 21.9% for 
Neospora Crassa laccase. 
Example 12 
Construction of pDSY67 and pDSY68 for heterologous expression of lcc1 in 
Aspergillus oryzae 
pDSY67 (FIG. 4) and pDSY68 (FIG. 5) are constructed for expression of 
Coprinus cinereus lcc1 gene. The Coprinus cinereus lcc1 gene is cloned 
into the expression vector pKS4 which contains the TAKA promoter, AMG 
terminator and the Aspergillus nidulans pyrg for selection. The lcc1 gene 
is inserted as 3 fragments into pKS4 digested with SwaI/NotI to obtain 
pDSY67 (FIG. 4). Sequencing of pDSY67 reveals the presence of 32 extra 
base pairs between the stop codon and the AMG terminator. pDSY68 is 
generated by removing the extra thirty-two base pairs. In order to remove 
the extra base pairs, pDSY67 is digested with PacI/NotI and the ends are 
blunted using T4 DNA polymerase. The blunt-end vector is ligated to 
itself, and the resulting plasmid pDSY68 is sequenced to confirm the extra 
base pairs are removed. 
Example 13 
Transformation of Aspergillus oryzae 
Aspergillus oryzae strains HowB712, JeRS316, and JeRS317 are grown for 18 
hours in YEG medium at 34.degree. C., and protoplasts are generated and 
transformed as described by Christensen et al. (1988, Biotechnology 6: 
1419-1422). The protoplasts are transformed with 10 .mu.g of either pDSY67 
or pDSY68. Transformants are selected on Minimal medium plates containing 
1.0 M sucrose. Minimal medium plates are comprised of 6.0 g of NaNO.sub.3, 
0.52 g of KCl, 1.52 g of KH.sub.2 PO.sub.4, 1.0 ml of trace metals 
solution, 20 g of Nobel Agar (Difco), 20 ml of 50% glucose, 20 ml of 
methionine (50 g/l), 20 ml of biotin (200 mg/l), 2.5 ml of 20% 
MgSO.sub.4.7H.sub.2 O, and 1.0 ml of mg/ml streptomycin per liter. The 
agar medium is adjusted to pH 6.5 prior to autoclaving and then glucose, 
methionine, biotin, MgSO.sub.4.7H.sub.2 O, and streptomycin are added as 
sterile solutions to the cooled autoclaved medium and poured into plates. 
The trace metals solution is comprised of 22 g of ZnSO.sub.4.7H.sub.2 O, 
11 g of H.sub.3 BO.sub.3, 5 g of MnCl.sub.2.4H.sub.2 O, 5 g of 
FeSO.sub.4.7H.sub.2 O, 1.6 g of CoCl.sub.2.5H.sub.2 O, 1.6 g of 
(NH.sub.4).sub.6 Mo.sub.7 O.sub.24, and 50 g of Na.sub.4 EDTA per liter. 
Example 14 
Screening of laccase transformants 
Primary transformants are screened first on Minimal medium plates 
containing 1% glucose as the carbon source and 1 mM 
2,2'-azinobis-(3-ethybenzthiazoline-6-sulfonic acid) (ABTS) to test for 
production of laccase. Transformants producing green zones on the ABTS 
plates are picked and spore purified before shake flask analysis. For 
shake flask analysis, the purified transformants are cultivated at 
37.degree. C. in MY51 medium comprised of 30 g of maltose, 2 g of 
MgSO.sub.4, 10 g of KH.sub.2 PO.sub.4, 2 g of K.sub.2 SO.sub.4, 2 g of 
citric acid, 10 g of yeast extract, 0.5 ml of trace metals solution, 1 g 
of urea, 2 g of (NH.sub.4).sub.2 SO.sub.4 pH 6.0 per liter. The trace 
metals solution is comprised of 14.3 g of ZnSO.sub.4.7H.sub.2 O, 2.5 g of 
CuSO.sub.4.5H.sub.2 O, 11 g of NiCl.sub.2.6H.sub.2 O, 13.8 g of 
FeSO.sub.4.7H.sub.2 O, 8.5 g of MnSO.sub.4.H.sub.2 O, and 3.0 g of citric 
acid per liter. Samples are taken at various intervals and centrifuged. 
The supernatants are diluted and assayed using ABTS as a substrate. 
Laccase activity is determined by syringaldazine oxidation. Specifically, 
60 .mu.l of syringaldazine stock solution (0.28 mM in 50% ethanol) and 20 
.mu.l of laccase sample are mixed with 0.8 ml of preheated 
Britton-Robinson buffer solution and incubated at 20.degree. C. The 
oxidation is monitored at 530 nm over 5 minutes and activity is expressed 
as "SOU" .mu.mole syringaldazine oxidized per minute ("SOU"). 
Britton-Robinson buffers with various pHs are used. ABTS oxidation assays 
are performed at 20.degree. C. using 1 mM ABTS, Britton-Robinson buffers 
(diluted 1.1-fold) by monitoring .DELTA.A405 in 96-well plates. 
For pDSY67, 3, 8, and 64 transformants, which are positive on ABTS, are 
obtained in Aspergillus oryzae JeRS316, JeRS317, and HowB712, 
respectively. For pDSY68, 34 and 56 transformants, which are positive on 
ABTS plates, are obtained in JeRS317 and HowB712, respectively. On average 
&gt;90% of the primary transformants are positive on ABTS plates. All of the 
transformants are spore purified and tested in shake flask for production 
of the laccase as described above. Laccase activity assays confirm that 
the transformants, which are positive on ABTS plates, are indeed producing 
laccase. 
Example 15 
Purification and characterization of recombinant Coprinus cinereus Lcc1 
Aspergillus oryzae JeRS317 (pDSY68, lcc1) is inoculated into a 10 liter lab 
fermentor containing medium comprised of Nutriose, yeast extract, 
(NH.sub.4).sub.2 HPO.sub.4, MgSO.sub.4.7H.sub.2 O, citric acid, K.sub.2 
SO.sub.4, CaCl.sub.2.H.sub.2 O, and trace metals solution and supplemented 
with CuSO.sub.4 and fermented at 31.degree. C., pH 7, 600-700 rpm for 7 
days. The broth is then recovered and filtered through cheesecloth. 
Cheesecloth filtered broth (pH 7.2, 15 mS) is filtered through Whatman #2 
filter paper, then concentrated and washed on a Spiral Concentrator 
(Amicon) with a S1Y30 membrane (16-fold, 0.8 mS). The broth is frozen 
overnight at -20.degree. C., thawed the next day, filtered again on 
Whatman #2 paper, and loaded onto a 120 ml Q-Sepharose XK26 column 
(Pharmacia, Uppsala, Sweden), pre-equilibrated with 10 mM Tris pH 7.7, 0.9 
mS (Buffer A). After loading and washing with Buffer A, a linear gradient 
with Buffer B (Buffer A plus 2 M NaCl) is applied and the active fractions 
are eluted around 7% Buffer B. The active fractions are dialyzed in Buffer 
A and then loaded onto a 40 ml Mono-Q 16/10 (Pharmacia, Uppsala, Sweden) 
column, pre-equilibrated with Buffer A. The active fractions pass through 
the column. 
The sequential ion-exchange chromatography on Q-Sepharose and Mono-Q yields 
a recombinant Coprinus cinereus laccase preparation with apparent 
homogeneity by SDS-PAGE analysis. An overall 64-fold purification and a 
recovery of 23% are achieved. 
A molecular weight of 66 kDa for the recombinant laccase is observed by 
SDS-PAGE analysis, similar to that of wild type laccase. The difference 
between the observed molecular weight and that derived from the DNA 
sequence (56 kDa) suggests the laccase is 18% glycosylated. The 
chromatographic elution pattern of recombinant laccase is very close to 
that of the recombinant Myceliophthora thermophila laccase under the same 
conditions, where the recombinant Coprinus cinereus laccase has a similar 
pI to the pI of 4.2 for recombinant Myceliophthora thermophila laccase, 
which is also close to the pI of wild type Coprinus cinereus laccase 
(3.7-4.0). 
Copper (Cu) titration of the purified recombinant laccase with 
2,2'-biquinoline is carried out as described by Felsenfeld, 1960, Archives 
of Biochemistry and Biophysics 87: 247-251. Photometric titration with 
2,2'-biquinoline gives a Cu to protein (subunit) stoichiometry of 
3.4.+-.0.2, indicating the four-Cu oxidase nature of recombinant Coprinus 
cinereus laccase. 
The purified recombinant Coprinus cinereus laccase shows a UV-visible 
spectrum with two maxima at 278 and 614 nm. The ratio of absorbance at 280 
nm to that at 600 nm is found as 22. 
The extinction coefficient for the enzyme is determined by amino acid 
analysis and the molecular weight derived from the DNA sequence. Amino 
acid analysis suggests an extinction coefficient of 1.6 l/(g*cm), similar 
to the predicted value of 1.2. 
The redox potential is measured by monitoring the recombinant Coprinus 
cinereus laccase's absorbance change at 600 nm with K.sub.3 Fe(CN).sub.6 
--K.sub.4 Fe(CN).sub.6 couple (0.433 V) and with I.sub.2 --NaI couple 
(0.536 V) in 9 mM MES-NaOH pH 5.3 buffer. At pH 5.3, a redox potential of 
0.55.+-.0.06 V is observed for the recombinant Coprinus cinereus laccase. 
The activity of recombinant Coprinus cinereus laccase is tested with 
syringaldazine and ABTS. With syringaldazine as the substrate, recombinant 
Coprinus cinereus laccase shows a LACU/A.sub.280 of 2.7 or a LACU/mg near 
4. The recombinant laccase exhibits a pH activity profile in the pH range 
from about 4 to about 9 with optimal activity at pH 6 to 7 similar to that 
of wild type Coprinus cinereus laccase (FIG. 6A), at which its 
SOU/A.sub.280 =5.6. At pH 5.3, syringaldazine shows a K.sub.m of 26.+-.6 
.mu.M and a k.sub.cat of 180.+-.20 min.sup.-1. With ABTS as the substrate, 
the recombinant laccase shows a pH activity profile in the pH range from 
about 2.7 to about 7 with optimal activity at pH 4 similar to wild type 
Coprinus cinereus laccase (FIG. 6B). At pH 5.3, a K.sub.m of 23.+-.3 .mu.M 
and a k.sub.cat of 1090.+-.30 min.sup.-1 are observed for ABTS oxidation. 
The values for K.sub.m and k.sub.cat are determined by fitting initial 
rates (v=.DELTA.A/.DELTA.t/.DELTA.e; .DELTA.e: extinction coefficient 
change), laccase concentration (E), and substrate concentration (S) into 
v=k.sub.cat *E*S/(K.sub.m +S) with the Prizm nonlinear regression software 
(GraphPad, San Diego, Calif.). Total amino acid analysis, from which the 
extinction coefficient is determined, is performed on a HP AminoQuant 
instrument. 
Example 16 
N-terminal sequencing 
Wild type Coprinus cinereus laccase is treated with a number of deblocking 
agents in order to remove the blocked N-terminus. Buffer exchange of 
samples is carried out in BioRad's BioSpin (P-6) device. Samples are 
treated with pyroglutamate aminopeptidase (Boehringer Mannheim, 
Indianapolis, Ind. and Sigma, St. Louis, Mo.), acylamino acid peptidase 
(Boehringer Mannheim, Indianapolis, Ind.), and acylase I (Sigma, St. 
Louis, Mo.) with deblocking protocols adapted from manufacturer's 
recommendations as follows. For pyroglutamate aminopeptidase treatment, a 
laccase sample is exchanged into 5% glycerol-10 mM EDTA-0.1 M sodium 
phosphate pH 8, then mixed with dithiothreitol (DTT) to 0.7 mM and horse 
liver peptidase (Sigma, St. Lois, Mo.) to 1/216 w/w laccase. The mixture 
(.about.6.2 mg/ml in laccase) is divided into three aliquots, of which one 
is adjusted 1 M urea and another is adjusted 0.5 M guanidine-HCl. Each 
sample is incubated at 4.degree. C. for 16 hours. For acylamino acid 
peptidase, a laccase sample is exchanged into 0.2 M NH.sub.4 HCO.sub.3 pH 
7.8, then mixed with EDTA to 1 mM, 2-mercaptoethanol to 1 mM, and 
peptidase to 1/5 w/w laccase. The mixture (.about.14 mg/ml in laccase) is 
divided into three aliquots, of which one is adjusted 0.01% in SDS, one is 
adjusted 0.08 M in guanidine-HCl, and another is adjusted 0.7 M in urea. 
Each sample is incubated at 37.degree. C. for 20 hours. For treatment with 
acylase I, a laccase sample is exchanged into 0.1 M sodium phosphate pH 7, 
then mixed with the acylase to 1/3 w/w of laccase. The mixture (.about.15 
mg/ml in laccase) is incubated at 37.degree. C. for 22 hours. 
The enzyme-treated laccase samples are concentrated using Amicon's 
Microcon-10 devices. The concentrated samples are run on SDS PAGE and 
electroblotted onto a PVDF membrane of sequencing grade (Novex, San Diego, 
Calif.). The PVDF membrane is stained with Coommassie blue R-250 to 
visualize the treated laccase bands. The PVDF membrane is cut to isolate 
the pieces containing the individual bands. Several lanes are combined and 
subjected directly to N-terminal sequencing on an ABI 476 Sequencer using 
a blot cartridge and liquid TFA delivery. 
The purified wild-type Coprinus cinereus laccase has a blocked N-terminus. 
However, treatment with both acylamino acid peptidase and acylase I leads 
to an identical sequenceable N-terminus. The resulting N-terminal sequence 
is shown below where it is uncertain whether S represents the actual 
N-terminus in the mature laccase, as, if this is the case, it would 
require an unexpected deacylase function by acylamino peptidase. 
SVDTMTLTNANVSPDGFTRAGI (SEQ ID NO:36) 
Under the conditions described, no deblocking is observed with 
pyroglutamate aminopeptidase. 
Direct N-terminal sequencing of the recombinant Coprinus cinereus laccase 
yields a blocked N-terminus, likely due to the same acylation at a Ser as 
observed in the wild-type laccase. 
Deposit of Biological Materials 
The following biological materials have been deposited under the terms of 
the Budapest Treaty with the Agricultural Research Service Patent Culture 
Collection, Northern Regional Research Center, 1815 University Street, 
Peoria, Ill., 61604, and given the following accession numbers: 
______________________________________ 
Accession 
Deposit Number Date of Deposit 
______________________________________ 
E. coli DH5.alpha. with pDSY71 
NRRL-B 21495 
August 18, 1995 
(lcc2 partial cDNA in pCRII) 
E. coli DH5.alpha. with pDSY72 NRRL-B 21496 August 18, 1995 
(lcc3 partial cDNA in pCRII) 
E. coli DH10B(ZL) with pDSY73 NRRL-B 21497 August 18, 1995 
(lcc1 genomic clone in pZL) 
E. coli DH5.alpha. with pDSY100 NRRL B-21589 June 21, 1996 
E. coli DH5.alpha. with pDSY105 NRRL B-21602 July 11, 1996 
______________________________________ 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 36 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Glu Val Asp Gly Gln Leu Thr Glu Pro His Th - #r Val Asp Arg Leu 
Gln 
1 5 - # 10 - # 15 
- - Ile Phe Thr Gly Gln Arg Tyr Ser Phe Val Le - #u Asp Ala Asn Gln Pro 
20 - # 25 - # 30 
- - Val Asp Asn Tyr Trp Ile Arg Ala 
35 - # 40 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
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1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
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1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
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1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
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(A) LENGTH: 17 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - Ile Leu Asp Pro Ala Xaa Pro Gly Ile Pro Th - #r Pro Gly Ala Ala Asp 
1 5 - # 10 - # 15 
- - Val 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
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1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
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(A) LENGTH: 7 amino - #acids 
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(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
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1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
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(D) TOPOLOGY: linear 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
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1 5 - # 10 - # 15 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
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1 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
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(A) LENGTH: 8 amino - #acids 
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(C) STRANDEDNESS: single 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
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1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
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1 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - Ala Gly Thr Phe 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
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1 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - Ile Pro Ala Pro Ser Ile Gln Gly Ala Ala Gl - #n Pro Asx Ala Thr 
1 5 - # 10 - # 15 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
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(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - ATCANTGGCA NGGNTNT - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
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(C) STRANDEDNESS: single 
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- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - ATCANTGGCA NGGTTNTTN - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- - TCATNTGNCA NTGANNAACC AGG - # - # 
23 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1176 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1176 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- - CAT TGG CAC GGT CTC TTC CAA CGA GGG ACC AA - #C TGG GCT GAT GGT GCA 
48 
His Trp His Gly Leu Phe Gln Arg Gly Thr As - #n Trp Ala Asp Gly Ala 
1 5 - # 10 - # 15 
- - GAT GGT GTC AAC CAG TGC CCG ATC TCT CCA GG - #C CAT GCT TTC CTC TAC 
96 
Asp Gly Val Asn Gln Cys Pro Ile Ser Pro Gl - #y His Ala Phe Leu Tyr 
20 - # 25 - # 30 
- - AAG TTC ACT CCA GCT GGC CAC GCT GGT ACT TT - #C TGG TAC CAT TCC CAC 
144 
Lys Phe Thr Pro Ala Gly His Ala Gly Thr Ph - #e Trp Tyr His Ser His 
35 - # 40 - # 45 
- - TTT GGC ACC CAA TAC TGC GAT GGT CTC CGT GG - #T CCA ATG GTC ATT TAC 
192 
Phe Gly Thr Gln Tyr Cys Asp Gly Leu Arg Gl - #y Pro Met Val Ile Tyr 
50 - # 55 - # 60 
- - GAC GAC AAT GAC CCA CAC GCT GCC CTC TAC GA - #C GAG GAT GAC GAG AAC 
240 
Asp Asp Asn Asp Pro His Ala Ala Leu Tyr As - #p Glu Asp Asp Glu Asn 
65 - # 70 - # 75 - # 80 
- - ACC ATC ATT ACC CTC GCC GAT TGG TAC CAT AT - #C CCC GCT CCC TCC ATT 
288 
Thr Ile Ile Thr Leu Ala Asp Trp Tyr His Il - #e Pro Ala Pro Ser Ile 
85 - # 90 - # 95 
- - CAG GGT GCT GCC CAG CCT GAC GCT ACG CTC AT - #C AAC GGT AAG GGT CGC 
336 
Gln Gly Ala Ala Gln Pro Asp Ala Thr Leu Il - #e Asn Gly Lys Gly Arg 
100 - # 105 - # 110 
- - TAC GTG GGC GGC CCA GCT GCC GAG CTT TCG AT - #C GTC AAT GTC GAG CAA 
384 
Tyr Val Gly Gly Pro Ala Ala Glu Leu Ser Il - #e Val Asn Val Glu Gln 
115 - # 120 - # 125 
- - GGG AAG AAG TAC CGA ATG CGT TTG ATC TCG CT - #G TCC TGC GAC CCC AAC 
432 
Gly Lys Lys Tyr Arg Met Arg Leu Ile Ser Le - #u Ser Cys Asp Pro Asn 
130 - # 135 - # 140 
- - TGG CAG TTC TCC ATT GAC GGA CAT GAG TTG AC - #G ATC ATT GAA GTC GAT 
480 
Trp Gln Phe Ser Ile Asp Gly His Glu Leu Th - #r Ile Ile Glu Val Asp 
145 1 - #50 1 - #55 1 - 
#60 
- - GGT CAG CTT ACT GAG CCG CAT ACG GTT GAT CG - #T CTC CAG ATC TTC 
ACT 528 
Gly Gln Leu Thr Glu Pro His Thr Val Asp Ar - #g Leu Gln Ile Phe Thr 
165 - # 170 - # 175 
- - GGT CAA AGG TAC TCC TTC GTT CTC GAC GCC AA - #C CAG CCG GTG GAC AAC 
576 
Gly Gln Arg Tyr Ser Phe Val Leu Asp Ala As - #n Gln Pro Val Asp Asn 
180 - # 185 - # 190 
- - TAC TGG ATC CGT GCT CAA CCC AAC AAG GGT CG - #A AAC GGA CTT GCT GGT 
624 
Tyr Trp Ile Arg Ala Gln Pro Asn Lys Gly Ar - #g Asn Gly Leu Ala Gly 
195 - # 200 - # 205 
- - ACC TTC GCC AAC GGT GTC AAC TCG GCC ATC CT - #T CGC TAT GCC GGC GCT 
672 
Thr Phe Ala Asn Gly Val Asn Ser Ala Ile Le - #u Arg Tyr Ala Gly Ala 
210 - # 215 - # 220 
- - GCC AAC GCT GAT CCA ACC ACC TCC GCC AAC CC - #C AAC CCC GCC CAA CTC 
720 
Ala Asn Ala Asp Pro Thr Thr Ser Ala Asn Pr - #o Asn Pro Ala Gln Leu 
225 2 - #30 2 - #35 2 - 
#40 
- - AAC GAA GCC GAC CTC CAT GCT CTC ATC GAC CC - #C GCT GCT CCC GGT 
ATC 768 
Asn Glu Ala Asp Leu His Ala Leu Ile Asp Pr - #o Ala Ala Pro Gly Ile 
245 - # 250 - # 255 
- - CCC ACT CCG GGC GCT GCA GAC GTC AAC CTC CG - #A TTC CAA TTG GGC TTC 
816 
Pro Thr Pro Gly Ala Ala Asp Val Asn Leu Ar - #g Phe Gln Leu Gly Phe 
260 - # 265 - # 270 
- - AGC GGC GGT CGA TTC ACG ATT AAC GGA ACC GC - #A TAC GAG AGT CCA AGC 
864 
Ser Gly Gly Arg Phe Thr Ile Asn Gly Thr Al - #a Tyr Glu Ser Pro Ser 
275 - # 280 - # 285 
- - GTT CCT ACG CTC TTG CAG ATT ATG AGT GGT GC - #G CAG AGT GCG AAC GAC 
912 
Val Pro Thr Leu Leu Gln Ile Met Ser Gly Al - #a Gln Ser Ala Asn Asp 
290 - # 295 - # 300 
- - TTG CTC CCT GCT GGA TCG GTG TAT GAG TTG CC - #C AGG AAC CAA GTT GTT 
960 
Leu Leu Pro Ala Gly Ser Val Tyr Glu Leu Pr - #o Arg Asn Gln Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- - GAG CTT GTT GTT CCT GCT GGT GTC CTC GGT GG - #T CCT CAT CCT TTC 
CAT 1008 
Glu Leu Val Val Pro Ala Gly Val Leu Gly Gl - #y Pro His Pro Phe His 
325 - # 330 - # 335 
- - CTC CAC GGT CAT GCG TTC AGT GTC GTC AGG AG - #T GCA GGC AGC AGC ACC 
1056 
Leu His Gly His Ala Phe Ser Val Val Arg Se - #r Ala Gly Ser Ser Thr 
340 - # 345 - # 350 
- - TAC AAC TTT GTC AAC CCC GTC AAG CGC GAT GT - #T GTT AGT CTT GGT GTT 
1104 
Tyr Asn Phe Val Asn Pro Val Lys Arg Asp Va - #l Val Ser Leu Gly Val 
355 - # 360 - # 365 
- - ACT GGA GAC GAA GTT ACC ATT CGA TTC GTC AC - #C GAT AAC CCA GGC CCG 
1152 
Thr Gly Asp Glu Val Thr Ile Arg Phe Val Th - #r Asp Asn Pro Gly Pro 
370 - # 375 - # 380 
- - TGG TTC TTC CAC TGC CAC ATT GAA - # - # 
1176 
Trp Phe Phe His Cys His Ile Glu 
385 3 - #90 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 392 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- - His Trp His Gly Leu Phe Gln Arg Gly Thr As - #n Trp Ala Asp Gly Ala 
1 5 - # 10 - # 15 
- - Asp Gly Val Asn Gln Cys Pro Ile Ser Pro Gl - #y His Ala Phe Leu Tyr 
20 - # 25 - # 30 
- - Lys Phe Thr Pro Ala Gly His Ala Gly Thr Ph - #e Trp Tyr His Ser His 
35 - # 40 - # 45 
- - Phe Gly Thr Gln Tyr Cys Asp Gly Leu Arg Gl - #y Pro Met Val Ile Tyr 
50 - # 55 - # 60 
- - Asp Asp Asn Asp Pro His Ala Ala Leu Tyr As - #p Glu Asp Asp Glu Asn 
65 - # 70 - # 75 - # 80 
- - Thr Ile Ile Thr Leu Ala Asp Trp Tyr His Il - #e Pro Ala Pro Ser Ile 
85 - # 90 - # 95 
- - Gln Gly Ala Ala Gln Pro Asp Ala Thr Leu Il - #e Asn Gly Lys Gly Arg 
100 - # 105 - # 110 
- - Tyr Val Gly Gly Pro Ala Ala Glu Leu Ser Il - #e Val Asn Val Glu Gln 
115 - # 120 - # 125 
- - Gly Lys Lys Tyr Arg Met Arg Leu Ile Ser Le - #u Ser Cys Asp Pro Asn 
130 - # 135 - # 140 
- - Trp Gln Phe Ser Ile Asp Gly His Glu Leu Th - #r Ile Ile Glu Val Asp 
145 1 - #50 1 - #55 1 - 
#60 
- - Gly Gln Leu Thr Glu Pro His Thr Val Asp Ar - #g Leu Gln Ile Phe 
Thr 
165 - # 170 - # 175 
- - Gly Gln Arg Tyr Ser Phe Val Leu Asp Ala As - #n Gln Pro Val Asp Asn 
180 - # 185 - # 190 
- - Tyr Trp Ile Arg Ala Gln Pro Asn Lys Gly Ar - #g Asn Gly Leu Ala Gly 
195 - # 200 - # 205 
- - Thr Phe Ala Asn Gly Val Asn Ser Ala Ile Le - #u Arg Tyr Ala Gly Ala 
210 - # 215 - # 220 
- - Ala Asn Ala Asp Pro Thr Thr Ser Ala Asn Pr - #o Asn Pro Ala Gln Leu 
225 2 - #30 2 - #35 2 - 
#40 
- - Asn Glu Ala Asp Leu His Ala Leu Ile Asp Pr - #o Ala Ala Pro Gly 
Ile 
245 - # 250 - # 255 
- - Pro Thr Pro Gly Ala Ala Asp Val Asn Leu Ar - #g Phe Gln Leu Gly Phe 
260 - # 265 - # 270 
- - Ser Gly Gly Arg Phe Thr Ile Asn Gly Thr Al - #a Tyr Glu Ser Pro Ser 
275 - # 280 - # 285 
- - Val Pro Thr Leu Leu Gln Ile Met Ser Gly Al - #a Gln Ser Ala Asn Asp 
290 - # 295 - # 300 
- - Leu Leu Pro Ala Gly Ser Val Tyr Glu Leu Pr - #o Arg Asn Gln Val Val 
305 3 - #10 3 - #15 3 - 
#20 
- - Glu Leu Val Val Pro Ala Gly Val Leu Gly Gl - #y Pro His Pro Phe 
His 
325 - # 330 - # 335 
- - Leu His Gly His Ala Phe Ser Val Val Arg Se - #r Ala Gly Ser Ser Thr 
340 - # 345 - # 350 
- - Tyr Asn Phe Val Asn Pro Val Lys Arg Asp Va - #l Val Ser Leu Gly Val 
355 - # 360 - # 365 
- - Thr Gly Asp Glu Val Thr Ile Arg Phe Val Th - #r Asp Asn Pro Gly Pro 
370 - # 375 - # 380 
- - Trp Phe Phe His Cys His Ile Glu 
385 3 - #90 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1170 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1170 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- - CAC TGG CAC GGC ATG TTC CAA AGG GGG ACT GC - #C TGG GCT GAT GGT CCT 
48 
His Trp His Gly Met Phe Gln Arg Gly Thr Al - #a Trp Ala Asp Gly Pro 
395 - # 400 - # 405 
- - GCT GGC GTC ACC CAA TGC CCT ATT TCC CCA GG - #G CAT TCG TTC TTG TAC 
96 
Ala Gly Val Thr Gln Cys Pro Ile Ser Pro Gl - #y His Ser Phe Leu Tyr 
410 - # 415 - # 420 
- - AAG TTC CAG GCT CTT AAC CAA GCC GGT ACT TT - #C TGG TAC CAC TCC CAT 
144 
Lys Phe Gln Ala Leu Asn Gln Ala Gly Thr Ph - #e Trp Tyr His Ser His 
425 4 - #30 4 - #35 4 - 
#40 
- - CAC GAA TCG CAA TAT TGT GAC GGT TTG CGT GG - #G GCT ATG GTC GTA 
TAT 192 
His Glu Ser Gln Tyr Cys Asp Gly Leu Arg Gl - #y Ala Met Val Val Tyr 
445 - # 450 - # 455 
- - GAC CCA GTC GAC CCA CAT CGC AAC TTG TAT GA - #C ATT GAC AAC GAG GCC 
240 
Asp Pro Val Asp Pro His Arg Asn Leu Tyr As - #p Ile Asp Asn Glu Ala 
460 - # 465 - # 470 
- - ACG ATC ATT ACG CTC GCA GAC TGG TAT CAC GT - #C CCT GCT CCC TCT GCA 
288 
Thr Ile Ile Thr Leu Ala Asp Trp Tyr His Va - #l Pro Ala Pro Ser Ala 
475 - # 480 - # 485 
- - GGT CTC GTT CCC ACC CCA GAT TCC ACG CTT AT - #C AAC GGT AAG GGC CGG 
336 
Gly Leu Val Pro Thr Pro Asp Ser Thr Leu Il - #e Asn Gly Lys Gly Arg 
490 - # 495 - # 500 
- - TAT GCT GGT GGC CCT ACC GTA CCT CTC GCG GT - #C ATT TCT GTA ACC CGA 
384 
Tyr Ala Gly Gly Pro Thr Val Pro Leu Ala Va - #l Ile Ser Val Thr Arg 
505 5 - #10 5 - #15 5 - 
#20 
- - AAC CGA CGA TAC CGG TTC CGC CTT GTT TCC CT - #T TCA TGC GAT CCT 
AAT 432 
Asn Arg Arg Tyr Arg Phe Arg Leu Val Ser Le - #u Ser Cys Asp Pro Asn 
525 - # 530 - # 535 
- - TAT GTA TTC TCT ATC GAT GGG CAT ACC ATG AC - #T GTT ATT GAG GTC GAC 
480 
Tyr Val Phe Ser Ile Asp Gly His Thr Met Th - #r Val Ile Glu Val Asp 
540 - # 545 - # 550 
- - GGA GTT AAC GTC CAA CCT CTC GTT GTC GAC TC - #G ATC CAG ATC TTC GCA 
528 
Gly Val Asn Val Gln Pro Leu Val Val Asp Se - #r Ile Gln Ile Phe Ala 
555 - # 560 - # 565 
- - GGT CAG CGC TAC TCG TTC GTT CTC AAC GCC AA - #C CGC CCC GTC GGC AAC 
576 
Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala As - #n Arg Pro Val Gly Asn 
570 - # 575 - # 580 
- - TAC TGG GTG CGA GCC AAC CCC AAC ATC GGT AC - #T ACG GGC TTC GTC GGT 
624 
Tyr Trp Val Arg Ala Asn Pro Asn Ile Gly Th - #r Thr Gly Phe Val Gly 
585 5 - #90 5 - #95 6 - 
#00 
- - GGA GTC AAT TCT GCG ATT CTG CGC TAT GTG GG - #C GCC TCC AAT ACA 
GAC 672 
Gly Val Asn Ser Ala Ile Leu Arg Tyr Val Gl - #y Ala Ser Asn Thr Asp 
605 - # 610 - # 615 
- - CCC ACT ACC ACC CAA ACT CCT TTC AGC AAC CC - #T CTC CTT GAG ACC AAT 
720 
Pro Thr Thr Thr Gln Thr Pro Phe Ser Asn Pr - #o Leu Leu Glu Thr Asn 
620 - # 625 - # 630 
- - CTC CAC CCC TTG ACC AAC CCT GCT GCT CCT GG - #C TTG CCT ACC CCA GGT 
768 
Leu His Pro Leu Thr Asn Pro Ala Ala Pro Gl - #y Leu Pro Thr Pro Gly 
635 - # 640 - # 645 
- - GGC GTC GAC GTC GCG ATC AAC CTT AAC ACG GT - #A TTC GAT TTC AGT AGT 
816 
Gly Val Asp Val Ala Ile Asn Leu Asn Thr Va - #l Phe Asp Phe Ser Ser 
650 - # 655 - # 660 
- - CTC ACC TTC TCC GTT AAC GGA GCC ACT TTC CA - #T CAA CCG CCC GTC CCT 
864 
Leu Thr Phe Ser Val Asn Gly Ala Thr Phe Hi - #s Gln Pro Pro Val Pro 
665 6 - #70 6 - #75 6 - 
#80 
- - GTC TTG CTT CAG ATC ATG AGC GGT GCA CAG AC - #T GCC CAG CAG CTT 
CTT 912 
Val Leu Leu Gln Ile Met Ser Gly Ala Gln Th - #r Ala Gln Gln Leu Leu 
685 - # 690 - # 695 
- - CCC TCC GGT TCG GTC TAC GTC CTT CCC CGT AA - #C AAA GTC ATC GAG CTT 
960 
Pro Ser Gly Ser Val Tyr Val Leu Pro Arg As - #n Lys Val Ile Glu Leu 
700 - # 705 - # 710 
- - TCT ATG CCT GGA GGC TCC ACT GGC AGT CCC CA - #T CCC TTC CAT CTC CAC 
1008 
Ser Met Pro Gly Gly Ser Thr Gly Ser Pro Hi - #s Pro Phe His Leu His 
715 - # 720 - # 725 
- - GGT CAC GAA TTT GCT GTG GTG AGA AGC GCG GG - #G AGT TCG ACC TAC AAC 
1056 
Gly His Glu Phe Ala Val Val Arg Ser Ala Gl - #y Ser Ser Thr Tyr Asn 
730 - # 735 - # 740 
- - TTC GCG AAC CCG GTA CGC AGG GAT GTC GTG AG - #T GCC GGT GTT GCT GGT 
1104 
Phe Ala Asn Pro Val Arg Arg Asp Val Val Se - #r Ala Gly Val Ala Gly 
745 7 - #50 7 - #55 7 - 
#60 
- - GAC AAC GTC ACC ATT CGA TTC CGT ACC GAT AA - #C CCT GGA CCA TGG 
ATT 1152 
Asp Asn Val Thr Ile Arg Phe Arg Thr Asp As - #n Pro Gly Pro Trp Ile 
765 - # 770 - # 775 
- - CTC CAT TGC CAT ATC GAC - # - # 
- #1170 
Leu His Cys His Ile Asp 
780 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 390 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- - His Trp His Gly Met Phe Gln Arg Gly Thr Al - #a Trp Ala Asp Gly Pro 
1 5 - # 10 - # 15 
- - Ala Gly Val Thr Gln Cys Pro Ile Ser Pro Gl - #y His Ser Phe Leu Tyr 
20 - # 25 - # 30 
- - Lys Phe Gln Ala Leu Asn Gln Ala Gly Thr Ph - #e Trp Tyr His Ser His 
35 - # 40 - # 45 
- - His Glu Ser Gln Tyr Cys Asp Gly Leu Arg Gl - #y Ala Met Val Val Tyr 
50 - # 55 - # 60 
- - Asp Pro Val Asp Pro His Arg Asn Leu Tyr As - #p Ile Asp Asn Glu Ala 
65 - # 70 - # 75 - # 80 
- - Thr Ile Ile Thr Leu Ala Asp Trp Tyr His Va - #l Pro Ala Pro Ser Ala 
85 - # 90 - # 95 
- - Gly Leu Val Pro Thr Pro Asp Ser Thr Leu Il - #e Asn Gly Lys Gly Arg 
100 - # 105 - # 110 
- - Tyr Ala Gly Gly Pro Thr Val Pro Leu Ala Va - #l Ile Ser Val Thr Arg 
115 - # 120 - # 125 
- - Asn Arg Arg Tyr Arg Phe Arg Leu Val Ser Le - #u Ser Cys Asp Pro Asn 
130 - # 135 - # 140 
- - Tyr Val Phe Ser Ile Asp Gly His Thr Met Th - #r Val Ile Glu Val Asp 
145 1 - #50 1 - #55 1 - 
#60 
- - Gly Val Asn Val Gln Pro Leu Val Val Asp Se - #r Ile Gln Ile Phe 
Ala 
165 - # 170 - # 175 
- - Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala As - #n Arg Pro Val Gly Asn 
180 - # 185 - # 190 
- - Tyr Trp Val Arg Ala Asn Pro Asn Ile Gly Th - #r Thr Gly Phe Val Gly 
195 - # 200 - # 205 
- - Gly Val Asn Ser Ala Ile Leu Arg Tyr Val Gl - #y Ala Ser Asn Thr Asp 
210 - # 215 - # 220 
- - Pro Thr Thr Thr Gln Thr Pro Phe Ser Asn Pr - #o Leu Leu Glu Thr Asn 
225 2 - #30 2 - #35 2 - 
#40 
- - Leu His Pro Leu Thr Asn Pro Ala Ala Pro Gl - #y Leu Pro Thr Pro 
Gly 
245 - # 250 - # 255 
- - Gly Val Asp Val Ala Ile Asn Leu Asn Thr Va - #l Phe Asp Phe Ser Ser 
260 - # 265 - # 270 
- - Leu Thr Phe Ser Val Asn Gly Ala Thr Phe Hi - #s Gln Pro Pro Val Pro 
275 - # 280 - # 285 
- - Val Leu Leu Gln Ile Met Ser Gly Ala Gln Th - #r Ala Gln Gln Leu Leu 
290 - # 295 - # 300 
- - Pro Ser Gly Ser Val Tyr Val Leu Pro Arg As - #n Lys Val Ile Glu Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Ser Met Pro Gly Gly Ser Thr Gly Ser Pro Hi - #s Pro Phe His Leu 
His 
325 - # 330 - # 335 
- - Gly His Glu Phe Ala Val Val Arg Ser Ala Gl - #y Ser Ser Thr Tyr Asn 
340 - # 345 - # 350 
- - Phe Ala Asn Pro Val Arg Arg Asp Val Val Se - #r Ala Gly Val Ala Gly 
355 - # 360 - # 365 
- - Asp Asn Val Thr Ile Arg Phe Arg Thr Asp As - #n Pro Gly Pro Trp Ile 
370 - # 375 - # 380 
- - Leu His Cys His Ile Asp 
385 3 - #90 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1161 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1161 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- - CAC TGG CAC GGT TTC TTG CAG GAG GGT ACA GC - #T TGG GCC GAC GGT CCT 
48 
His Trp His Gly Phe Leu Gln Glu Gly Thr Al - #a Trp Ala Asp Gly Pro 
395 - # 400 - # 405 
- - GCG GGT GTT ACT CAA TGC CCC ATT GCC CCT GG - #T CAC TCT TTC CTC TAT 
96 
Ala Gly Val Thr Gln Cys Pro Ile Ala Pro Gl - #y His Ser Phe Leu Tyr 
410 - # 415 - # 420 
- - AAG TTC CAG GCC AAA AAC CAA GCT GGT ACC TT - #C TGG TAC CAT TCC CAC 
144 
Lys Phe Gln Ala Lys Asn Gln Ala Gly Thr Ph - #e Trp Tyr His Ser His 
425 - # 430 - # 435 
- - CAC ATG TCT CAG TAT TGT GAC GGC CTG AGA GG - #C GTC ATG GTC GTT TAC 
192 
His Met Ser Gln Tyr Cys Asp Gly Leu Arg Gl - #y Val Met Val Val Tyr 
440 - # 445 - # 450 
- - GAT CCC CTA GAT CCC CAT CGT CAC CTG TAT GA - #C GTT GAT AAC GAG AAT 
240 
Asp Pro Leu Asp Pro His Arg His Leu Tyr As - #p Val Asp Asn Glu Asn 
455 4 - #60 4 - #65 4 - 
#70 
- - ACT ATC ATC ACG CTC GCG GAC TGG TAT CAC GA - #T CCC GCC CCT TCT 
GCT 288 
Thr Ile Ile Thr Leu Ala Asp Trp Tyr His As - #p Pro Ala Pro Ser Ala 
475 - # 480 - # 485 
- - GGA CTC GTC CCA ACC CCC TGG TCG ACT TTG AT - #C AAT GGC AAG GGC CGT 
336 
Gly Leu Val Pro Thr Pro Trp Ser Thr Leu Il - #e Asn Gly Lys Gly Arg 
490 - # 495 - # 500 
- - TAC CCA GGC GGA CCC GTC GTG CCC TTG GCC GT - #C ATT CAC GTC AGC CGC 
384 
Tyr Pro Gly Gly Pro Val Val Pro Leu Ala Va - #l Ile His Val Ser Arg 
505 - # 510 - # 515 
- - GGA AAG CGC TAC CGC TTC CGC CTC GTC TCC CT - #T TCG TGC GAC CCT AAC 
432 
Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Le - #u Ser Cys Asp Pro Asn 
520 - # 525 - # 530 
- - TAT GTA TTC TCT ATT GAC GGT CAC ACC ATG AC - #G GTC ATT GAA GTC GAT 
480 
Tyr Val Phe Ser Ile Asp Gly His Thr Met Th - #r Val Ile Glu Val Asp 
535 5 - #40 5 - #45 5 - 
#50 
- - GGT GTC AAC CAT GAA CCG TTG GTT GTC GAC CA - #C ATT CAA ATC TTT 
GCT 528 
Gly Val Asn His Glu Pro Leu Val Val Asp Hi - #s Ile Gln Ile Phe Ala 
555 - # 560 - # 565 
- - GGT CAA CGG TAC TCG TTT GTC TTG AAC GCC AA - #C CGG CCC GTC AAC AAC 
576 
Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala As - #n Arg Pro Val Asn Asn 
570 - # 575 - # 580 
- - TAC TGG GTC AGG GCT AAC CCC AAC CTC GGC TC - #T GTC GGC TTC GGT GGC 
624 
Tyr Trp Val Arg Ala Asn Pro Asn Leu Gly Se - #r Val Gly Phe Gly Gly 
585 - # 590 - # 595 
- - GGT ATT AAT TCC GCA ATT CTG CGA TAT GTT GG - #A GCT CCT GCC GTC GAC 
672 
Gly Ile Asn Ser Ala Ile Leu Arg Tyr Val Gl - #y Ala Pro Ala Val Asp 
600 - # 605 - # 610 
- - CCA ACC ACC TCC CAA TTG CCT TTC AGC AAC CC - #A CTC CTC GAG ACC AAC 
720 
Pro Thr Thr Ser Gln Leu Pro Phe Ser Asn Pr - #o Leu Leu Glu Thr Asn 
615 6 - #20 6 - #25 6 - 
#30 
- - TTG CAC CCT CTC GTA AAT CCT GCT GCA CCT GG - #C GGC CCT TCC CCC 
GGT 768 
Leu His Pro Leu Val Asn Pro Ala Ala Pro Gl - #y Gly Pro Ser Pro Gly 
635 - # 640 - # 645 
- - GAC GTC GAT GTC GCC ATC AAC CTG GAT ATC TT - #G TTC GAC GTC TCA ATC 
816 
Asp Val Asp Val Ala Ile Asn Leu Asp Ile Le - #u Phe Asp Val Ser Ile 
650 - # 655 - # 660 
- - CTC AAG TTC ACT GTC AAC GGT GCT ACC TTC GA - #T GAA CCA CCC GTT CCG 
864 
Leu Lys Phe Thr Val Asn Gly Ala Thr Phe As - #p Glu Pro Pro Val Pro 
665 - # 670 - # 675 
- - GTC CTT CTC CAG ATT TTG AGC GGT GCA CAT AC - #C GCC TCA TCT CTT CTC 
912 
Val Leu Leu Gln Ile Leu Ser Gly Ala His Th - #r Ala Ser Ser Leu Leu 
680 - # 685 - # 690 
- - CCC TCT GGC AGC GTC TAC ACT CTT CCC CCT AA - #C AAG GTC ATT GAG CTC 
960 
Pro Ser Gly Ser Val Tyr Thr Leu Pro Pro As - #n Lys Val Ile Glu Leu 
695 7 - #00 7 - #05 7 - 
#10 
- - ACT ATT CCC GGT GGT GGT ATC GGT GCT CCT CA - #C CCC ATC CAT CTT 
CAC 1008 
Thr Ile Pro Gly Gly Gly Ile Gly Ala Pro Hi - #s Pro Ile His Leu His 
715 - # 720 - # 725 
- - GGC CAT ACC TTC AAG GTT GTC CGT AGC GCA GG - #C AGC TCG ACT TAC AAC 
1056 
Gly His Thr Phe Lys Val Val Arg Ser Ala Gl - #y Ser Ser Thr Tyr Asn 
730 - # 735 - # 740 
- - TTC GTC AAT CCC GTT GAG CGA GAT GTT GTC AA - #C GTT GGT CAA GCT GGC 
1104 
Phe Val Asn Pro Val Glu Arg Asp Val Val As - #n Val Gly Gln Ala Gly 
745 - # 750 - # 755 
- - GAC AAT GTC ACC ATT CGA TTC GTC ACT GAT AA - #T GCT GGT CCC TGG ATT 
1152 
Asp Asn Val Thr Ile Arg Phe Val Thr Asp As - #n Ala Gly Pro Trp Ile 
760 - # 765 - # 770 
- - CTT CAC TGC - # - # 
- # 1161 
Leu His Cys 
775 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 387 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- - His Trp His Gly Phe Leu Gln Glu Gly Thr Al - #a Trp Ala Asp Gly 
Pro 
1 5 - # 10 - # 15 
- - Ala Gly Val Thr Gln Cys Pro Ile Ala Pro Gl - #y His Ser Phe Leu Tyr 
20 - # 25 - # 30 
- - Lys Phe Gln Ala Lys Asn Gln Ala Gly Thr Ph - #e Trp Tyr His Ser His 
35 - # 40 - # 45 
- - His Met Ser Gln Tyr Cys Asp Gly Leu Arg Gl - #y Val Met Val Val Tyr 
50 - # 55 - # 60 
- - Asp Pro Leu Asp Pro His Arg His Leu Tyr As - #p Val Asp Asn Glu Asn 
65 - # 70 - # 75 - # 80 
- - Thr Ile Ile Thr Leu Ala Asp Trp Tyr His As - #p Pro Ala Pro Ser Ala 
85 - # 90 - # 95 
- - Gly Leu Val Pro Thr Pro Trp Ser Thr Leu Il - #e Asn Gly Lys Gly Arg 
100 - # 105 - # 110 
- - Tyr Pro Gly Gly Pro Val Val Pro Leu Ala Va - #l Ile His Val Ser Arg 
115 - # 120 - # 125 
- - Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Le - #u Ser Cys Asp Pro Asn 
130 - # 135 - # 140 
- - Tyr Val Phe Ser Ile Asp Gly His Thr Met Th - #r Val Ile Glu Val Asp 
145 1 - #50 1 - #55 1 - 
#60 
- - Gly Val Asn His Glu Pro Leu Val Val Asp Hi - #s Ile Gln Ile Phe 
Ala 
165 - # 170 - # 175 
- - Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala As - #n Arg Pro Val Asn Asn 
180 - # 185 - # 190 
- - Tyr Trp Val Arg Ala Asn Pro Asn Leu Gly Se - #r Val Gly Phe Gly Gly 
195 - # 200 - # 205 
- - Gly Ile Asn Ser Ala Ile Leu Arg Tyr Val Gl - #y Ala Pro Ala Val Asp 
210 - # 215 - # 220 
- - Pro Thr Thr Ser Gln Leu Pro Phe Ser Asn Pr - #o Leu Leu Glu Thr Asn 
225 2 - #30 2 - #35 2 - 
#40 
- - Leu His Pro Leu Val Asn Pro Ala Ala Pro Gl - #y Gly Pro Ser Pro 
Gly 
245 - # 250 - # 255 
- - Asp Val Asp Val Ala Ile Asn Leu Asp Ile Le - #u Phe Asp Val Ser Ile 
260 - # 265 - # 270 
- - Leu Lys Phe Thr Val Asn Gly Ala Thr Phe As - #p Glu Pro Pro Val Pro 
275 - # 280 - # 285 
- - Val Leu Leu Gln Ile Leu Ser Gly Ala His Th - #r Ala Ser Ser Leu Leu 
290 - # 295 - # 300 
- - Pro Ser Gly Ser Val Tyr Thr Leu Pro Pro As - #n Lys Val Ile Glu Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Thr Ile Pro Gly Gly Gly Ile Gly Ala Pro Hi - #s Pro Ile His Leu 
His 
325 - # 330 - # 335 
- - Gly His Thr Phe Lys Val Val Arg Ser Ala Gl - #y Ser Ser Thr Tyr Asn 
340 - # 345 - # 350 
- - Phe Val Asn Pro Val Glu Arg Asp Val Val As - #n Val Gly Gln Ala Gly 
355 - # 360 - # 365 
- - Asp Asn Val Thr Ile Arg Phe Val Thr Asp As - #n Ala Gly Pro Trp Ile 
370 - # 375 - # 380 
- - Leu His Cys 
385 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- - ACTGCGATGG TCTCCGTGGT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- - GGGGCCTGGG TTATCGGTGA C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3327 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(726..85 - #1, 907..1023, 1101..1247, 
1316..1696, - #1752..2240, 2321..2494, 2548..2607, 
2670..2793) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- - CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AA - 
#CCATCACC 60 
- - CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CT - 
#AAAGGGAG 120 
- - CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AA - 
#GGGAAGAA 180 
- - AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GC - 
#GTAACCAC 240 
- - CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCC CATTCGCCAT TC - 
#AGGCTGCG 300 
- - CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC TG - 
#GCGAAAGG 360 
- - GGGATGTGCT GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT CA - 
#CGACGTTG 420 
- - TAAAACGACG GCCAGTGAAT TGAATTTAGG TGACACTATA GAAGAGCTAT GA - 
#CGTCGCAT 480 
- - GCACGCGTAC GTAAGCTTGG ATCCTCTAGA GCGACCGCCG ACTAGTGAGC TC - 
#GTCGACCC 540 
- - GGGAATTGCA GCGTCCCTGG TCGTACGTTA GCCTACGCTT TACAGCACCG AA - 
#AGAAGTAT 600 
- - AAAATCTGTA TGAAAGTTGG CGAAGAAACC TCAGACTACT CTCGTCGTCT AT - 
#CTTCACTC 660 
- - CTCTGCTCCT CTCTCCTCCA CAGACTCTCC TTGACAGCCT CGTCGTATCA GA - 
#GAACAGAA 720 
- - CAACA ATG TTC AAG AAC CTC CTC TCG TTC GCC - #CTT CTG GCG ATT AGC 
767 
Met Phe Lys Asn Leu Leu Ser - #Phe Ala Leu Leu Ala Ile Ser 
1 - # 5 - # 10 
- - GTT GCC AAC GCT CAG ATC GTC AAT TCG GTC GA - #T ACC ATG ACC CTC ACC 
815 
Val Ala Asn Ala Gln Ile Val Asn Ser Val As - #p Thr Met Thr Leu Thr 
15 - # 20 - # 25 - # 30 
- - AAC GCG AAC GTC AGT CCC GAC GGT TTC ACT CG - #A GCT GTAAGTATAG 
861 
Asn Ala Asn Val Ser Pro Asp Gly Phe Thr Ar - #g Ala 
35 - # 40 
- - GTCTTCAGCA CACTGTTGAT TATCCATTAC TTACCAACTT AACAG GGT - #ATC CTC 
915 
- # - # Gly Ile L - #eu 
- # - # - # 45 
- - GTC AAT GGA GTT CAT GGA CCT CTT ATT CGA GG - #T GGA AAG AAC GAC AAC 
963 
Val Asn Gly Val His Gly Pro Leu Ile Arg Gl - #y Gly Lys Asn Asp Asn 
50 - # 55 - # 60 
- - TTT GAG CTC AAC GTC GTT AAC GAC TTG GAC AA - #C CCC ACT ATG CTT CGG 
1011 
Phe Glu Leu Asn Val Val Asn Asp Leu Asp As - #n Pro Thr Met Leu Arg 
65 - # 70 - # 75 
- - CCT ACC AGT ATC GTGAGTTCTA CAGAAATAAA CACTGATCCA TC - #ATGATCCA 
1063 
Pro Thr Ser Ile 
80 
- - GAACACTGAC AACGTTCTGA TTTTGGTTTG CTTGTAG CAT TGG CAC - #GGT CTC TTC 
1118 
- # - # His Trp His Gly Leu Phe 
- # - # - #85 
- - CAA CGA GGG ACC AAC TGG GCT GAT GGT GCA GA - #T GGT GTC AAC CAG TGC 
1166 
Gln Arg Gly Thr Asn Trp Ala Asp Gly Ala As - #p Gly Val Asn Gln Cys 
90 - # 95 - # 100 
- - CCG ATC TCT CCA GGC CAT GCT TTC CTC TAC AA - #G TTC ACT CCA GCT GGC 
1214 
Pro Ile Ser Pro Gly His Ala Phe Leu Tyr Ly - #s Phe Thr Pro Ala Gly 
105 - # 110 - # 115 
- - CAC GCT GGT ACT TTC TGG TAC CAT TCC CAC TT - #T GTAAGCCCGA CCCCCCGAC 
T 1267 
His Ala Gly Thr Phe Trp Tyr His Ser His Ph - #e 
120 1 - #25 1 - #30 
- - ATGATCATCT TGACTGGAGT CCTGATTGAT GTCCAACTAA TTTACTAG GGC - #ACC CAA 
1324 
- # - # Gly - # Thr 
Gln 
- - TAC TGC GAT GGT CTC CGT GGT CCA ATG GTC AT - #T TAC GAC GAC AAT 
GAC 1372 
Tyr Cys Asp Gly Leu Arg Gly Pro Met Val Il - #e Tyr Asp Asp Asn Asp 
135 - # 140 - # 145 
- - CCA CAC GCT GCC CTC TAC GAC GAG GAT GAC GA - #G AAC ACC ATC ATT ACC 
1420 
Pro His Ala Ala Leu Tyr Asp Glu Asp Asp Gl - #u Asn Thr Ile Ile Thr 
150 1 - #55 1 - #60 1 - 
#65 
- - CTC GCC GAT TGG TAC CAT ATC CCC GCT CCC TC - #C ATT CAG GGT GCT 
GCC 1468 
Leu Ala Asp Trp Tyr His Ile Pro Ala Pro Se - #r Ile Gln Gly Ala Ala 
170 - # 175 - # 180 
- - CAG CCT GAC GCT ACG CTC ATC AAC GGT AAG GG - #T CGC TAC GTG GGC GGC 
1516 
Gln Pro Asp Ala Thr Leu Ile Asn Gly Lys Gl - #y Arg Tyr Val Gly Gly 
185 - # 190 - # 195 
- - CCA GCT GCC GAG CTT TCG ATC GTC AAT GTC GA - #G CAA GGG AAG AAG TAC 
1564 
Pro Ala Ala Glu Leu Ser Ile Val Asn Val Gl - #u Gln Gly Lys Lys Tyr 
200 - # 205 - # 210 
- - CGA ATG CGT TTG ATC TCG CTG TCC TGC GAC CC - #C AAC TGG CAG TTC TCC 
1612 
Arg Met Arg Leu Ile Ser Leu Ser Cys Asp Pr - #o Asn Trp Gln Phe Ser 
215 - # 220 - # 225 
- - ATT GAC GGA CAT GAG TTG ACG ATC ATT GAA GT - #C GAT GGT CAG CTT ACT 
1660 
Ile Asp Gly His Glu Leu Thr Ile Ile Glu Va - #l Asp Gly Gln Leu Thr 
230 2 - #35 2 - #40 2 - 
#45 
- - GAG CCG CAT ACG GTT GAT CGT CTC CAG ATC TT - #C ACT GTAAGCATTG 
1706 
Glu Pro His Thr Val Asp Arg Leu Gln Ile Ph - #e Thr 
250 - # 255 
- - AAATCGGTGT GTTTCCGTTG AGAAAGCACA CTCACCTTTA ATCAG GGT - #CAA AGG 
1760 
- # - # Gly Gln A - #rg 
- # - # - # 260 
- - TAC TCC TTC GTT CTC GAC GCC AAC CAG CCG GT - #G GAC AAC TAC TGG ATC 
1808 
Tyr Ser Phe Val Leu Asp Ala Asn Gln Pro Va - #l Asp Asn Tyr Trp Ile 
265 - # 270 - # 275 
- - CGT GCT CAA CCC AAC AAG GGT CGA AAC GGA CT - #T GCT GGT ACC TTC GCC 
1856 
Arg Ala Gln Pro Asn Lys Gly Arg Asn Gly Le - #u Ala Gly Thr Phe Ala 
280 - # 285 - # 290 
- - AAC GGT GTC AAC TCG GCC ATC CTT CGC TAT GC - #C GGC GCT GCC AAC GCT 
1904 
Asn Gly Val Asn Ser Ala Ile Leu Arg Tyr Al - #a Gly Ala Ala Asn Ala 
295 - # 300 - # 305 
- - GAT CCA ACC ACC TCC GCC AAC CCC AAC CCC GC - #C CAA CTC AAC GAA GCC 
1952 
Asp Pro Thr Thr Ser Ala Asn Pro Asn Pro Al - #a Gln Leu Asn Glu Ala 
310 - # 315 - # 320 
- - GAC CTC CAT GCT CTC ATC GAC CCC GCT GCT CC - #C GGT ATC CCC ACT CCG 
2000 
Asp Leu His Ala Leu Ile Asp Pro Ala Ala Pr - #o Gly Ile Pro Thr Pro 
325 3 - #30 3 - #35 3 - 
#40 
- - GGC GCT GCA GAC GTC AAC CTC CGA TTC CAA TT - #G GGC TTC AGC GGC 
GGT 2048 
Gly Ala Ala Asp Val Asn Leu Arg Phe Gln Le - #u Gly Phe Ser Gly Gly 
345 - # 350 - # 355 
- - CGA TTC ACG ATT AAC GGA ACC GCA TAC GAG AG - #T CCA AGC GTT CCT ACG 
2096 
Arg Phe Thr Ile Asn Gly Thr Ala Tyr Glu Se - #r Pro Ser Val Pro Thr 
360 - # 365 - # 370 
- - CTC TTG CAG ATT ATG AGT GGT GCG CAG AGT GC - #G AAC GAC TTG CTC CCT 
2144 
Leu Leu Gln Ile Met Ser Gly Ala Gln Ser Al - #a Asn Asp Leu Leu Pro 
375 - # 380 - # 385 
- - GCT GGA TCG GTG TAT GAG TTG CCC AGG AAC CA - #A GTT GTT GAG CTT GTT 
2192 
Ala Gly Ser Val Tyr Glu Leu Pro Arg Asn Gl - #n Val Val Glu Leu Val 
390 - # 395 - # 400 
- - GTT CCT GCT GGT GTC CTC GGT GGT CCT CAT CC - #T TTC CAT CTC CAC GGT 
2240 
Val Pro Ala Gly Val Leu Gly Gly Pro His Pr - #o Phe His Leu His Gly 
405 4 - #10 4 - #15 4 - 
#20 
- - GTACGTCAAG TTTTCTTTTC TCTTCTTTTT TTCATGGGTG GTCAAGTGTA CA - 
#TGAGCTTA 2300 
- - CCAAGGATTG AATTGTGTAG CAT GCG TTC AGT GTC GTC AG - #G AGT GCA GGC 
2350 
- # His Ala Phe Ser Val Val Arg Ser - #Ala Gly 
- # - # 425 - # 430 
- - AGC AGC ACC TAC AAC TTT GTC AAC CCC GTC AA - #G CGC GAT GTT GTT AGT 
2398 
Ser Ser Thr Tyr Asn Phe Val Asn Pro Val Ly - #s Arg Asp Val Val Ser 
435 - # 440 - # 445 
- - CTT GGT GTT ACT GGA GAC GAA GTT ACC ATT CG - #A TTC GTC ACC GAT AAC 
2446 
Leu Gly Val Thr Gly Asp Glu Val Thr Ile Ar - #g Phe Val Thr Asp Asn 
450 - # 455 - # 460 
- - CCA GGC CCG TGG TTC TTC CAC TGC CAC ATT GA - #A TTC CAT CTC ATG AAC 
2494 
Pro Gly Pro Trp Phe Phe His Cys His Ile Gl - #u Phe His Leu Met Asn 
465 - # 470 - # 475 
- - GTAAGTCTTC ATATCCATCG TTGTATACTC CAGAGTCTAA CCCACCTCCA CA - #G GGC 
2550 
- # - # - # Gly 
- - TTG GCG ATC GTC TTT GCT GAA GAC ATG GCG AA - #C ACG GTT GAT GCT AAC 
2598 
Leu Ala Ile Val Phe Ala Glu Asp Met Ala As - #n Thr Val Asp Ala Asn 
480 4 - #85 4 - #90 4 - 
#95 
- - AAC CCA CCT GTACGTCCCC TCCTATTGAC TCAAATACTA ATTTCCGAA - #G 
2647 
Asn Pro Pro 
- - CTAACTTCGG CATCAATTAC AG GTC GAG TGG GCC CAG CTT - # TGC GAG ATT 
TAC 2699 
- # Val Glu Trp Ala Gln Leu C - #ys Glu Ile Tyr 
- # 500 - # 505 
- - GAT GAC CTG CCG CCT GAG GCG ACC TCG ATT CA - #A ACC GTT GTG CGT CGC 
2747 
Asp Asp Leu Pro Pro Glu Ala Thr Ser Ile Gl - #n Thr Val Val Arg Arg 
510 - # 515 - # 520 
- - GCT GAG CCC ACC GGC TTT TCG GCC AAG TTC CG - #C AGG GAG GGC TTG 
2792 
Ala Glu Pro Thr Gly Phe Ser Ala Lys Phe Ar - #g Arg Glu Gly Leu 
525 5 - #30 5 - #35 
- - TAGATAATAT TATAGTTGAC CAGAGGGCCA GTGGTAGGAG GCTGCTATAG TC - 
#AAAGTTGG 2852 
- - TCACAGAGGG AAGAGTTAGT CGCAGAGAAG TCGTTTGAGT ACTACTAGTT AT - 
#TCATCGTG 2912 
- - TTGTTATTTA TCGTGGTTGT TACATACTTA TTAACTATCG TTATGTGTGC TT - 
#GAGTTTGG 2972 
- - AATGACAATG TATTTGATTG TCGAGTTGGA ATCCTTGTTG AAGTGCTAGT AA - 
#CCTTTTGA 3032 
- - TGGACCTCCG ACCTGCCTTT TCCCCGCACT TCCTCGATTG AATATTTGAG CG - 
#CGAGGCAC 3092 
- - GAATCGAACC CACGACCCGC GAATGTCCAA ATTCCGGACC GGTACCTGCA GG - 
#CGTACCAG 3152 
- - CTTTCCCTAT AGTGAGTCGT ATTAGAGCTT GGCGTAATCA TGGTCATAGC TG - 
#TTTCCTGT 3212 
- - GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TA - 
#AAGTGTAA 3272 
- - AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT CA - #CTG 
3327 
- - - - (2) INFORMATION FOR SEQ ID NO:27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 539 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- - Met Phe Lys Asn Leu Leu Ser Phe Ala Leu Le - #u Ala Ile Ser Val Ala 
1 5 - # 10 - # 15 
- - Asn Ala Gln Ile Val Asn Ser Val Asp Thr Me - #t Thr Leu Thr Asn Ala 
20 - # 25 - # 30 
- - Asn Val Ser Pro Asp Gly Phe Thr Arg Ala Gl - #y Ile Leu Val Asn Gly 
35 - # 40 - # 45 
- - Val His Gly Pro Leu Ile Arg Gly Gly Lys As - #n Asp Asn Phe Glu Leu 
50 - # 55 - # 60 
- - Asn Val Val Asn Asp Leu Asp Asn Pro Thr Me - #t Leu Arg Pro Thr Ser 
65 - # 70 - # 75 - # 80 
- - Ile His Trp His Gly Leu Phe Gln Arg Gly Th - #r Asn Trp Ala Asp Gly 
85 - # 90 - # 95 
- - Ala Asp Gly Val Asn Gln Cys Pro Ile Ser Pr - #o Gly His Ala Phe Leu 
100 - # 105 - # 110 
- - Tyr Lys Phe Thr Pro Ala Gly His Ala Gly Th - #r Phe Trp Tyr His Ser 
115 - # 120 - # 125 
- - His Phe Gly Thr Gln Tyr Cys Asp Gly Leu Ar - #g Gly Pro Met Val Ile 
130 - # 135 - # 140 
- - Tyr Asp Asp Asn Asp Pro His Ala Ala Leu Ty - #r Asp Glu Asp Asp Glu 
145 1 - #50 1 - #55 1 - 
#60 
- - Asn Thr Ile Ile Thr Leu Ala Asp Trp Tyr Hi - #s Ile Pro Ala Pro 
Ser 
165 - # 170 - # 175 
- - Ile Gln Gly Ala Ala Gln Pro Asp Ala Thr Le - #u Ile Asn Gly Lys Gly 
180 - # 185 - # 190 
- - Arg Tyr Val Gly Gly Pro Ala Ala Glu Leu Se - #r Ile Val Asn Val Glu 
195 - # 200 - # 205 
- - Gln Gly Lys Lys Tyr Arg Met Arg Leu Ile Se - #r Leu Ser Cys Asp Pro 
210 - # 215 - # 220 
- - Asn Trp Gln Phe Ser Ile Asp Gly His Glu Le - #u Thr Ile Ile Glu Val 
225 2 - #30 2 - #35 2 - 
#40 
- - Asp Gly Gln Leu Thr Glu Pro His Thr Val As - #p Arg Leu Gln Ile 
Phe 
245 - # 250 - # 255 
- - Thr Gly Gln Arg Tyr Ser Phe Val Leu Asp Al - #a Asn Gln Pro Val Asp 
260 - # 265 - # 270 
- - Asn Tyr Trp Ile Arg Ala Gln Pro Asn Lys Gl - #y Arg Asn Gly Leu Ala 
275 - # 280 - # 285 
- - Gly Thr Phe Ala Asn Gly Val Asn Ser Ala Il - #e Leu Arg Tyr Ala Gly 
290 - # 295 - # 300 
- - Ala Ala Asn Ala Asp Pro Thr Thr Ser Ala As - #n Pro Asn Pro Ala Gln 
305 3 - #10 3 - #15 3 - 
#20 
- - Leu Asn Glu Ala Asp Leu His Ala Leu Ile As - #p Pro Ala Ala Pro 
Gly 
325 - # 330 - # 335 
- - Ile Pro Thr Pro Gly Ala Ala Asp Val Asn Le - #u Arg Phe Gln Leu Gly 
340 - # 345 - # 350 
- - Phe Ser Gly Gly Arg Phe Thr Ile Asn Gly Th - #r Ala Tyr Glu Ser Pro 
355 - # 360 - # 365 
- - Ser Val Pro Thr Leu Leu Gln Ile Met Ser Gl - #y Ala Gln Ser Ala Asn 
370 - # 375 - # 380 
- - Asp Leu Leu Pro Ala Gly Ser Val Tyr Glu Le - #u Pro Arg Asn Gln Val 
385 3 - #90 3 - #95 4 - 
#00 
- - Val Glu Leu Val Val Pro Ala Gly Val Leu Gl - #y Gly Pro His Pro 
Phe 
405 - # 410 - # 415 
- - His Leu His Gly His Ala Phe Ser Val Val Ar - #g Ser Ala Gly Ser Ser 
420 - # 425 - # 430 
- - Thr Tyr Asn Phe Val Asn Pro Val Lys Arg As - #p Val Val Ser Leu Gly 
435 - # 440 - # 445 
- - Val Thr Gly Asp Glu Val Thr Ile Arg Phe Va - #l Thr Asp Asn Pro Gly 
450 - # 455 - # 460 
- - Pro Trp Phe Phe His Cys His Ile Glu Phe Hi - #s Leu Met Asn Gly Leu 
465 4 - #70 4 - #75 4 - 
#80 
- - Ala Ile Val Phe Ala Glu Asp Met Ala Asn Th - #r Val Asp Ala Asn 
Asn 
485 - # 490 - # 495 
- - Pro Pro Val Glu Trp Ala Gln Leu Cys Glu Il - #e Tyr Asp Asp Leu Pro 
500 - # 505 - # 510 
- - Pro Glu Ala Thr Ser Ile Gln Thr Val Val Ar - #g Arg Ala Glu Pro Thr 
515 - # 520 - # 525 
- - Gly Phe Ser Ala Lys Phe Arg Arg Glu Gly Le - #u 
530 - # 535 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2940 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(588..70 - #4, 758..823, 877..945, 999..1145, 
1200..1271, - #1324..1338, 1409..1438, 1488..1685, 
1749..2276, - #2340..2360, 2413..2562, 2619..2642, 
2694..2750, - #2804..2859) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- - ACTCACTATA GGGAAAGCTG GTACGCCTGC AGGTACCGGT CCGGAATTCC TT - 
#TCACCCCA 60 
- - GATCCTGGTA TAGGATAGAC CCAGATACTC TTACTAAGGT GGCACGAATG AC - 
#CGACCGAA 120 
- - TCTCGCGAGA AATCTTTCAA CTTTTCCAGA CACTTGATGA GTCGAAAACA AT - 
#GCGTTTAC 180 
- - CCCTGGAGTT ACGGATTGGG TCTCAAGTGA CTGTTACAAC AAGCGCTCAG GA - 
#TCCCCTAG 240 
- - TATGTCTAAT CGTGACGTCT CTACCGACGC TTGGCGCTCA TTGACAGCTA TC - 
#GCGACAGA 300 
- - TTCTTACATT TTTGTCAACG CCATCCTTTC TCGTTTACGT AGCTTTCTGC TA - 
#CGGTGCTG 360 
- - TCCTTTGTCA GAGATCCCTC CAGCACGACG ATTGATAACG AGATCTCAGT CG - 
#ACGGAACG 420 
- - GCTCCCTGGA CCTGATGCAC TTATCCTCTT ACTCATTGCA GTCATTACAA TC - 
#GAGTCTCG 480 
- - TTCGCACGTT GTCACGGAAC GGGACCTGAA AAATGAAGGA TATAAACCCC CA - 
#AGTGCCGC 540 
- - CCTGAAACTT TCAGACTTTT TGAGTCGACA AGCTCGAGGT CTCCAAC ATG - #CAA TTG 
596 
- # - # Met - #Gln Leu 
- # - # - #1 
- - CTT GCC TTC GTC CTC GCT GCT TTA CCC CTC GC - #A CGG GCT GCC ATT GGC 
644 
Leu Ala Phe Val Leu Ala Ala Leu Pro Leu Al - #a Arg Ala Ala Ile Gly 
5 - # 10 - # 15 
- - CCT GTT GGC AAC CTA GTC ATC GCC AAC GCG AA - #C GTC TCA CCA GAC GGC 
692 
Pro Val Gly Asn Leu Val Ile Ala Asn Ala As - #n Val Ser Pro Asp Gly 
20 - # 25 - # 30 - # 35 
- - TTC GTT CGC TCG GTGAGTGGGC CCGCGGCCTT TCACCATTTC TT - #TTCATTAA 
744 
Phe Val Arg Ser 
- - CTCTCCTCTG CAG GCT GTC CTT GCC GGC GCT ACA GG - #T ACC AGC CTT GAG 
793 
Ala Val - #Leu Ala Gly Ala Thr Gly Thr Ser Leu Glu 
40 - # 45 - # 50 
- - CAC CCA GGG CCT GTT ATC GTG GGC CAG AAG GT - #AACACTAT TGACGTCCCT 
843 
His Pro Gly Pro Val Ile Val Gly Gln Lys 
55 - # 60 
- - TGGTCAGAAT CCTTCCTTAC ACCCTTTATC TAG GGC GAC ACT TT - #C CAC ATC 
AAT 897 
- # - # Gly Asp Thr Phe His Ile Asn 
- # - # 65 
- - GTC ATC GAT GAC CTT ACT GAC CCC ACT ATG CT - #T CGA ACA ACC AGT ATT 
945 
Val Ile Asp Asp Leu Thr Asp Pro Thr Met Le - #u Arg Thr Thr Ser Ile 
70 - # 75 - # 80 
- - GTAAAGCAAA TTTGCTTGGC ATCCTTCAAA CTTCACACTG ACGTTCATGT CA - #G CAC 
1001 
- # - # - # His 
- # - # - # 85 
- - TGG CAC GGT TTC TTG CAG GAG GGT ACA GCT TG - #G GCC GAC GGT CCT GCG 
1049 
Trp His Gly Phe Leu Gln Glu Gly Thr Ala Tr - #p Ala Asp Gly Pro Ala 
90 - # 95 - # 100 
- - GGT GTT ACT CAA TGC CCC ATT GCC CCT GGT CA - #C TCT TTC CTC TAT AAG 
1097 
Gly Val Thr Gln Cys Pro Ile Ala Pro Gly Hi - #s Ser Phe Leu Tyr Lys 
105 - # 110 - # 115 
- - TTC CAG GCC AAA AAC CAA GCT GGT ACC TTC TG - #G TAC CAT TCC CAC CAC 
1145 
Phe Gln Ala Lys Asn Gln Ala Gly Thr Phe Tr - #p Tyr His Ser His His 
120 - # 125 - # 130 
- - GTGAGAGCGA TGCTGGTAAC GGACCTTGGG TCAATACTGA CTCTTGACTT AC - #AG ATG 
1202 
- # - # - # Met 
- - TCT CAG TAT TGT GAC GGC CTG AGA GGC GTC AT - #G GTC GTT TAC GAT CCC 
1250 
Ser Gln Tyr Cys Asp Gly Leu Arg Gly Val Me - #t Val Val Tyr Asp Pro 
135 1 - #40 1 - #45 1 - 
#50 
- - CTA GAT CCC CAT CGT CAC CTG GTGCGTACGC CTATCTATG - #A CTCTCACCTT 
1301 
Leu Asp Pro His Arg His Leu 
155 
- - CGTACTCATT CCACCTACAC AG TAT GAC GTT GAT AAC GTA - #ATCCTTC 
1348 
- # Tyr Asp Val Asp Asn 
- # 160 
- - CAACCCTTAC GTCTCCGCTA AAGCTTACAT TCAATCTTCA TTGTTTCCTC AT - 
#TTTCTCAG 1408 
- - GAG AAT ACT ATC ATC ACG CTC GCG GAC TGG GT - #AAGCGCGC AAATAACCTA 
1458 
Glu Asn Thr Ile Ile Thr Leu Ala Asp Trp 
165 - # 170 
- - CGAAAGTTCC AGTATCTGAC TGTTTTCAG TAT CAC GAT CCC GCC - # CCT TCT GCT 
1511 
- # Tyr His A - #sp Pro Ala Pro Ser Ala 
- # - # 175 - # 180 
- - GGA CTC GTC CCA ACC CCC TGG TCG ACT TTG AT - #C AAT GGC AAG GGC CGT 
1559 
Gly Leu Val Pro Thr Pro Trp Ser Thr Leu Il - #e Asn Gly Lys Gly Arg 
185 - # 190 - # 195 
- - TAC CCA GGC GGA CCC GTC GTG CCC TTG GCC GT - #C ATT CAC GTC AGC CGC 
1607 
Tyr Pro Gly Gly Pro Val Val Pro Leu Ala Va - #l Ile His Val Ser Arg 
200 - # 205 - # 210 
- - GGA AAG CGC TAC CGC TTC CGC CTC GTC TCC CT - #T TCG TGC GAC CCT AAC 
1655 
Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Le - #u Ser Cys Asp Pro Asn 
215 - # 220 - # 225 
- - TAT GTA TTC TCT ATT GAC GGT CAC ACC ATG GT - #TCGTAACC CTCCCATAAT 
1705 
Tyr Val Phe Ser Ile Asp Gly His Thr Met 
230 - # 235 
- - CCACTCCTCC CCTGCCTCAT ATTTTACGTT TTGCGACTGT TAG ACG GT - #C ATT GAA 
1760 
- # - # Thr Val Ile Gl - 
#u 
- # - # 240 
- - GTC GAT GGT GTC AAC CAT GAA CCG TTG GTT GT - #C GAC CAC ATT CAA 
ATC 1808 
Val Asp Gly Val Asn His Glu Pro Leu Val Va - #l Asp His Ile Gln Ile 
245 - # 250 - # 255 
- - TTT GCT GGT CAA CGG TAC TCG TTT GTC TTG AA - #C GCC AAC CGG CCC GTC 
1856 
Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu As - #n Ala Asn Arg Pro Val 
260 - # 265 - # 270 
- - AAC AAC TAC TGG GTC AGG GCT AAC CCC AAC CT - #C GGC TCT GTC GGC TTC 
1904 
Asn Asn Tyr Trp Val Arg Ala Asn Pro Asn Le - #u Gly Ser Val Gly Phe 
275 2 - #80 2 - #85 2 - 
#90 
- - GGT GGC GGT ATT AAT TCC GCA ATT CTG CGA TA - #T GTT GGA GCT CCT 
GCC 1952 
Gly Gly Gly Ile Asn Ser Ala Ile Leu Arg Ty - #r Val Gly Ala Pro Ala 
295 - # 300 - # 305 
- - GTC GAC CCA ACC ACC TCC CAA TTG CCT TTC AG - #C AAC CCA CTC CTC GAG 
2000 
Val Asp Pro Thr Thr Ser Gln Leu Pro Phe Se - #r Asn Pro Leu Leu Glu 
310 - # 315 - # 320 
- - ACC AAC TTG CAC CCT CTC GTA AAT CCT GCT GC - #A CCT GGC GGC CCT TCC 
2048 
Thr Asn Leu His Pro Leu Val Asn Pro Ala Al - #a Pro Gly Gly Pro Ser 
325 - # 330 - # 335 
- - CCC GGT GAC GTC GAT GTC GCC ATC AAC CTG GA - #T ATC TTG TTC GAC GTC 
2096 
Pro Gly Asp Val Asp Val Ala Ile Asn Leu As - #p Ile Leu Phe Asp Val 
340 - # 345 - # 350 
- - TCA ATC CTC AAG TTC ACT GTC AAC GGT GCT AC - #C TTC GAT GAA CCA CCC 
2144 
Ser Ile Leu Lys Phe Thr Val Asn Gly Ala Th - #r Phe Asp Glu Pro Pro 
355 3 - #60 3 - #65 3 - 
#70 
- - GTT CCG GTC CTT CTC CAG ATT TTG AGC GGT GC - #A CAT ACC GCC TCA 
TCT 2192 
Val Pro Val Leu Leu Gln Ile Leu Ser Gly Al - #a His Thr Ala Ser Ser 
375 - # 380 - # 385 
- - CTT CTC CCC TCT GGC AGC GTC TAC ACT CTT CC - #C CCT AAC AAG GTC ATT 
2240 
Leu Leu Pro Ser Gly Ser Val Tyr Thr Leu Pr - #o Pro Asn Lys Val Ile 
390 - # 395 - # 400 
- - GAG CTC ACT ATT CCC GGT GGT GGT ATC GGT GC - #T CCT GTAGGTCTTT 
2286 
Glu Leu Thr Ile Pro Gly Gly Gly Ile Gly Al - #a Pro 
405 - # 410 
- - CTTCTTCATC TTTCTCTCGA TCTCGATGGT GTTCACTCAC TATTTGAAAC CA - #G CAC 
2342 
- # - # - # His 
- # - # - # 415 
- - CCC ATC CAT CTT CAC GGC GTGAGTATCC ATCCGTTAAG CT - #TCATTAAG 
2390 
Pro Ile His Leu His Gly 
420 
- - TCCCATGCTG ACCGTTTGAC AG CAT ACC TTC AAG GTT GTC - # CGT AGC GCA 
GGC 2442 
- # His Thr Phe Lys Val Val A - #rg Ser Ala Gly 
- # - # 425 - # 430 
- - AGC TCG ACT TAC AAC TTC GTC AAT CCC GTT GA - #G CGA GAT GTT GTC AAC 
2490 
Ser Ser Thr Tyr Asn Phe Val Asn Pro Val Gl - #u Arg Asp Val Val Asn 
435 - # 440 - # 445 
- - GTT GGT CAA GCT GGC GAC AAT GTC ACC ATT CG - #A TTC GTC ACT GAT AAT 
2538 
Val Gly Gln Ala Gly Asp Asn Val Thr Ile Ar - #g Phe Val Thr Asp Asn 
450 - # 455 - # 460 
- - GCT GGT CCC TGG ATT CTT CAC TGC GTGCGCTATT TC - #TTTAGGCA TTCAACGTGT 
2592 
Ala Gly Pro Trp Ile Leu His Cys 
465 - # 470 
- - CAGAGTCTTA CCCCCGTTCT TTTCAG CAC ATT GAC TGG CAT - #TTG GTT TTG 
2642 
- # His Ile Asp Trp - #His Leu Val Leu 
- # - # 475 
- - GTAAGTTCAC GTTTTGACGC ATCAGGCGAA TGGTACTCTA ACTTCCTCCA G - #GGC CTG 
2699 
- # - # - # Gly 
Leu 
- # - # - # 480 
- - TCT GTC GTC TTC GCG GAA GAT GTC CCC ACC AT - #C GAT AGC TCC GTT CAA 
2747 
Ser Val Val Phe Ala Glu Asp Val Pro Thr Il - #e Asp Ser Ser Val Gln 
485 - # 490 - # 495 
- - CCT GTAAGTTCTG CGTGCCTCTG CTCGATATCA TTTGGCTGAC TTCTTGGCT - #T TAG 
2803 
Pro 
- - CCC GCC TGG CAT GAT CTG TGC CCC ATC TAT GA - #C GCT CTT CCC CCC GGC 
2851 
Pro Ala Trp His Asp Leu Cys Pro Ile Tyr As - #p Ala Leu Pro Pro Gly 
500 - # 505 - # 510 
- - ACG AGG TAATCTCGCC CATGACATAC TGGCACGGTA TGACTTGGAC AG - #GTTACGGA 
2907 
Thr Arg 
515 
- - AATCAAAGTA AATGTTGGAT AAGAAGAATA ACA - # - # 
2940 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 516 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- - Met Gln Leu Leu Ala Phe Val Leu Ala Ala Le - #u Pro Leu Ala Arg Ala 
1 5 - # 10 - # 15 
- - Ala Ile Gly Pro Val Gly Asn Leu Val Ile Al - #a Asn Ala Asn Val Ser 
20 - # 25 - # 30 
- - Pro Asp Gly Phe Val Arg Ser Ala Val Leu Al - #a Gly Ala Thr Gly Thr 
35 - # 40 - # 45 
- - Ser Leu Glu His Pro Gly Pro Val Ile Val Gl - #y Gln Lys Gly Asp Thr 
50 - # 55 - # 60 
- - Phe His Ile Asn Val Ile Asp Asp Leu Thr As - #p Pro Thr Met Leu Arg 
65 - # 70 - # 75 - # 80 
- - Thr Thr Ser Ile His Trp His Gly Phe Leu Gl - #n Glu Gly Thr Ala Trp 
85 - # 90 - # 95 
- - Ala Asp Gly Pro Ala Gly Val Thr Gln Cys Pr - #o Ile Ala Pro Gly His 
100 - # 105 - # 110 
- - Ser Phe Leu Tyr Lys Phe Gln Ala Lys Asn Gl - #n Ala Gly Thr Phe Trp 
115 - # 120 - # 125 
- - Tyr His Ser His His Met Ser Gln Tyr Cys As - #p Gly Leu Arg Gly Val 
130 - # 135 - # 140 
- - Met Val Val Tyr Asp Pro Leu Asp Pro His Ar - #g His Leu Tyr Asp Val 
145 1 - #50 1 - #55 1 - 
#60 
- - Asp Asn Glu Asn Thr Ile Ile Thr Leu Ala As - #p Trp Tyr His Asp 
Pro 
165 - # 170 - # 175 
- - Ala Pro Ser Ala Gly Leu Val Pro Thr Pro Tr - #p Ser Thr Leu Ile Asn 
180 - # 185 - # 190 
- - Gly Lys Gly Arg Tyr Pro Gly Gly Pro Val Va - #l Pro Leu Ala Val Ile 
195 - # 200 - # 205 
- - His Val Ser Arg Gly Lys Arg Tyr Arg Phe Ar - #g Leu Val Ser Leu Ser 
210 - # 215 - # 220 
- - Cys Asp Pro Asn Tyr Val Phe Ser Ile Asp Gl - #y His Thr Met Thr Val 
225 2 - #30 2 - #35 2 - 
#40 
- - Ile Glu Val Asp Gly Val Asn His Glu Pro Le - #u Val Val Asp His 
Ile 
245 - # 250 - # 255 
- - Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Va - #l Leu Asn Ala Asn Arg 
260 - # 265 - # 270 
- - Pro Val Asn Asn Tyr Trp Val Arg Ala Asn Pr - #o Asn Leu Gly Ser Val 
275 - # 280 - # 285 
- - Gly Phe Gly Gly Gly Ile Asn Ser Ala Ile Le - #u Arg Tyr Val Gly Ala 
290 - # 295 - # 300 
- - Pro Ala Val Asp Pro Thr Thr Ser Gln Leu Pr - #o Phe Ser Asn Pro Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Leu Glu Thr Asn Leu His Pro Leu Val Asn Pr - #o Ala Ala Pro Gly 
Gly 
325 - # 330 - # 335 
- - Pro Ser Pro Gly Asp Val Asp Val Ala Ile As - #n Leu Asp Ile Leu Phe 
340 - # 345 - # 350 
- - Asp Val Ser Ile Leu Lys Phe Thr Val Asn Gl - #y Ala Thr Phe Asp Glu 
355 - # 360 - # 365 
- - Pro Pro Val Pro Val Leu Leu Gln Ile Leu Se - #r Gly Ala His Thr Ala 
370 - # 375 - # 380 
- - Ser Ser Leu Leu Pro Ser Gly Ser Val Tyr Th - #r Leu Pro Pro Asn Lys 
385 3 - #90 3 - #95 4 - 
#00 
- - Val Ile Glu Leu Thr Ile Pro Gly Gly Gly Il - #e Gly Ala Pro His 
Pro 
405 - # 410 - # 415 
- - Ile His Leu His Gly His Thr Phe Lys Val Va - #l Arg Ser Ala Gly Ser 
420 - # 425 - # 430 
- - Ser Thr Tyr Asn Phe Val Asn Pro Val Glu Ar - #g Asp Val Val Asn Val 
435 - # 440 - # 445 
- - Gly Gln Ala Gly Asp Asn Val Thr Ile Arg Ph - #e Val Thr Asp Asn Ala 
450 - # 455 - # 460 
- - Gly Pro Trp Ile Leu His Cys His Ile Asp Tr - #p His Leu Val Leu Gly 
465 4 - #70 4 - #75 4 - 
#80 
- - Leu Ser Val Val Phe Ala Glu Asp Val Pro Th - #r Ile Asp Ser Ser 
Val 
485 - # 490 - # 495 
- - Gln Pro Pro Ala Trp His Asp Leu Cys Pro Il - #e Tyr Asp Ala Leu Pro 
500 - # 505 - # 510 
- - Pro Gly Thr Arg 
515 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- - AGCTCGATGA CTTTGTTACG G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- - CAGCGCTACT CGTTCGTTCT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3566 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(456..57 - #8, 631..696, 746..814, 869..1015, 
1069..1140, - #1199..1213, 1271..1300, 1366..1563, 
1622..2149, - #2213..2233, 2303..2452, 2514..2537, 
2598..2654, - #2725..2776) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- - TGAAGGAGAA TCCCTCGAAG TGGAATTTTC TTTCCAGAAG ATGCAATCTG GT - 
#TTTGTCTC 60 
- - ATCCATTTTT GTGACGTTTA CTCACCATTT CGAATCTAGG ATCGTTCGCC GA - 
#TTTGCTCA 120 
- - TATCTTTGCG ACCACTCAAT ATTGCTTTAC GTACCCCCTC GTGAGAGGCA CA - 
#AATGCATT 180 
- - CCTTGCGATG CCCGATTCCA ATCTCAATGC AGGTACGTCC CTGGTTTCAT AC - 
#CAATGCGT 240 
- - GTTTTGGACT GGCATTCCTG ACCTTTGTTC CGGTTGACGT TTCTAGTTAT TT - 
#CGTGTGAC 300 
- - CTGTATGATT AATCGTACAG CCTGAATCTT GTCCTCAAAG TGCACAAATT AG - 
#GGCTCAAG 360 
- - CTACCAGGCG AGGCAGGTAT AAAGCGCTCT ACTCTCCATC CGACGTTCCC CA - 
#CTCACCAC 420 
- - CAGCCGGCTG AGTTCACCCG TTCTTGAAAC TCGTT ATG TTG CTT - #TTA GCG ACT 
473 
- # - # Met Leu Leu Leu Ala Thr 
- # - # 1 - # 5 
- - GCT CTC GCT ACA TCC CTC TTA CCT TTC GTG CT - #G GGA GCC ATT GGC CCC 
521 
Ala Leu Ala Thr Ser Leu Leu Pro Phe Val Le - #u Gly Ala Ile Gly Pro 
10 - # 15 - # 20 
- - AGT ACC AAC CTT GTC GTC GCG AAC AAG GTC AT - #C GCT CCC GAC GGC TTC 
569 
Ser Thr Asn Leu Val Val Ala Asn Lys Val Il - #e Ala Pro Asp Gly Phe 
25 - # 30 - # 35 
- - AGT CGA TCT GTGAGCCTTT TCTGTGGACT GGACGCTTCT TCAGTGACT - #G 
618 
Ser Arg Ser 
40 
- - ATCATGTCGC AG GCT GTC CTC GCT GGC GCT ACC CAG - # CCA ACG GTG CAG 
666 
Ala Val Le - #u Ala Gly Ala Thr Gln Pro Thr Val Gln 
- # 45 - # 50 
- - TTC CCT GGC CCC GTC ATT CAA GGG AAT AAG GT - #AGGCAGAT TTCAACCGTT 
716 
Phe Pro Gly Pro Val Ile Gln Gly Asn Lys 
55 - # 60 
- - TCCTGTCACA TCATGTTGAG TCTTTGTAG AAC AGT TTC TTT GCG - # ATC AAC GTC 
769 
- # Asn Ser P - #he Phe Ala Ile Asn Val 
- # - # 65 - # 70 
- - ATT GAC GCT CTG ACC GAC CCC ACT ATG CTG AG - #G ACT ACG AGT ATC 
81 - #4 
Ile Asp Ala Leu Thr Asp Pro Thr Met Leu Ar - #g Thr Thr Ser Ile 
75 - # 80 - # 85 
- - GTAAGTCAGT TCTATTGATG CTGCGATCAG CGGAAGCTCA CCATCTTTTA AC - #AG CAC 
871 
- # - # - # His 
- - TGG CAC GGC ATG TTC CAA AGG GGG ACT GCC TG - #G GCT GAT GGT CCT GCT 
919 
Trp His Gly Met Phe Gln Arg Gly Thr Ala Tr - #p Ala Asp Gly Pro Ala 
90 - # 95 - # 100 
- - GGC GTC ACC CAA TGC CCT ATT TCC CCA GGG CA - #T TCG TTC TTG TAC AAG 
967 
Gly Val Thr Gln Cys Pro Ile Ser Pro Gly Hi - #s Ser Phe Leu Tyr Lys 
105 - # 110 - # 115 
- - TTC CAG GCT CTT AAC CAA GCC GGT ACT TTC TG - #G TAC CAC TCC CAT CAC 
1015 
Phe Gln Ala Leu Asn Gln Ala Gly Thr Phe Tr - #p Tyr His Ser His His 
120 1 - #25 1 - #30 1 - 
#35 
- - GTAACTACAA TCTATCTGTA CTGACGTGAC GATGTTGACT CAGTCATTCT CA - #G GAA 
1071 
- # - # - # Glu 
- - TCG CAA TAT TGT GAC GGT TTG CGT GGG GCT AT - #G GTC GTA TAT GAC CCA 
11195 
Ser Gln Tyr Cys Asp Gly Leu Arg Gly Ala Me - #t Val Val Tyr Asp Pro 
140 - # 145 - # 150 
- - GTC GAC CCA CAT CGC AAC TTG GTGAGCATCC TTTACTTTA - #T TCCCAAGGAA 
1170 
Val Asp Pro His Arg Asn Leu 
155 
- - GCCATCAGTC TAATGACTTG CCATTTAG TAT GAC ATT GAC AAC - #GTATGTAACC 
1223 
- # Tyr Asp Ile - #Asp Asn 
- # 160 
- - TCCGGCGTTT GGTCGTCTTG TGATCCGCAG TTCACCTTGT TTTACAG GAG - #GCC ACG 
1279 
- # - # Glu - #Ala Thr 
- # - # 165 
- - ATC ATT ACG CTC GCA GAC TGG GTAAGAATCT AATTACTTT - #C GATTACCTTC 
1330 
Ile Ile Thr Leu Ala Asp Trp 
170 
- - GAGCATACCT AACTCGGGGC CCTTCTGTTC GCCAG TAT CAC GTC - #CCT GCT CCC 
1383 
- # - # Tyr His Val Pro Ala Pro 
- # - # 175 - # 180 
- - TCT GCA GGT CTC GTT CCC ACC CCA GAT TCC AC - #G CTT ATC AAC GGT AAG 
1431 
Ser Ala Gly Leu Val Pro Thr Pro Asp Ser Th - #r Leu Ile Asn Gly Lys 
185 - # 190 - # 195 
- - GGC CGG TAT GCT GGT GGC CCT ACC GTA CCT CT - #C GCG GTC ATT TCT GTA 
1479 
Gly Arg Tyr Ala Gly Gly Pro Thr Val Pro Le - #u Ala Val Ile Ser Val 
200 - # 205 - # 210 
- - ACC CGA AAC CGA CGA TAC CGG TTC CGC CTT GT - #T TCC CTT TCA TGC GAT 
1527 
Thr Arg Asn Arg Arg Tyr Arg Phe Arg Leu Va - #l Ser Leu Ser Cys Asp 
215 - # 220 - # 225 
- - CCT AAT TAT GTA TTC TCT ATC GAT GGG CAT AC - #C ATG GTACGCACTA 
1573 
Pro Asn Tyr Val Phe Ser Ile Asp Gly His Th - #r Met 
230 - # 235 - # 240 
- - GTTCCCATCC CTGTAAAACG GGTGCTAACG ACGTGTATCA TCCCTTAG ACT - #GTT ATT 
1630 
- # - # Thr - # Val 
Ile 
- - \GAG GTC GAC GGA GTT AAC GTC CAA CCT CTC G - #TT GTC GAC TCG ATC 
CAG 1678 
Glu Val Asp Gly Val Asn Val Gln Pro Leu Va - #l Val Asp Ser Ile Gln 
245 - # 250 - # 255 
- - ATC TTC GCA GGT CAG CGC TAC TCG TTC GTT CT - #C AAC GCC AAC CGC CCC 
1726 
Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Le - #u Asn Ala Asn Arg Pro 
260 2 - #65 2 - #70 2 - 
#75 
- - GTC GGC AAC TAC TGG GTG CGA GCC AAC CCC AA - #C ATC GGT ACT ACG 
GGC 1774 
Val Gly Asn Tyr Trp Val Arg Ala Asn Pro As - #n Ile Gly Thr Thr Gly 
280 - # 285 - # 290 
- - TTC GTC GGT GGA GTC AAT TCT GCG ATT CTG CG - #C TAT GTG GGC GCC TCC 
1822 
Phe Val Gly Gly Val Asn Ser Ala Ile Leu Ar - #g Tyr Val Gly Ala Ser 
295 - # 300 - # 305 
- - AAT ACA GAC CCC ACT ACC ACC CAA ACT CCT TT - #C AGC AAC CCT CTC CTT 
1870 
Asn Thr Asp Pro Thr Thr Thr Gln Thr Pro Ph - #e Ser Asn Pro Leu Leu 
310 - # 315 - # 320 
- - GAG ACC AAT CTC CAC CCC TTG ACC AAC CCT GC - #T GCT CCT GGC TTG CCT 
1918 
Glu Thr Asn Leu His Pro Leu Thr Asn Pro Al - #a Ala Pro Gly Leu Pro 
325 - # 330 - # 335 
- - ACC CCA GGT GGC GTC GAC GTC GCG ATC AAC CT - #T AAC ACG GTA TTC GAT 
1966 
Thr Pro Gly Gly Val Asp Val Ala Ile Asn Le - #u Asn Thr Val Phe Asp 
340 3 - #45 3 - #50 3 - 
#55 
- - TTC AGT AGT CTC ACC TTC TCC GTT AAC GGA GC - #C ACT TTC CAT CAA 
CCG 2014 
Phe Ser Ser Leu Thr Phe Ser Val Asn Gly Al - #a Thr Phe His Gln Pro 
360 - # 365 - # 370 
- - CCC GTC CCT GTC TTG CTT CAG ATC ATG AGC GG - #T GCA CAG ACT GCC CAG 
2062 
Pro Val Pro Val Leu Leu Gln Ile Met Ser Gl - #y Ala Gln Thr Ala Gln 
375 - # 380 - # 385 
- - CAG CTT CTT CCC TCC GGT TCG GTC TAC GTC CT - #T CCC CGT AAC AAA GTC 
2110 
Gln Leu Leu Pro Ser Gly Ser Val Tyr Val Le - #u Pro Arg Asn Lys Val 
390 - # 395 - # 400 
- - ATC GAG CTT TCT ATG CCT GGA GGC TCC ACT GG - #C AGT CCC GTAAGTCTTA 
2159 
Ile Glu Leu Ser Met Pro Gly Gly Ser Thr Gl - #y Ser Pro 
405 - # 410 - # 415 
- - ATTGTCTTCA TTTCCAACAA GTCGGTGATT AACGCTGGAT CATTCGCTGA CA - #G CAT 
2215 
- - - # - # - # 
His 
- - CCC TTC CAT CTC CAC GGT GTATGTAGGC CTCTGTCTGA TC - #TCATTCGG 
2263 
Pro Phe His Leu His Gly 
420 
- - AAGCGTTACT GACGGTGCTT CTTTGTTTCG ATCTGATAG CAC GAA TTT - # GCT GTG 
2317 
- - - # - # His Glu Phe Ala 
Val 
- # - # 425 
- - GTG AGA AGC GCG GGG AGT TCG ACC TAC AAC TT - #C GCG AAC CCG GTA 
CGC 2365 
Val Arg Ser Ala Gly Ser Ser Thr Tyr Asn Ph - #e Ala Asn Pro Val Arg 
430 - # 435 - # 440 
- - AGG GAT GTC GTG AGT GCC GGT GTT GCT GGT GA - #C AAC GTC ACC ATT CGA 
2413 
Arg Asp Val Val Ser Ala Gly Val Ala Gly As - #p Asn Val Thr Ile Arg 
445 4 - #50 4 - #55 4 - 
#60 
- - TTC CGT ACC GAT AAC CCT GGA CCA TGG ATT CT - #C CAT TGC GTGCGTCAAG 
2462 
Phe Arg Thr Asp Asn Pro Gly Pro Trp Ile Le - #u His Cys 
465 - # 470 
- - TCATCGTCCT CGTGCTGAAT TGATTGTCTA ACCAAGATAT CACATACTTA G - #CAT ATC 
2519 
- # - # - # His 
Ile 
- # - # - # 
475 
- - GAC TGG CAC CTT GTT TTG GTAAGTCTTC GCTTCTTCCA GA - #CGTGATTA 
2567 
Asp Trp His Leu Val Leu 
480 
- - ACTTTACTGA TCGCGATGAT GGGAATACAG GGG TTG GCT GTA GT - #G TTC GCT 
GAG 2621 
- # Gly Leu - #Ala Val Val Phe Ala Glu 
- # - # 485 
- - GAC GCT CCT ACT GTT GCA ACC ATG GAT CCC CC - #T GTGAGTAGCG CCCGTGCTT 
T 2674 
Asp Ala Pro Thr Val Ala Thr Met Asp Pro Pr - #o 
490 4 - #95 5 - #00 
- - TGAGGAGTTG TGAAACCCGA GCTCAACGTG AAACGTTTTC CACTTTACAG CC - #T GCT 
2730 
- # - # - # Pro Ala 
- - TGG GAC CAA CTT TGC CCG ATC TAC GAT GCT CT - #C CCT CCC AAC ACA 
2775 
Trp Asp Gln Leu Cys Pro Ile Tyr Asp Ala Le - #u Pro Pro Asn Thr 
505 - # 510 - # 515 
- - TAAGTCGTTC AATTCAAGGC TGTTGACGTG AAGGGAGCAA GAAGGAAAGT AA - 
#GAGAAAGG 2835 
- - CAGTCACATC CCGTCGGTTT GCCTCTGAAA TATCGATTAA TCACGCTTTT TA - 
#TCACTTGT 2895 
- - AATTATCTTT CTTTGTTACA GTGGCTCTTT GACGCTGGCT CTCCAGTGCG TT - 
#AGAGTCGA 2955 
- - TAATAATAGC AATTCTCTAC TTTTAGGCAG ATTTTTAGGC AGGGCTGTGG TA - 
#CGCTTTAT 3015 
- - ATTAAGTTAA AAGAGCACCA ATAATGTCGC CCTCAGCTGG GCTCTTGTCG GC - 
#CGACTAGC 3075 
- - TCAGTTGGTT AGAGCGTCGT GCTAATAACG CGAAGGTCTT GGGTTCGATC CC - 
#CACGTTGG 3135 
- - CCAGTAGCCC CCTTTTTGTT AATCCTGGCA CTTTCCTGTT CCTACTAACC CT - 
#TTTGAGAG 3195 
- - TCCAGAAAAA TCACCATGAC TTAATTTTTT CTTTTCATAG AAGTCCTGGA AG - 
#GGTAAGGA 3255 
- - AGTGATATAA CTAGATGACC CAACATTCAG TGCTGGTCGT CAGATGCAGG TG - 
#TCTTTTCG 3315 
- - ACCAATCGAA GCATTCGGCG AAGATTCGAT CCAATTGCGC CTGCCTGTCC GC - 
#AGCATCTT 3375 
- - CGAACGGCGA AGGACTGTCG AAGAACGTTA CGTACGCGCG GATTGTCAGT TT - 
#ACGAAGGC 3435 
- - GAGGAAACCC CATTGAGAGT AGATCGTCAA GCGTCTTCCA TTGGCCCAGG TC - 
#CACATTCA 3495 
- - GATCGCAGCC GATTTGAACG ATAGGGATGA TATTGAGTCC TCCAGAACGT TC - 
#TGTCCCTG 3555 
- - CATCAAAGCG A - # - # 
- # 3566 
- - - - (2) INFORMATION FOR SEQ ID NO:33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 517 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
- - Met Leu Leu Leu Ala Thr Ala Leu Ala Thr Se - #r Leu Leu Pro Phe Val 
1 5 - # 10 - # 15 
- - Leu Gly Ala Ile Gly Pro Ser Thr Asn Leu Va - #l Val Ala Asn Lys Val 
20 - # 25 - # 30 
- - Ile Ala Pro Asp Gly Phe Ser Arg Ser Ala Va - #l Leu Ala Gly Ala Thr 
35 - # 40 - # 45 
- - Gln Pro Thr Val Gln Phe Pro Gly Pro Val Il - #e Gln Gly Asn Lys Asn 
50 - # 55 - # 60 
- - Ser Phe Phe Ala Ile Asn Val Ile Asp Ala Le - #u Thr Asp Pro Thr Met 
65 - # 70 - # 75 - # 80 
- - Leu Arg Thr Thr Ser Ile His Trp His Gly Me - #t Phe Gln Arg Gly Thr 
85 - # 90 - # 95 
- - Ala Trp Ala Asp Gly Pro Ala Gly Val Thr Gl - #n Cys Pro Ile Ser Pro 
100 - # 105 - # 110 
- - Gly His Ser Phe Leu Tyr Lys Phe Gln Ala Le - #u Asn Gln Ala Gly Thr 
115 - # 120 - # 125 
- - Phe Trp Tyr His Ser His His Glu Ser Gln Ty - #r Cys Asp Gly Leu Arg 
130 - # 135 - # 140 
- - Gly Ala Met Val Val Tyr Asp Pro Val Asp Pr - #o His Arg Asn Leu Tyr 
145 1 - #50 1 - #55 1 - 
#60 
- - Asp Ile Asp Asn Glu Ala Thr Ile Ile Thr Le - #u Ala Asp Trp Tyr 
His 
165 - # 170 - # 175 
- - Val Pro Ala Pro Ser Ala Gly Leu Val Pro Th - #r Pro Asp Ser Thr Leu 
180 - # 185 - # 190 
- - Ile Asn Gly Lys Gly Arg Tyr Ala Gly Gly Pr - #o Thr Val Pro Leu Ala 
195 - # 200 - # 205 
- - Val Ile Ser Val Thr Arg Asn Arg Arg Tyr Ar - #g Phe Arg Leu Val Ser 
210 - # 215 - # 220 
- - Leu Ser Cys Asp Pro Asn Tyr Val Phe Ser Il - #e Asp Gly His Thr Met 
225 2 - #30 2 - #35 2 - 
#40 
- - Thr Val Ile Glu Val Asp Gly Val Asn Val Gl - #n Pro Leu Val Val 
Asp 
245 - # 250 - # 255 
- - Ser Ile Gln Ile Phe Ala Gly Gln Arg Tyr Se - #r Phe Val Leu Asn Ala 
260 - # 265 - # 270 
- - Asn Arg Pro Val Gly Asn Tyr Trp Val Arg Al - #a Asn Pro Asn Ile Gly 
275 - # 280 - # 285 
- - Thr Thr Gly Phe Val Gly Gly Val Asn Ser Al - #a Ile Leu Arg Tyr Val 
290 - # 295 - # 300 
- - Gly Ala Ser Asn Thr Asp Pro Thr Thr Thr Gl - #n Thr Pro Phe Ser Asn 
305 3 - #10 3 - #15 3 - 
#20 
- - Pro Leu Leu Glu Thr Asn Leu His Pro Leu Th - #r Asn Pro Ala Ala 
Pro 
325 - # 330 - # 335 
- - Gly Leu Pro Thr Pro Gly Gly Val Asp Val Al - #a Ile Asn Leu Asn Thr 
340 - # 345 - # 350 
- - Val Phe Asp Phe Ser Ser Leu Thr Phe Ser Va - #l Asn Gly Ala Thr Phe 
355 - # 360 - # 365 
- - His Gln Pro Pro Val Pro Val Leu Leu Gln Il - #e Met Ser Gly Ala Gln 
370 - # 375 - # 380 
- - Thr Ala Gln Gln Leu Leu Pro Ser Gly Ser Va - #l Tyr Val Leu Pro Arg 
385 3 - #90 3 - #95 4 - 
#00 
- - Asn Lys Val Ile Glu Leu Ser Met Pro Gly Gl - #y Ser Thr Gly Ser 
Pro 
405 - # 410 - # 415 
- - His Pro Phe His Leu His Gly His Glu Phe Al - #a Val Val Arg Ser Ala 
420 - # 425 - # 430 
- - Gly Ser Ser Thr Tyr Asn Phe Ala Asn Pro Va - #l Arg Arg Asp Val Val 
435 - # 440 - # 445 
- - Ser Ala Gly Val Ala Gly Asp Asn Val Thr Il - #e Arg Phe Arg Thr Asp 
450 - # 455 - # 460 
- - Asn Pro Gly Pro Trp Ile Leu His Cys His Il - #e Asp Trp His Leu Val 
465 4 - #70 4 - #75 4 - 
#80 
- - Leu Gly Leu Ala Val Val Phe Ala Glu Asp Al - #a Pro Thr Val Ala 
Thr 
485 - # 490 - # 495 
- - Met Asp Pro Pro Pro Ala Trp Asp Gln Leu Cy - #s Pro Ile Tyr Asp Ala 
500 - # 505 - # 510 
- - Leu Pro Pro Asn Thr 
515 
- - - - (2) INFORMATION FOR SEQ ID NO:34: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
- - AGAATTGACT CCACCGACGA A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:35: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
- - GAATTCTGGC ATTCCTGACC TTTGTTC - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:36: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
- - Ser Val Asp Thr Met Thr Leu Thr Asn Ala As - #n Val Ser Pro Asp Gly 
1 5 - # 10 - # 15 
- - Phe Thr Arg Ala Gly Ile 
20 
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