Cassette for the expression of an endothiapepsin precursor in Cryphonectria parasitica

The present invention relates to a new cassette for the expression of an endothiapepsin precursor in Cryphonectria parasitica, to a strain of this species transformed with this cassette, to a process for preparing endothiapepsin using this strain and also to a process for preparing such a strain devoid of a dominant selection marker.

The present invention relates to a new cassette for the expression of an 
endothiapepsin precursor in Cryphonectria parasitica, to a strain of this 
species transformed with this cassette, to a process for preparing 
endothiapepsin using this strain and also to a process for preparing such 
a strain devoid of a dominant selection marker. 
The filamentous fungus Cryphonectria parasitica, also known as Endothia 
parasitica, which belongs to the Ascomycetes group, naturally secretes an 
aspartic protease, namely endothiapepsin, capable of curdling milk by 
specifically hydrolysing the micelles of casein, and which is hence useful 
in the manufacture of certain cheeses, especially cooked cheeses of the 
Emmental type. This enzyme, whose sequence of 330 amino acids has been 
described by Barkolt V., 1987, Eur. J. Biochem. 167, 327-338, then 
replaces chymosin. 
This enzyme is currently produced on an industrial scale by processes of 
fermentation of Cryphonectria parasitica strains chosen for their good 
level of expression of endothiapepsin. 
These processes possess drawbacks, and in particular that of an 
immobilisation of a large reactor volume, since the quantity of 
endothiapepsin per unit of biomass, and hence the quantity of 
endothiapepsin per unit of reactor volume for a given quantity of biomass, 
is low. 
Conventional mutation/selection techniques enable the quantity of 
endothiapepsin produced per unit of biomass to be increased by only 
approximately 30%. 
There is hence a need for genetic engineering tools enabling Cryphonectria 
parasitica strains to be obtained which are overproductive of 
endothiapepsin with a large overproduction factor. The latter is defined 
here as the ratio of the quantity of endothiapepsin produced by the 
overproductive transformed strain to the quantity of this protein produced 
by the untransformed control strain, for the same quantity of biomass. 
Cryphonectria parasitica is a filamentous fungus whose genetics is still 
very little known, in contrast to that of other filamentous fungi of the 
Ascomycetes group such as those of the genus Aspergillus, Neurospora or 
Trichoderma. Only very recently have the first transformations of 
Cryphonectria been described, by Churchill et al. 1990, Curr. Gen, 17, 
25-31. These authors transformed laboratory strains of Cryphonectria using 
plasmids carrying markers for resistance to hygromycin, benomyl or G 418. 
No protein of commercial interest has ever been produced hitherto using a 
Cryphonectria recombined by genetic engineering. 
Recently, at the Congress: "Annual Meeting of the American 
Phytopathological Society and the Canadian Phytopathological Society", 
Aug. 4th to 8th 1990, G. H. Choi et al. presented--apparently (according 
to the published abstract of their communication)--the coding sequence of 
endothiapepsin, an assumed prepro sequence of 88 amino acids and a portion 
of the endothiapepsin promoter comprising the TATA box but not the 
activator regions necessary for the promoter to be able to function. Such 
a genomic DNA fragment, but containing a prepro sequence of 89 amino 
acids, had already been isolated and sequenced by the Applicant in 1989, 
who had observed that it does not permit the expression of endothiapepsin 
(see Sections 1 and 6 below). 
The present invention relates to a new cassette for the expression of an 
endothiapepsin precursor in Cryphonectria parasitica, characterised in 
that it comprises a functional promoter upstream of a sequence coding for 
the endothiapepsin precursor, endothiapepsin having the following sequence 
(P1)(SEQ ID NO:1): 
Ser Thr Gly Ser Ala Thr Thr Thr Pro Ile Asp Ser Leu Asp Asp Ala Tyr 
- Ile Thr Pro Val Gln Ile Gly 
Thr Pro Ala Gln Thr Leu Asn Leu 
Asp Phe 
- Asp Thr Gly Ser Ser Asp Leu Trp Val Phe Ser Ser Glu Thr Thr Ala Ser 
- Glu Val Asp Gly Gln Thr Ile 
Tyr Thr Pro Ser Lys Ser Thr Thr 
Ala Lys 
- Leu Leu Ser Gly Ala Thr Trp Ser Ile Ser Tyr Gly Asp Gly Ser Ser Ser 
- Ser Gly Asp Val Tyr Thr Asp 
Thr Val Ser Val Gly Gly Leu Thr 
Val Thr 
- Gly Gln Ala Val Glu Ser Ala Lys Lys Val Ser Ser Ser Phe Thr Glu Asp 
- Ser Thr Ile Asp Gly Leu Leu 
Gly Leu Ala Phe Ser Thr Leu Asn 
Thr Val 
- Ser Pro Thr Gln Gln Lys Thr Phe Phe Asp Asn Ala Lys Ala Ser Leu Asp 
- Ser Pro Val Phe Thr Ala Asp 
Leu Gly Tyr His Ala Pro Gly Thr 
Tyr Asn 
- Phe Gly Phe Ile Asp Thr Thr Ala Tyr Thr Gly Ser Ile Thr Tyr Thr Ala 
- Val Ser Thr Lys Gln Gly Phe 
Trp Glu Trp Thr Ser Thr Gly Tyr 
Ala Val 
- Gly Ser Gly Thr Phe Lys Ser Thr Ser Ile Asp Gly Ile Ala Asp Thr Gly 
- Thr Thr Leu Leu Tyr Leu Pro 
Ala Thr Val Val Ser Ala Tyr Trp 
Ala Gln 
- Val Ser Gly Ala Lys Ser Ser Ser Ser Val Gly Gly Tyr Val Phe Pro Cys 
- Ser Ala Thr Leu Pro Ser Phe 
Thr Phe Gly Val Gly Ser Ala Arg 
Ile Val 
- Ile Pro Gly Asp Tyr Ile Asp Phe Gly Pro Ile Ser Thr Gly Ser Ser Ser 
- Cys Phe Gly Gly Ile Gln Ser 
Ser Ala Gly Ile Gly Ile Asn Ile 
Phe Gly 
- Asp Val Ala Leu Lys Ala Ala Phe Val Val Phe Asn Gly Ala Thr Thr Pro 
- Thr Leu Gly Phe Ala Ser Lys. 
A cassette for the expression of an endothiapepsin precursor denotes here a 
DNA sequence comprising the sequence coding for this precursor, flanked by 
signals enabling this coding sequence in Cryphonectria parasitica to be 
transcribed and translated. 
Endothiapepsin precursor is understood to mean a protein capable of being 
secreted and of generating endothiapepsin in the culture medium after one 
or more maturation steps. 
The natural precursor of endothiapepsin, referred to as 
preproendothiapepsin and having the following sequence (P4)(SEQ ID NO:2), 
will preferably be used: 
Met Ser Ser Pro Leu Lys Asn Ala Leu Val Thr Ala Met Leu Ala Gly Gly 
- Ala Leu Ser Ser Pro Thr Lys 
Gln His Val Gly Ile Pro Val Asn 
Ala Ser 
- Pro Glu Val Gly Pro Gly Lys Tyr Ser Phe Lys Gln Val Arg Asn Pro Asn 
- Tyr Lys Phe Asn Gly Pro Leu 
Ser Val Lys Lys Thr Tyr Leu Lys 
Tyr Gly 
- Val Pro Ile Pro Ala Trp Leu Glu Asp Ala Val Gln Asn Ser Thr Ser Gly 
- Leu Ala Glu Arg Ser Thr Gly 
Ser Ala Thr Thr Thr Pro Ile Asp 
Ser Leu 
- Asp Asp Ala Tyr Ile Thr Pro Val Gln Ile Gly Thr Pro Ala Gln Thr Leu 
- Asn Leu Asp Phe Asp Thr Gly 
Ser Ser Asp Leu Trp Val Phe Ser 
Ser Glu 
- Thr Thr Ala Ser Glu Val Asp Gly Gln Thr Ile Tyr Thr Pro Ser Lys Ser 
- Thr Thr Ala Lys Leu Leu Ser 
Gly Ala Thr Trp Ser Ile Ser Tyr 
Gly Asp 
- Gly Ser Ser Ser Ser Gly Asp Val Tyr Thr Asp Thr Val Ser Val Gly Gly 
- Leu Thr Val Thr Gly Gln Ala 
Val Glu Ser Ala Lys Lys Val Ser 
Ser Ser 
- Phe Thr Glu Asp Ser Thr Ile Asp Gly Leu Leu Gly Leu Ala Phe Ser Thr 
- Leu Asn Thr Val Ser Pro Thr 
Gln Gln Lys Thr Phe Phe Asp Asn 
Ala Lys 
- Ala Ser Leu Asp Ser Pro Val Phe Thr Ala Asp Leu Gly Tyr His Ala Pro 
- Gly Thr Tyr Asn Phe Gly Phe 
Ile Asp Thr Thr Ala Tyr Thr Gly 
Ser Ile 
- Thr Tyr Thr Ala Val Ser Thr Lys Gln Gly Phe Trp Glu Trp Thr Ser Thr 
- Gly Tyr Ala Val Gly Ser Gly 
Thr Phe Lys Ser Thr Ser Ile Asp 
Gly Ile 
- Ala Asp Thr Gly Thr Thr Leu Leu Tyr Leu Pro Ala Thr Val Val Ser Ala 
- Tyr Trp Ala Gln Val Ser Gly 
Ala Lys Ser Ser Ser Ser Val Gly 
Gly Tyr 
- Val Phe Pro Cys Ser Ala Thr Leu Pro Ser Phe Thr Phe Gly Val Gly Ser 
- Ala Arg Ile Val Ile Pro Gly 
Asp Tyr Ile Asp Phe Gly Pro Ile 
Ser Thr 
- Gly Ser Ser Ser Cys Phe Gly Gly Ile Gln Ser Ser Ala Gly Ile Gly Ile 
- Asn Ile Phe Gly Asp Val Ala 
Leu Lys Ala Ala Phe Val Val Phe 
Asn Gly 
- Ala Thr Thr Pro Thr Leu Gly Phe Ala Ser Lys. 
By anology with what is known for other aspartic proteases such as calf 
chymosin (Foltmann, 1970, Methods in Enzymol., 19, 421-435), swine 
pepsinogen (James and Sielecki, 1986, Nature, 319, 33-38) and S. 
cerevisiae protease A (Woolford et al., 1986 Mol. Cel. Biol. 6, 
2500-2510), it may be assumed that the natural precursor of endothiapepsin 
generates an inactive secreted form, referred to as proendothiapepsin, 
which self-activates to mature endothiapepsin. 
As a result of the degeneracy of the genetic code, there is a large number 
of DNA sequences coding for a protein whose sequence P4 (SEQ ID NO:2) 
corresponds to the formula given above. Among these, a suitable sequence 
is that which comprises the following sequence (N4a)(SEQ ID NO:3): 
ATGTCTT CCCCTCTCAA GAACGCCTTG GTGACCGCCA TGTTGGCTGG 
- TGGTGCTCTC AGCTCGCCTA CAAAGCAACA CGTTGGAATT CCCGTCAACG 
- CCTCTCCTGA AGTTGGCCCC GGAAAGTACT CGTTCAAGCA AGTCCGGAAC 
- CCCAACTACA AGTTCAACGG GCCTCTGTCG GTCAAGAAGA CGTACCTCAA 
- GTACGGCGTG CCGATCCCAG CCTGGCTGGA GGATGCTGTC CAGAACTCTA 
- CCTCGGGCCT GGCTGAGCGC TCGACCGGTT CTGCGACCAC AACTCCCATC 
- GACAGCCTCG ATGATGCTTA CATCACTCCG GTTCAGATCG GCACCCCTGC 
- GCAGACTCTG AACCTGGACT TTGACACTGG ATCTTCGGAT CTGTGGGTCT 
- TCAGCAGCGA GACTACAGCC AGCGAGGTCG ATGGGCAGAC CATCTACACC 
- CCCAGCAAGA GCACCACCGC CAAGCTGCTG TCGGCGCTAC CTGGTCCATC 
- TCCTACGGAG ACGGTAGCTC TTCCAGCGGC GATGTCTACA CTGACACCGT 
- CTCGGTTGGA GGCCTTACCG TGACGGGCCA GGCTGTCGAG TCGGCCAAGA 
- AGGTTTCTTC CAGCTTCACC GAGGACTCGA CCATTGACGG TCTCCTGGGC 
- CTGGCCTTCA GCACCCTGAA CACTGTGTCG CCTACCCAGC AAAAGACTTT 
- CTTCGACAAT GCGAAGGCGT CCTTGGACTC GCCTGTGTTC ACGGCTGATC 
- TTGGCTACCA TGCCCCTGGT ACCTACAACT TCGGCTTCAT CGATACCACT 
- GCCTACACGG GCTCCATCAC CTACACCGCT GTCTCGACCA AGCAAGGGTT 
- CTGGGAGTGG ACTTCGACCG GCTACGCCGT CGGCTCCGGC ACCTTCAAGT 
- CGACTTCCAT CGACGGCATC GCTGACACTG GCACGACCCT CCTGTACCTC 
- CCTGCCACCG TCGTGTCGGC CTACTGGGCC CAGGTCTCGG GCGCCAAGTC 
- CAGCTCTTCC GTCGGCGGCT ACGTCTTCCC CTGCAGCGCG ACCCTGCCTT 
- CCTTCACCTT CGGCGTTGGC TCAGCTCGCA TTGTGATTCC TGGCGACTAC 
- ATTGATTTCG GCCCCATCTC CACTGGAAGC TCGTCTTGCT TTGGCGGCAT 
- CCAGTCCAGC GCTGGTATCG GCATCAACAT CTTCGGTGAT GTCGCTCTGA 
- AGGCTTTGTC GTCTTCAACG GGGCTACAAC TCCCACTCTT GGCTTTGCTT 
- CCAAG 
It is preferable for the sequence coding for the endothiapepsin precursor 
to be interrupted by at least one intron. It is known, in effect, that the 
presence of introns in the coding portion of a gene can in some cases 
increase the expression of the latter (see, for example, the work of J. 
Callis et al., 1987, Genes and Development, 1, 1183-1200). 
An advantageous sequence coding for preproendothiapepsin is hence that 
which comprises the sequence (N4a)(SEQ ID NO:3) interrupted by at least 
one intron. An especially valued sequence of this type is that which 
comprises the following sequence (N4b)(SEQ ID NO:4): 
AT GTCTTCCCCT CTCAAGAACG 
- CCTTGGTGAC CGCCATGTTG GCTGGTGGTG CTCTCAGCTC GCCTACAAAG 
- CAACACGTTG GAATTCCCGT CAACGCCTCT CCTGAAGTTG GCCCCGGAAA 
- GTACTCGTTC AAGCAAGGTG AGTAGAGCTG CTTCTGTGTG TTGCAACAGA 
- AGACCAACGC AAAAAGAAGA GGTCAAGGCA AGACGGATAT TTTACTGACA 
- ATTATACTTT TGAAGTCCGG AACCCCAACT ACAAGTTCAA CGGGCCTCTG 
- TCGGTCAAGA AGACGTACCT CAAGTACGGC GTGCCGATCC CAGCCTGGCT 
- GGAGGATGCT GTCCAGAACT CTACCTCGGG CCTGGCTGAG CGCTCGACCG 
- GTTCTGCGAC CACAACTCCC ATCGACAGCC TCGATGATGC TTACATCACT 
- CCGGTTCAGA TCGGCACCCC TGCGCAGACT CTGAACCTGG ACTTTGACAC 
- TGGATCTTCG GATCTGTGGG TCTTCAGCAG CGAGACTACA GCCAGCGAGG 
- TTGGTCAACC CTCGCCCGCA TTTTATTGCA TACATTTTTA GTTTTTTTGG 
- TAATCAGAAT ACTAACATTG GGAATTTCCC AACTGTAGGT CGATGGGCAG 
- ACCATCTACA CCCCCAGCAA GAGCACCACC GCCAAGCTGC TGTCGGGCGC 
- TACCTGGTCC ATCTCCTACG GAGACGGTAG CTCTTCCAGC GGCGATGTCT 
- ACACTGACAC CGTCTCGGTT GGAGGCCTTA CCGTGACGGG CCAGGCTGTC 
- GAGTCGGCCA AGAAGGTTTC TTCCAGCTTC ACCGAGGACT CGACCATTGA 
- CGGTCTCCTG GGCCTGGCCT TCAGCACCCT GAACACTGTG TCGCCTACCC 
- AGCAAAAGAC TTTCTTCGAC AATGCGAAGG CGTCCTTGGA CTCGCCTGTG 
- TTCACGGCTG ATCTTGGCTA CCATGCCCGT GAGTGACCCC TCTTGATACA 
- TATACTTTTT GATGAATCTT GTTGGAGAAG CATTCCCCAC TAATATGGAA 
- ATTGTTTGTA TCTACAGCTG GTACCTACAA CTTCGGCTTC ATCGATACCA 
- CTGCCTACAC GGGCTCCATC ACCTACACCG CTGTCTCGAC CAAGCAAGGG 
- TTCTGGGAGT GGACTTCGAC CGGCTACGCC GTCGGCTCCG GCACCTTCAA 
- GTCGACTTCC ATCGACGGCA TCGCTGACAC TGGCACGACC CTCCTGTACC 
- TCCCTGCCAC CGTCGTGTCG GCCTACTGGG CCCAGGTCTC GGGCGCCAAG 
- TCCAGCTCTT CCGTCGGCGG CTACGTCTTC CCCTGCAGCG CGACCCTGCC 
- TTCCTTCACC TTCGGCGTTG GCTCAGCTCG CATTGTGATT CCTGGCGACT 
- ACATTGATTT CGGCCCCATC TCCACTGGAA GCTCGTCTTG CTTTGGCGGC 
- ATCCAGTCCA GCGCTGGTAT CGGCATCAAC ATCTTCGGTG ATGTCGCTCT 
- GAAGGCCGCC TTTGTCGTCT TCAACGGGGC TACAACTCCC ACTCTTGGCT 
- TTGCTTCCAA G 
A functional promoter means here a constitutive or regulable promoter 
capable of producing in Cryphonectria parasitica transcription of the 
sequence coding for the endothiapepsin precursor. This promoter contains a 
TATA element located in a zone rich in AT, a transcription initiation 
region downstream of this element and, upstream of the latter, sequences 
referred to as upstream activating sequences UAS or upstream repressing 
sequences URS which regulate the strength of the promoter through the 
effect of regulatory proteins. 
To determine the functionality of a DNA sequence as a promoter, the method 
described in Section 11 will conveniently be used. This consists in 
transforming a Cryphonectria parasitica strain rendered deficient in the 
production of endothiapepsin by a mutation of the structural gene for this 
protein, with the expression cassette carrying the test sequence and a 
selection marker, and then in identifying among the transformants those 
which are producers of endothiapepsin using the selection test on agar 
medium containing casein described in Section 7. 
A valued promoter is the promoter of the gene coding for 
preproendothiapepsin or a functional portion of this promoter. The portion 
in question comprises, for example, a portion carrying the TATA box of the 
following sequence (N5)(SEQ ID NO:5): 
AAGCTTATCC GCCGCCGGCG GGGGAATTCT ATTGAACTTG TTCGAATCAT 
- TGGTCCGTGG TCTTTTCGTC CATGCGGGCT CCGCTGGCGG ATGAATGACC 
- TTCTGGCTTC TAGCCTGGCG AAGCGATGTT ACTCTGTTGT CTATACTATA 
- CGATATGGTC AAGAGAGCAC ATGTGCCGCC AGATGAAGAC ATGTATATAA 
- AAGGAGTGGC CTCGACGGTT GCTCAACCAT CTTCTGTCTG TCCCAACGCC 
- ATCGACTCTT CAACTTCTCC TTCGTGTTCC ACCACCATCA CCTTGCTCCA 
- GACTTAGGAC TTTCAGCAAC CTTCAAAG 
and, upstream of the sequence (N5)(SEQ ID NO:5), a segment X of the 
fragment C bounded by the 5' end of the fragment A and the 5' end of the 
fragment C, chosen so that the segment X contains an activator region. The 
fragment C is a portion of the genomic DNA of Cryphonectria parasitica 
contained in the E. coli strain deposited with the CNCM on 31.08.1990 
under No. I-998. Its restriction map, as well as that of the fragment A 
contained in the fragment C, are shown in FIG. 4. The nucleotide sequence 
of the fragment A, which comprises the genomic DNA of Cryphonectria 
parasitica coding for preproendothiapepsin, is shown in FIG. 2, and is 
identified in the Sequence Listing as SEQ ID NO:32. 
A more precise localisation of this activator region may be effected by 
obtaining a series of segments of the fragment C (prepared, for example, 
by digestion using endonucleases or exonucleases) comprising the fragment 
A flanked on the 5' side by segments of different sizes of the portion of 
the fragment C bounded by the 5' end of the fragment A and the 5' end of 
the fragment C, and determining the functionality of the promoter obtained 
using the method mentioned above. 
An example of a segment X of the fragment C containing an activator region 
is the fragment of sequence below (SEQ ID NO:6): 
GCATGCTTGG CTCTTTAACG TCCTGCCCAT TCAGGGCCTT CAGCCGGCAC 
- TGGTCCTTCA TCAAGGGGGA CCTCATGACC ATGAACTAAT CTGTGATATC 
- TGATATATTC TAGAAGGCTT GGCTCCTCAA AGTTTCCAGC TAATGAATCA 
- GCGGCCCGCC GCCCTTAAAC CGCATCAGGC AAGTCGTTTG GTGTTGCCAG 
- GCGATGGCGA CAGGAGAGTG GTGTTGATGG GACAAGGGGA GGGAGGCTTA 
- GCCGACTTCA TCCATAGCAC CCACCTGCTT GGCGCCGATA AGTCTGACGA 
- TCCGCTTGAG CTGCAAAACG GCTCCTTGAC CTTTGTTTGG TCGACCGAGG 
- GAAATAGTCT CTTTTTGCGT GATCGTGCGC GCTTCGTATA GCAATAGCAG 
- CCAGCACCAG CAGGACGGGC CGTTGTCACG GTCACATCGT TCGCAACATG 
- CCGAGCGTAG GGATGAACGA ATGACTCGAG CCTTGCCTGA CAGTCTGGCA 
- ATCAATCTAT GGTCACGCAC GATCACAAGC CAATCGCTGT GACTGCGTTA 
- CTAGCCCAAT AATCCCTTGT TCGATCAGAG TGTTCTACAG ACTTCAAGTG 
- AGGTTCAC 
Examples of functional portions of the promoter of the gene coding for 
preproendothiapepsin are the BglII-ScaI and BamHI-ScaI segments of the 
fragment C (see FIG. 4). 
It can also be advantageous to use a promoter originating from another gene 
known to be expressed in Cryphonectria parasitica or in another 
filamentous fungus of the Ascomycetes group, for example the promoter of 
the gene coding for glyceraldehyde-3-phosphate dehydrogenase of 
Cryphonectria parasitica described by Choi et al., 1990, Nucleic Acids 
Research, 18, 18, Oxford University Press, or that of the gene coding for 
glyceraldehyde-3-phosphate dehydrogenase of Aspergillus nidulans described 
by Mullaney et al., 1985, Mol. Gen. Genet., 199, 37-45. 
The expression cassette is introduced into Cryphonectria parasitica 
preferably by cotransformation with a vector carrying the selection gene. 
The expression cassette is itself carried by a vector, or preferably in 
the form of a linear fragment. The expression cassette is preferably 
maintained in the integrated state in the chromosome. The vector carrying 
the selection gene is maintained either by integration in the chromosome, 
or in extrachromosomal linear form using sequences of the telomeric 
sequence type. After sporulation, transformants which have lost the 
selection marker are preferably selected. 
The invention hence also relates to a Cryphonectria parasitica strain 
productive of endothiapepsin, characterised in that it is transformed with 
the cassette defined above and overproduces endothiapepsin compared with 
the untransformed strain, that is to say secretes more endothiapepsin than 
the latter. 
For use of this strain in the agri-foodstuffs industry, it is advantageous 
for this strain to be devoid of a dominant selection marker such as, for 
example, an antibiotic resistance gene. 
Preferably, the host strain is the strain SEBR103 obtained as a result of a 
conventional mutation/selection process and deposited with the CNCM at the 
Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris C e dex 15, France, on 
31.08.1990 under No. I-997. It is then perceived that the transformed 
strain overproduces endothiapepsin compared with the strain SEBR103 with 
an overproduction ratio equal to at least two. 
The invention also relates to a process for preparing endothiapepsin, 
characterised in that it comprises a step of culturing of the strain 
defined above, followed by a step of isolation and purification of this 
protein. This process will advantageously replace the current processes 
for producing endothiapepsin. 
The invention also relates to a process for obtaining a Cryphonectria 
parasitica strain over-productive of endothiapepsin, transformed with the 
cassette defined above and devoid of a dominant selection marker, 
characterised in that it comprises at least one cycle entailing a step of 
cotransformation with a cassette according to one of Claims 1 to 12, and a 
dominant selection marker, followed by a step of purification by 
sporulation enabling the dominant selection marker to be removed. 
Preferably, this process comprises at least two cycles of this type. 
A better understanding of the invention will be gained from the description 
below, divided into sections, which comprises experimental results and a 
discussion of the latter. Some of these sections relate to experiments 
performed with the aim of carrying out the invention, others to examples 
of embodiment of the invention, naturally given purely by way of 
illustration. 
A large part of the collective techniques below, which are well known to 
those skilled in the art, is described in detail in the work by Sambrook 
and Maniatis: "Molecular cloning: a Laboratory manual" published in 1989 
by Cold Spring Harbor Press publications, New York (2nd edition): 
A better understanding of the description below will be gained by reference 
to FIGS. 1 to 12.

Section 1 
Isolation of the fragment A, an approximately 2.1-kb fragment of genomic 
DNA containing the coding sequence of the endothiapepsin precursor. 
1) Preparation of the genomic DNA 
The strain referred to as SEBR 103 was identified by the Centraal Bureau 
Voor Schimellcultures as belonging to the species Cryphonectria parasitica 
and deposited with the Collection Nationale de Culture de Microorganismes 
(National Collection of Microorganism Cultures) C.N.C.M. under No. I-997. 
From conidiospores of Cryphonectria parasitica strain SEBR 103 harvested at 
the surface of a Petri dish containing an agar medium, referred to as 
medium G and whose composition is specified in Table 3 below, previously 
inoculated with a mycelial implant or with a suspension of conidiospores 
of this same strain and then incubated for 4 weeks at room temperature and 
in the light, a Petri dish containing 25 ml of an agar medium, referred to 
as medium A and whose composition is given in Table 1 below, is inoculated 
by plating out one drop of conidiospore suspension. After 3 days' 
incubation at 30.degree. C., the mycelium obtained is used to inoculate a 
1--1 flask containing 100 ml of a liquid medium, referred to as medium B 
and whose composition is specified in Table 2 below. After 24 h of 
incubation at 28.degree. C. with stirring at 220 rpm, 25 ml of the culture 
broth are used for inoculating 250 ml of fresh medium B contained in a 
1--1 flask. The following day, after incubation under the conditions 
already defined, the whole of the culture broth (250 ml) is centrifuged at 
5,000 g for 5 min. The pellet is resuspended in 0.15M sodium acetate 
solution containing 1 mM EDTA. The mycelium is recovered by filtration on 
Whatman 3MM paper, frozen in liquid nitrogen and reduced to a powder using 
a mortar and pestle. 
10 g of mycelium powder are taken up in 40 ml of 0.15M sodium acetate 
solution containing 4% of lauroylsarcosine sodium salt, pH 5. After 30 min 
of gentle stirring at room temperature, sodium chloride is added to a 
final concentration of 0.1M. After 30 min of gentle stirring at room 
temperature, the proteins are extracted with a phenol solution (49% v/v) 
containing isoamyl alcohol (2% v/v) and chloroform (49% v/v). After 3 
extractions with this mixture, extraction is performed with a chloroform 
solution containing isoamyl alcohol (96% of chloroform/4% of isoamyl 
alcohol). After 2 extractions of this type, the aqueous phase is subjected 
to the addition of 2 volumes of ethanol. The 2 solutions are mixed gently 
by inversion, which causes the appearance of a filament of precipitated 
DNA. This filament is removed using a Pasteur pipette and deposited in an 
Eppendorf tube. This tube is centrifuged for 5 min at 100 g. The pellet is 
washed in 70% ethanol solution and then dried under vacuum. The pellet is 
taken up in 1 ml of buffer, referred to as TE buffer, of composition [10 
mM Tris-HCl, pH 8; 1 mM EDTA]. 
TABLE 1 
______________________________________ 
Composition of medium A 
Glucose, anhydrous 50 g/l 
Soybean flour (Soyoptim of Societe 20 g/l 
Industrielle des Oleagineux) 
Calcium nitrate 9 g/l 
Agar (Difco Bacto-agar) 15 g/l 
Saline solution 1 (composition 0.5 g/l 
specified below) 
Check or adjust the pH to 6.0 using 
1N HCl or 1N NaOH 
Composition of saline solution 1 
H.sub.3 BO.sub.3 30 ml 
MnCl.sub.2.4H.sub.2 O 79 ml 
ZnCl.sub.2 200 ml 
Na.sub.2 MoO.sub.4.2H.sub.2 O 20 ml 
FeCl.sub.3, anhydrous 50 ml 
CuSO.sub.4.5H.sub.2 O 200 ml 
Distilled water qs 500 ml 
(This suspension is stored for not 
more than one month at +4.degree. C.) 
______________________________________ 
TABLE 2 
______________________________________ 
Composition of medium B 
Glucose 10 g/l 
Thiamine 2 mg/l 
Saline solution 2 (composition given 62.5 ml/l 
below) 
Yeast extract (Difco) 2.5 g/l 
Malt extract (Difco) 7.5 g/l 
Check or adjust the pH to 6.0 using 
1N HCl or 1N NaOH 
Sterilise for 30 min at 110.degree. C. 
Composition of saline solution 2 
NH.sub.4 NO.sub.3 24 g/l 
KH.sub.2 PO.sub.4 16 g/l 
NaH.sub.2 SO.sub.4 4 g/l 
KCl 8 g/l 
MgSO.sub.4.7H.sub.2 O 2 g/l 
CaCl.sub.2 1 g/l 
Saline solution 1 8 ml/l 
(composition specified in Table 1) 
______________________________________ 
TABLE 3 
______________________________________ 
Composition of medium G 
______________________________________ 
Part A 
Malt extract 20 g 
Yeast extract 2 g 
Agar 16 g 
Water 1 l 
Adjust to pH 5.5 
Autoclave for 20 min at 120.degree. C. 
Part B 
Aspartic acid 1,000 mg 
Biotin 10 mg 
Water 1 l 
Dilute part B to 1/10, distribute it 
while filtering at a concentration of 
0.2M in 1 ml fractions and store 
at -20.degree. C. 
______________________________________ 
At the time of use, add 1 ml of diluted part B to 1 l of part A 
supercooled to 45.degree. C., then distribute the mixture in Petri dishes 
2) Preparation of probe 1 and probe 2 
These probes are pools of synthetic oligonucleotides, comprising the 
collective coding sequences for two peptides chosen from within the amino 
acid sequence of mature endothiapepsin exported into the culture medium of 
C. parasitica, described by V. BARKHOLT, 1987, Eur. J. Biochem., 167, 
327-338, shown in FIG. 1. The peptides chosen from within this sequence, 
referred to as peptide 1 and peptide 2 and corresponding, respectively, to 
probe 1 and probe 2, are as follows: 
Peptide 1(SEQ ID NO:7): Val-Asp-Gly-Gln-Thr 
Peptide 2(SEQ ID NO:8): Gly-Phe-Trp-Glu-Trp-Thr 
To these peptides there correspond, respectively, 256 (4.sup.3 
.times.2.sup.2) and 64 (4.sup.2 .times.2.sup.2) oligonucleotides coding 
for the latter, represented by the formulae below. 
A A A 
C C C G C 
GT GA GG CA AC probe 1(SEQ ID NO:9) 
G T G A G 
T T T 
A A 
C C A C 
GG TT TGGGA TGGAC probe 2(SEQ ID NO:10) 
G T G G 
T T 
3) Labelling of probe 1 and probe 2 
The probes are labelled with terminal deoxynucleotidyl transferase (TdT) 
(marketed by Stratagene, ref.: 600 132). 
The reaction is performed on 100 ng of a mixture of oligonucleotides 
dissolved in "Cobalt" reaction buffer (supplied at 10-fold concentration 
by IBI Inc.): 1.4M potassium cacodylate pH 7.2, 300 mM dithiothreitol, 1 
.mu.l of terminal deoxynucleotidyl transferase enzyme (Stratagene) and 50 
.mu.Ci of .sup.32 P-labelled deoxycytidine triphosphate dCTP. 
The reaction is carried out at 37.degree. C. for 10 minutes and is then 
stopped by adding 1 .mu.l of 0.5M EDTA. 
The products are extracted with phenol and the mixture is dialysed on a 
Biogel P 10 polyacrylamide column (Biorad: 150-1050). 
Radiolabelled probe 1 and radiolabelled probe 2 are thereby obtained. 
4) Hydrolysis of Cryphonectria parasitica genomic DNA 
The genomic DNA obtained at the end of 1) was subjected separately to a 
digestion with each of the following restriction enzymes: EcoRI, HindIII 
and BamHI. In each case, 10 .mu.g of the digestion product were spotted on 
0.8% agarose gel and subjected to electrophoresis in the presence of a 
series of radiolabelled size markers (Amersham ref. SJ5000). The DNA was 
then transferred onto a nitrocellulose filter (Biorad, ref. 162-0117) 
according to the technique well known to those skilled in the art under 
the name of Southern blotting, described in Maniatis, op. cit., this 
operation being repeated so as to obtain two nitrocellulose filters 
intended for hybridisation, one with probe 1, the other with probe 2. 
5) Hybridisation 
Each nitrocellulose filter treated according to the usual techniques 
(Maniatis et al., op. cit.) was first washed in a prehybridisation 
solution containing 6.times.SSC, 10.times.Denhardt's and 100 .mu.g/ml of 
sonicated and denatured salmon sperm DNA (Sigma D9156) for a few hours at 
42.degree. C., and then incubated in the same solution and under the same 
conditions as those stated above in the presence of one of the labelled 
probes 1 and 2. The hybridisation is left to proceed overnight. The 
6.times.SSC solution is obtained by dilution of a 20.times.SSC solution. 
The preparation of the 20.times.SSC buffer is described in Maniatis, op. 
cit. In brief, this buffer contains 175.3 g/l of NaCl and 88.2 g/l of 
sodium citrate, and is adjusted to pH 7 with a few drops of 1N NaOH. The 
10.times.Denhardt's solution contains 1 g of Ficoll, 1 g of 
polyvinylpyrrolidone and 1 g of bovine serum albumin per 500 ml final 
volume. 
After the hybridisation, each of the filters is washed individually in a 
solution containing 0.5 SSC at 42.degree. C. The filters are then exposed 
to a photographic film (Kodak XAR5) overnight. Analysis of the developed 
film shows, in the case of the hydrolysate obtained with the enzyme 
HindIII, that a band whose molecular weight corresponds approximately to a 
fragment of size slightly greater than 2.1 kb responds positively with 
both radio-labelled probes. 
6) Cloning of an approximately 2.1-kb HindIII-HindIII DNA- fragment which 
hybridises with the radio-labelled probes 1 and 2 
a) Formation of a library of genomic DNA. 
100 .mu.g of Cryphonectria parasitica DNA are hydrolysed with the enzyme 
HindIII and the fragments are separated by electrophoresis on 0.8% agarose 
gel. The region which corresponds to the fragments of size slightly 
greater than 2.1 kb is cut out and the DNA is purified by adsorption on 
silica milk (Geneclean Tm, Biorad P.O. Box 2284, La Jolla, Calif. 
92038-2284). These fragments are ligated using phage T4 DNA ligase (Gibco 
BRL) in pBR322 which has been hydrolysed with HindIII and dephosphorylated 
(Biolabs Ref. 321). Competent cells (that is to say cells capable of being 
transformed) (RRI cells, Gibco BRL, Ref. 520-8261 SA) are transformed, as 
directed by the supplier, with the ligation solution and plated out on 
agar medium, referred to as LB agar medium (Maniatis, op. cit.), of 
composition specified in Table 4 below. The collection of colonies 
obtained is the library of genomic DNA. 
TABLE 4 
______________________________________ 
Composition of LB agar medium 
______________________________________ 
Bacto tryptone casein hydrolysate (Difco) 
10 g/l 
Bacto yeast extract (Difco) 5 g/l 
NaCl 10 g/l 
Adjust the pH to 7.0 using 5N NaOH 
Bacto agar (Difco) 15 g/l 
Autoclave for 20 min at 120.degree. C. 
______________________________________ 
b) Selection of clones carrying the HindIII--HindIII fragment which 
hybridises with the radiolabelled probes 1 and 2, referred to as fragment 
A. 
The colonies obtained after transformation are adsorbed on a nitrocellulose 
filter (Schleicher and Schull, Ref. 40117) and replicated on two other 
nitrocellulose filters. A first series of filters is hybridised with 
radiolabelled probe 1 and a second series of filters is hybridised with 
radiolabelled probe 2. The prehybridisation, hybridisation and washing 
conditions are the same as those used previously (see 5 above). 
The clones which respond positively with both probes are purified. The 
plasmid DNA of one of these clones is isolated. The corresponding plasmid 
is referred to as p472. After checking that it did indeed hybridise with 
each of the two radiolabelled probes 1 and 2, the 2.1-kb HindIII--HindIII 
fragment carried on this plasmid was isolated and subcloned into M13mpl9 
(Pharmacia). This fragment is referred to as fragment A. It was sequenced 
by the cyclone technique (IBI "Cyclone I Biosystem"). 
The nucleotide sequence of the fragment A is shown in FIG. 2, which also 
indicates the numbering of the nucleotides, chosen arbitrarily so as to 
assign No. 1 to the nucleotide of the 5' end of the fragment A, as well as 
the amino acid sequence translated. 
7) Description of the sequence of the frament A (see FIG. 2 (SEQ ID NO:32)) 
The fragment A carries the nucleotide sequence coding for the peptide 
sequence of mature endothiapepsin exported into the culture medium, 
described by Barkolt (see FIG. 1), which begins at nucleotide 694 and ends 
at nucleotide 1861 and is interrupted by two introns located at 
nucleotides 850-938 and 1279-1367. Upstream of nucleotide 694, the open 
reading frame continues as far as nucleotide 566, where there is a signal 
characteristic of the end of an intron: AG. At nucleotides 468-469, there 
is a signal characteristic of the beginning of an intron: GT. It will be 
noted that the sequence straddling the beginning of the intron and the end 
of the exon, namely AAGGTGAGT, corresponds to the 5' consensus sequence of 
the splicing junction described by Mount S. M., 1982, Nucl. Ac. Res., 10, 
459-472. Upstream of nucleotide 468, there is only a single open reading 
frame (frame not interrupted by a stop codon) containing at least one ATG. 
This frame contains an ATG at position 365-367 (the nucleotide environment 
of which is compatible with M. Kozak, 1984, Nucl. Ac. Res. 12, p. 2) and 
an ATG at position 329-331, this reading frame being interrupted by the 
stop codon TAG at position 305-307. University of Wisconsin software 
U.W.G.C.G.: Devereux et al., 1984, Nucl. Ac. Res., 12, 8711-8721-Option: 
Testing for a signal peptide according to the method of G. von Heijne, 
1986, Nucl. Ac. Res., 14, 483-490, predicts in this open reading frame a 
single sequence coding for a signal peptide, the sequence below (SEQ ID 
NO:11), referred to as a pre nucleotide sequence (beginning at nucleotide 
329): 
ATGTCTT CCCCTCTCAA GAACGCCTTG GTGACCGCCA TGTTGGCTGG 
- TGGTGCTCTC AGC 
coding for the signal peptide of 20 amino acids of the following sequence, 
referred to as a pre peptide sequence (SEQ ID NO:12): 
Met Ser Ser Pro Leu Lys Asn Ala Leu Val Thr Ala Met Leu Ala Gly Gly Ala Leu 
Ser 
A signal peptide is expected by those skilled in the art, since 
endothiapepsin is a secreted protein, which requires the presence of a 
signal peptide. 
Between the sequence coding for the above signal peptide and that coding 
for the mature protein, there is the following nucleotide sequence (SEQ ID 
NO:13), referred to as a pro nucleotide sequence (beginning at nucleotide 
389): 
TCGCCTA CAAAGCAACA CGTTGGAATT CCCGTCAACG 
- CCTCTCCTGA AGTTGGCCCC GGAAAGTACT CGTTCAAGCA AGTCCGGAAC 
- CCCAACTACA AGTTCAACGG GCCTCTGTCG GTCAAGAAGA CGTACCTCAA 
- GTACGGCGTG CCGATCCCAG CCTGGCTGGA GGATGCTGTC CAGAACTCTA 
- CCTCGGGCCT GGCTGAGCGC 
coding for the following peptide sequence, referred to as a pro peptide 
sequence (SEQ ID NO:14). 
Ser Pro Thr Lys Gln His Val Gly Ile Pro Val Asn Ala Ser Pro Glu Val 
- Gly Pro Gly Lys Tyr Ser Phe 
Lys Gln Val Arg Asn Pro Asn Tyr 
Lys Phe 
- Asn Gly Pro Leu Ser Val Lys Lys Thr Tyr Leu Lys Tyr Gly Val Pro Ile 
- Pro Ala Trp Leu Glu Asp Ala 
Val Gln Asn Ser Thr Ser Gly Leu 
Ala Glu 
- Arg 
A pro peptide sequence (389-693) is also expected by those skilled in the 
art, since it has the function of inhibiting endothiapepsin, which is 
probably toxic to C. parasitica, before it is exported to the outer 
medium, following which this sequence is cleaved off. 
There is hence upstream of the sequence coding for mature endothiapepsin 
the sequence coding for the following prepro peptide sequence (SEQ ID 
NO:15): 
Met Ser Ser Pro Leu Lys Asn Ala Leu Val Thr Ala Met Leu Ala Gly Gly 
- Ala Leu Ser Ser Pro Thr Lys 
Gln His Val Gly Ile Pro Val Asn 
Ala Ser 
- Pro Glu Val Gly Pro Gly Lys Tyr Ser Phe Lys Gln Val Arg Asn Pro Asn 
- Tyr Lys Phe Asn Gly Pro Leu 
Ser Val Lys Lys Thr Tyr Leu Lys 
Tyr Gly 
- Val Pro Ile Pro Ala Trp Leu Glu Asp Ala Val Gln Asn Ser Thr Ser Gly 
- Leu Ala Glu Arg 
The nucleotide sequence beginning at nucleotide 329 (FIG. 2) and ending at 
nucleotide 1861 (FIG. 2), interrupted by three introns, is shown in FIG. 
5, the introns being underlined, and hence codes for preproendothiapepsin, 
the amino acid sequence of which is shown in FIG. 3. 
The fragment A comprises in addition a sequence of 328 nucleotides on the 
5' side of the initiation ATG (329-331) and a sequence of 275 nucleotides 
on the 3' side of the TAA stop codon (1862-1864), which contains several 
potential polyadenylation sites. 
The portion of the fragment A on the 5' side of the initiation ATG 
comprises, on the one hand the untranslated 5' end of the messenger RNA, 
and on the other hand, upstream of this end, a TATAA sequence (187-191), 
usually referred to as a TATA box, a consensus sequence present in most 
promoters of eukaryotes (Ballance D. J., 1986, Yeast, 2, 229-236). In 
contrast, this portion does not comprise a sequence of the type usually 
referred to as an upstream activating sequence (UAS) or an upstream 
regulatory sequence (URS), which are present, for example, in 
Saccharomyces (Guarente L., 1988, all, 52, 303-305) and in Neurospora 
(Frederick G. D., 1990,Mol. Gen. Gent., 221, 148-154). There is hence no 
promoter-activating region upstream of the TATA box in the fragment A. The 
promoter is hence not functional, as will be shown in Section 6. 
Section 2 
Isolation of the fragment B, an approximately 32.6-kb fragment of genomic 
DNA containing the coding sequence of the endothiapepsin precursor. 
1) Preparation of C. parasitica DNA 
Genomic DNA of C. parasitica SEBR 103 was prepared according to a protocol 
similar to that described by B. TURCQ (University Thesis: specialist field 
Life Sciences, examined orally on Jan. 6, 1989 at the University of 
BORDEAUX II), summarised below. 
Preparation of protoplasts 
The mycelium originating from 250 ml of culture broth of Cryphonectria 
parasitica strain SEBR 103, prepared as described in Section 1, is 
filtered off on gauze and then rinsed with 50 ml of 1M MgSO.sub.4. After 
incubation for 30 min at 37.degree. C., the mycelium is again filtered off 
on gauze and taken up in 20 ml of 1M MgSO.sub.4. 20 ml of 1M MgSO.sub.4 
containing 10 mg/l of the enzyme mixture CAYLASE C3 (company CAYLA), 
consisting of chitinases and .beta.-1,3-, .beta.-1,6-, .alpha.-1,3- and 
.alpha.-1,4-glucanases as well as other polysaccharidases, are then added 
and the resulting mixture is incubated for 1 h 30 min at 37.degree. C. 
with gentle stirring. After filtration of the mixture, the filtrate is 
centrifuged for 10 min at 3,000 g, and the protoplast pellet obtained is 
taken up in 20 ml of a buffer, referred to as ST buffer, of composition 
0.8M sorbitol, 100 mM Tris-HCl, pH 7.5. 
Extraction of genomic DNA from the protoplasts 
After a further centrifugation for 10 min at 3,000 g, the protoplast pellet 
is taken up with 14 ml of lysis buffer (100 mM Tris-HCl pH 9, 35 mM EDTA 
pH 8; 4% (weight/volume) SDS; proteinase K (Sigma) 600 .mu.g/ml), and the 
mixture is then incubated for 1 h at 50.degree. C. After a centrifugation 
for 10 min at 12,000 g, the supernatant volume is adjusted to 15.5 ml with 
TE buffer of composition 10 mM Tris-HCl pH 8, 1 mM EDTA, and 19.53 g of 
CsCl are then added. After an ultracentrifugation (16 h at 50,000 rpm in a 
vertical rotor), the gradient is collected in fractions, which are 
dialysed against buffer of composition 10 mM Tris-HCl pH 8, 1 mM EDTA, and 
analysed on 0.8% agarose gel. The fraction possessing a spectrometric 
ratio between the absorption at 260 nm and the absorption at 280 nm in the 
region of 1.8 was retained. 
Construction of the cosmid library 
Approximately 10 .mu.g of genomic DNA of the above fraction were subjected 
to a partial digestion with the restriction enzyme MboI and ligated using 
T4 ligase to cosmid pHC79-ura5, an approximately 8-kb cosmid vector 
constructed by inserting the EcoRI-EcoRI fragment containing the ura5 gene 
(Begueret et al., 1984, Gene, 32 487-492) at the EcoRI site of the 
commercially available cosmid pHC79 (marketed by BRL and constructed by 
HOHN B. et al., 1980, Gene, 11, 291-298), the cosmid pHC79-ura5 having 
been linearised beforehand with the endonuclease BamHI and 
dephosphorylated with alkaline phosphatase (Promega ref. CIP--M 204). 
The ligation mixture was packaged in phage particles using the Stratagene 
"Gigapack plus" kit, and was used for transforming the E. coli receptor 
strain LE 392 (Murray et al., 1977,Mol. gen. Genet. 150, p. 53), 
com-mercially available and distributed by Genofit. After plating out on 
LB agar medium (see Table 4) supplemented with 100 mg/l of ampicillin, 
approximately 4,500 ampicillin-resistant clones thereby obtained were 
subcultured individually on microtitration plates containing LB liquid 
medium, of composition specified in Table 4 -but without agar, and stored 
at -80.degree. C. It was shown by digestion of cosmids extracted from some 
12 clones taken at random that the average size of the inserts of the 
library was approximately 37 kb. 
2) Screening of the library by hybridisation with the fragment A 
The fragment A containing the endothiapepsin structural gene was used as a 
probe for hybridisation experiments. Initially, the clones contained in 
the microtitration plates were subcultured in Petri dishes containing LB 
agar medium (see Table 4) with the addition of ampicillin, and then 
transferred onto nylon membranes (Hybond N.sup.+, Amersham). The bacteria 
were then lysed using a solution containing 1.5M NaCl and 0.5M NaOH. After 
treatment with a solution of proteinase K (Sigma) for 30 min at 37.degree. 
C., the filters are washed with 2.times.SSC (NaCl 17.5 g/l, sodium citrate 
8.82 g/l, pH 7) and pre-hybridised at 42.degree. C. for 20 min. The 
filters are hybridised at 42.degree. C. overnight with the fragment A 
isolated in Example 1, labelled with horseradish peroxidase (Amersham), 
and are revealed with the "Gene detection system" ECL chemiluminescent 
probe kit (RPN 2101, Amersham). The hybridisation signals obtained are 
visualised on a suitable film. Of the 4,500 clones of the library, 2 
clones gave a positive signal. These two clones, hereinafter designated 
8H12 and 41H7 and containing, respectively, the cosmids referred to as 
p8H12 and p41H7, were subcultured on LB liquid medium containing 100 mg/l 
of ampicillin. After culturing overnight at 37.degree. C., the cosmids are 
extracted by the method of lysis in an alkaline medium and purified by 
ultracentrifugation with caesium chloride and ethidium bromide according 
to the techniques described in Maniatis, op. cit. The cosmids thus 
purified were digested with the enzyme HindIII and the fragments obtained 
were subjected to electrophoresis on 0.8% agarose gel. Southern blotting 
on a nylon membrane was performed, and the filter was hybridised with the 
fragment A using the technique described above. The presence of this 
fragment in each of the cosmids p8H12 and p41H7 was thereby confirmed. 
3) Physical analysis of the Positive cosmids p8H12 and p41H7 
It was found that the restriction profiles of cosmids p8H12 and p41H7, 
obtained using the enzymes NotI, SmaI, SfiI, XbaI, BamHI and PvuI, were 
identical, thereby indicating that this region of the genomic DNA 
containing the gene coding for endothiapepsin of C. parasitica was cloned 
without rearrangement for these two clones. The restriction profile for 
cosmid p8H12, the cosmid adopted for the next part of the study, is given 
in Table 5 below. 
TABLE 5 
______________________________________ 
Restriction profile of cosmid p8H12 
Number of 
Restriction cleavage TOTAL 
enzyme sites Size in kb in kb 
______________________________________ 
NotI 1 not determined 
-- 
SmaI 2 not determined -- 
SfiI 3 20; 16; 4.1 40.1 
XbaI 4 23; 14; 2.9; 1.2 41.1 
BamHI 5 15; 9; 7; 7; 2.6 40.6 
PvuI 5 23; 7.4; 6.4; 2.1; 40.4 
1.5 
Average 40.6 
______________________________________ 
This profile makes it possible to calculate the average size of cosmid 
p8H12, equal to approximately 40.6 kb, and hence that of the genomic 
insert, equal to approximately 40.6-8.0=32.6 kb. This genomic insert is 
referred to as fragment B. 
It was shown, moreover, by Southern blotting on a nylon membrane, that the 
approximately 9-kb BamHI--BamHI fragment (see Table 5), hereinafter 
referred to as fragment C, was the only BamHI--BamHI fragment to be 
hybridised with the fragment A used as a probe; it hence contained the 
whole of this fragment. 
Section 3 
Cloning of the fragment C, an approximately 9-kb fragment of genomic DNA 
containing the coding sequence of the endothiapepsin precursor. 
10 .mu.g of cosmid p8H12 were digested with the endonuclease BamHI and the 
different fragments were separated on 0.8% agarose gel. 
The product of digestion with the enzyme BamHI, containing the fragment C, 
was ligated using T4 DNA ligase (Gibco BRL) to plasmid pBR322 opened at 
the BamHI site and dephosphorylated (marketed by Biolabs--ref. 320). The 
ligation product was used to transform competent cells of E. coli strain 
K12 RR1 (Gibco BRL ref. 520-8261A). After the transformation mixture has 
been plated out on Petri dishes containing LB agar medium supplemented 
with ampicillin (100 .mu.g/ml), and incubation of the dishes at 37.degree. 
C. for 24 h, the colonies are replicated on nylon membranes; the bacteria 
are then lysed and the membranes are thereafter hybridised with the 
fragment A, as described above in Section 2.2). 18 colonies containing DNA 
which hybridises with the fragment A were thereby detected. Their plasmid 
DNA content was extracted and analysed on 0.8% agarose gel after digestion 
with the endonuclease BamHI. It was thus verified that all these colonies 
contained a plasmid derived from pBR322 which had an approximately 9-kb 
fragment inserted at the BamHI site. A clone referred to as SEBR 3104, 
containing the plasmid designated pEp1, was chosen for the next part of 
the study. The clone SEBR 3104 was deposited with the CNCM under No. 
I-998. 
Plasmid pEp1 was subjected to single and/or multiple digestions using the 
enzymes BamHI, HindIII, PstI, SacI, SphI, BglII and ScaI. The restriction 
map obtained is shown in FIG. 4, the symbols B, H. P, Sp, C, G and S 
representing, respectively, BamHI, HindIII, PstI, SphI, SacI, BglII and 
ScaI, the initiation codon of the endothiapepsin gene of C. parasitica 
being indicated by an i and the arrow indicating the direction of 
transcription of the endothiapepsin gene. The fragment A described in 
Section 2, as well as the fragments D, E and F described in Sections 4 and 
5, are also shown in this figure. 
It is apparent that the BamHI site (B) which forms the 5' end of the 
fragment C is located approximately 4 kb from the initiation codon, and 
that the BglII site (G) on the 5' side is located approximately 3 kb from 
the initiation codon. It hence appears to be advantageous to isolate and 
clone the approximately 5.1-kb BglII-BglII fragment containing the whole 
of the fragment A, referred to as fragment D, and which probably contains 
the information necessary for expression of the endothiapepsin precursor. 
Section 4 
Cloning of the fragment D, an approximately 5.2-kb fragment of genomic DNA 
containing the coding sequence of the endothiapepsin precursor. 
20 .mu.g of plasmid pEp1 were digested with the enzyme BglII (see FIG. 4) 
and the digestion products were separated on 0.8% low-melting-temperature 
agarose (Sigma --Ref. A9414) gel. After staining of the agarose gel with 
ethidium bromide, the agarose band containing the approximately 5.2-kb 
fragment D is cut out with a scalpel under ultraviolet light at 310 nm. 
The DNA is then extracted according to the instructions in the NACS. 
52PRE kit (Gibco BRL), and thereafter diluted in 10 .mu.l of TE buffer 
of composition 10 mM Tris-HCl pH 8, 1 mM EDTA; 1 .mu.l of the suspension 
obtained was ligated using T4 DNA ligase (Gibco BRL) to plasmid pBT6, 
derived from plasmid pBT3 by the insertion of a BglII linker at the SmaI 
site of the polylinker of pUC12. Plasmid pBT3, described by Orbach M. J. 
et al., 1986,Mol. Cell. Biol., 6, 2452-2461, carries a mutated gene for 
.beta.-tubulin of Neurospora crassa (a filamentous ascomycete fungus) 
conferring benomyl resistance (dominant selection marker). Before 
ligation, plasmid pBT6 was linearised with the endonuclease BglII and 
dephosphorylated with alkaline phosphatase (Promega, Ref. CIP-M204). 
The ligation product was used to transform competent cells of E. coli 
strain K12 RR1 (Gibco BRL Ref. 530-8261SA). After the transformation 
mixture has been plated out on Petri dishes containing LB agar medium 
supplemented with ampicillin (100 .mu.g/ml), and incubation of the dishes 
at 37.degree. C. for 24 h, the colonies are replicated on nylon membranes. 
The bacteria are then lysed, and the membranes are thereafter hybridised 
with the fragment A, as described in Section 2.2). Two colonies containing 
DNA which hybridises with the fragment A were thereby detected. Their 
plasmid DNA content was extracted and analysed on 0.8% agarose gel after 
digestion with the endonuclease BglII. It was thus verified that these two 
colonies contained a plasmid derived from pBT6 which had the approximately 
5.2-kb fragment D inserted in both possible orientations at the BglII 
site. A clone containing this plasmid, designated plasmid pEp2, was chosen 
for the next part of the study. 
Section 5 
Cloning of the fragment F, an approximately 3.5-kb fragment of genomic DNA 
containing the coding sequence of the endothiapepsin precursor. 
The cloning was carried out in two steps. First, the approximately 3.7-kb 
fragment E containing the whole of the fragment F was cloned into plasmid 
pUC18 at the SphI site of the polylinker. Plasmid pEp3 thereby obtained 
enabled the fragment F to be purified, which fragment was then subcloned 
in its turn into plasmid pUC18 at the BamHI site of the polylinker. 
Plasmid pEp4 was thereby obtained. 
1) Construction of plasmid pEp3 
1 .mu.g of plasmid pEp1 was digested with the enzyme SphI (see FIG. 4), and 
the DNA was then purified with 0.1 volume of 3M sodium acetate and 2 
volumes of ethanol. An approximately 3.7-kb SphI fragment, referred to as 
fragment E, was thereby obtained. The DNA was then dissolved in 40 .mu.l 
of TE buffer of composition (10 mM Tris-HCl pH 8, 1 mM EDTA) and 
thereafter dialysed on a P10 column (Pharmacia). 1 .mu.l of the mixture 
obtained above was then ligated using T4 DNA ligase (Gibco BRL) with 
approximately 25 ng of plasmid pUC18 previously linearised with the 
endonuclease SphI and dephosphorylated with alkaline phosphatase (Promega, 
Ref. CIP-M204). The ligation product was used to transform competent cells 
of E. coli strain DH5a (Gibco BRL, Ref. 530-8263 SA). After the 
transformation mixture had been plated out on Petri dishes containing LB 
agar medium supplemented with ampicillin (100 .mu.g/ml), X-gal (40 
.mu.g/ml) and IPTG (2 .mu.g/ml), and incubation of the dishes at 
37.degree. C. for 24 h, 350 white colonies were subcultured on the same 
medium. The colonies were then replicated on nylon membranes, the bacteria 
were thereafter lysed and finally the membranes were hybridised with the 
fragment A as described above in Section 2.2). 37 colonies containing DNA 
which hybridises with the fragment A were thereby detected. 
The plasmid DNA of 30 clones was extracted and analysed on 0.8% agarose gel 
after digestion with the endonuclease SphI. Two colonies containing a 
plasmid derived from pUC18 which had the 3.7-kb fragment E inserted (in 
both possible orientations) at the SphI site were retained. These plasmids 
were referred to as pEp3(a) and pEp3(b). In plasmid pEp3(b), the BglII 
site of the fragment E is at a distance of approximately 3.5 kb from the 
BamHI site located on the polylinker of pUC18. 
2) Construction of plasmid pEp4 
10 .mu.g of plasmid pEp3(b) were subjected to three successive digestions 
with the endonucleases BglII, BamHI and PvuI, and the digestion products 
were separated on 0.8% agarose gel. After staining of the gel with 
ethidium bromide, the agarose band containing the approximately 3.5-kb 
SphI fragment, designated fragment E, mixed with an SphI-BamHI fragment of 
the polylinker of pUC18, was cut out with a scalpel under ultraviolet 
light at 310 nm. The DNA is then extracted and thereafter dissolved in 20 
.mu.l of TE buffer of composition (10 mM Tris-HCl pH 8, 1 mM EDTA). 5 
.mu.l of the suspension obtained were ligated to approximately 750 ng of 
plasmid pUC18 previously linearised with the endonuclease BamHI and 
dephosphorylated with alkaline phosphatase. The ligation product was used 
to transform competent cells of E. coli strain DH5.alpha. according to the 
protocol described above. The plasmid DNA of 30 while colonies was 
extracted and analysed on 0.8% agarose gel after digestion with the 
endonucleases BamHI, BglII or SmaI. A colony was adopted which contains a 
plasmid derived from pUC18 which has the approximately 3.5-kb fragment F, 
mixed with an SphI-BamHI fragment of the polylinker of pUC18, inserted at 
the BamHI site. This plasmid was designated pEp4. 
3) Determination of the sequence of the SphI-HindIII segment of the segment 
F 
This sequence, determined as above (see Section 1), is shown in FIG. 12. It 
contains signals for activation of the promoter of the gene coding for 
preproendothiapepsin, as will be shown in Section 10. 
Section 6 
Transformation of C. parasitica with each of the vectors containing one of 
the fragments A, B and C. 
Preparation of protoplasts 
The mycelium originating from 250 ml of the culture broth of C. parasitica 
strain SEBR 103, prepared as described in Section 1, is rinsed with 50 ml 
of 1M MgSO.sub.4. After incubation for 30 min at 37.degree. C., the 
mycelium is filtered off on gauze and taken up in 20 ml of 1M MgSO.sub.4. 
20 ml of 1M MgSO4 containing 10 mg/l of the enzyme mixture CAYLASE C3 
(company CAYLA), consisting of chitinases and .beta.-1,3-, .beta.-1,6-, 
.alpha.-1,3- and .alpha.-1,4-glucanases as well as other 
polysaccharidases, are then added and the resulting mixture is incubated 
for 1 h 30 min at 37.degree. C. with gentle stirring. After filtration of 
the mixture, the filtrate is centrifuged for 10 min at 3,000 g, and the 
protoplast pellet obtained is then taken up in 15 ml of the buffer, 
referred to as ST buffer, of composition 0.8M sorbitol, 100 mM Tris-HCl, 
pH 7.5. After a further centrifugation for 10 min at 3,000 g, the pellet 
is taken up in 10 ml of a buffer, referred to as STC10 buffer, of 
composition 0.8M sorbitol, 100 mM Tris-HCl pH 7.5, 10 mM CaCl.sub.2. The 
protoplasts are then counted using a Malassez cell, so as to adjust their 
concentration to 10.sup.8 /ml after centrifugation for 10 min at 3,000 g 
and taking up the pellet in a buffer, referred to as STC50 buffer, of 
composition 0.8M sorbitol, 100 mM Tris-HCl pH 7.5, 50 mM CaCl.sub.2. 
Cotransformation of protoplasts with cosmid p8H12 and plasmid pBT3 
Cosmid p8H12 which contains the fragment B (see Section 2) does not carry a 
dominant selection marker (usable for direct selection). Plasmid pBT3, 
described by Orbach M. J. et al., 1986,Mol. Cell. Biol., 6, 2452-2461, 
which carries a mutated gene for .beta.-tubulin of Neurospora crassa 
(filamentous Ascomycete fungus) conferring resistance to benomyl (dominant 
selection marker) was hence used jointly with cosmid p8H12 
(cotransformation method: see the work on cotransformation of Aspergillus 
niger of Wernars K. et al., 1987,Mol. Gen. Genet., 209, 71-77). 
A mixture composed of 1 .mu.g of plasmid pBT3, previously purified by 
ultracentrifugation in a buffer containing caesium chloride and ethidium 
bromide according to the techniques described in Sambrook, op. cit., and 4 
.mu.g of cosmid p8H12, purified in the same manner, in 10 ml of TE buffer 
of composition (10 mM Tris-HCl pH 8, 1 mM EDTA), is incubated for 20 
minutes at 0.degree. C. with 100 ml of the protoplast preparation prepared 
above (equivalent to 10.sup.7 protoplasts). After the addition of 1 ml of 
a solution consisting of 60% (weight/volume) of PEG 4000 (polyethylene 
glycol of molecular mass 4000) and buffer of composition 20 mM Tris-HCl pH 
7.5, 100 mM CaCl.sub.2, and incubation of the mixture for 10 min at room 
temperature, 1 ml of STC10 buffer (defined above) is mixed with the 
mixture. The protoplasts thus treated are included in 60 ml of agar medium 
containing 1 mg/l of Benlate (antifungal marketed by Dupont Nemours), 
referred to as medium D and whose composition is specified in Table 6 
below, maintained supercooled at 45.degree. C. The supercooled mixture is 
plated out on Petri dishes containing an agar medium supplemented with 1 
mg/l of Benlate (antifungal marketed by Dupont Nemours which contains 50% 
of benomyl), referred to as medium C and whose composition is specified in 
Table 7 below. The Petri dishes are incubated at 28.degree. C. for the 
time necessary for the appearance of benomyl-resistant regenerated 
protoplasts. The regenerated transformants thereby obtained are referred 
to as 29Pn, n designating the number of the clone under consideration. The 
benomyl resistance of the clones is confirmed by subculturing mycelial 
implants of each clone on medium B made into an agar medium by adding 20 
g/l of agar and supplemented with 1 mg/l of Benlate. 
Only the clones which grow on this medium are set up for sporulation 
according to the method described in Section 1.1). The conidiospres 
obtained are harvested in bulk in a buffer containing at least 15% of 
glycerol and stored at -80.degree. C. 
TABLE 6 
______________________________________ 
Composition of medium D 
______________________________________ 
Sucrose 250 g/l 
Glucose 20 g/l 
Thiamine 2 g/l 
Asparagine 100 mg/l 
Malt extract 0.2 g/l 
Agar 20 g/l 
Saline solution 1 (composition 62.5 ml 
specified in Table 2) 
Adjust the pH to 6.0 using 1N HCl or 
1N NaOH, 
Autoclave for 30 min at 110.degree. C., then 
Add 1 mg/l of Benlate to the medium 
cooled to 60.degree. C. 
______________________________________ 
TABLE 7 
______________________________________ 
Composition of medium C 
______________________________________ 
Medium D 750 ml/l 
STC10 buffer 250 ml/l 
(of composition 0.8M sorbitol, 
100 mM Tris-HCl pH 7.5, 10 mM CaCl.sub.2) 
Adjust the pH to 6.0 
Add 1 mg/l of Benlate to the medium 
autoclaved at 60.degree. C. 
______________________________________ 
Cotransformation of protoplasts with plasmid p472 and plasmid PBT3 on the 
one hand, as well as with plasmid pEp1 and plasmid pBT3 on the other hand 
Neither plasmid p472 which contains the fragment A (see Section 1) nor 
plasmid pEp1 which contains the fragment C (see Section 3) carries a 
dominant selection marker. 
C. parasitica strain SEBR 103 was cotransformed according to a protocol 
identical to that described in the subsection above, with the following 
mixtures of plasmids: 4 .mu.g of pEp1 and 1 .mu.g of pBT3, 4 .mu.g of p472 
and 1 .mu.g of pBT3. The transformants thereby obtained are referred to as 
30Pn for the cotransformation with plasmids pEp1 and pBT3, and 31Pn for 
the cotransformation with plasmids p472 and pBT3, n designating the number 
of the clone under consideration. 
Section 7 
Selection of transformed strains over-productive of endothiapepsin. 
1) General method 
a) Selection on agar medium containing casein 
Mycelial implants of approximately 100 benomylresistant colonies were 
subcultured on an agar medium containing casein, referred to as medium E 
and whose composition is specified in Table 8 below. On this medium, 
Cryphonectria parasitica colonies which produce the protease give rise to 
a halo of precipitation whose area is proportional to the quantity of 
endothiapepsin secreted. 
The overproductive strains are adopted on the basis of a ratio of the 
diameter of the halo of precipitation to the diameter of the colony, the 
ratio being significantly higher than that for the untransformed control 
strain. A preparation of conidiospores of these overproductive strains is 
made according to the method used in Section 1.1). In addition, it was 
verified by adding 5 .mu.g/ml of pepstatin, a substance specifically 
inhibiting aspartic proteases, to medium E that the increase in the halos 
observed in the overproductive strains was reduced. This result shows that 
the observed effect is indeed due to an overproduction of an aspartic 
protease. 
b) Selection in liquid medium 
.alpha.) Study in flasks: 
To confirm this result, tests of production in flasks were carried out in 
the following manner: inoculation of 250-ml flasks containing 40 ml of 
medium F whose composition is specified below. The flasks are then 
incubated at 28.degree. C. on an eccentric rotary agitator adjusted to 220 
rpm for 48 h. For each strain, culturing was carried out in 3 different 
flasks and the mean of the results of assay of coagulant activity for the 
3 flasks was calculated. The control consists of untransformed 
Cryphonectria parasitica strain SEBR 103. The assay of coagulant activity 
is carried out according to the official method of determination of the 
enzyme content of coagulant solutions, published in the Journal Officiel 
de la Republique Fran.cedilla.aise (Official Journal of the French 
Republic) of Mar. 20, 1981 (section C), summarised below: 
1 ml of culture supernatant diluted with water so as to obtain a 
coagulation time of between 5 and 10 min is added to 10 ml of standardised 
milk (supplied by INRA--Experimental Dairy Station-39800 POLIGNY), placed 
in a suitable bottle; 
the coagulation time, identified by the appearance of a flocculation of the 
milk on the wall of the bottle when rotated in a waterbath at 30.degree. 
C., is measured; 
the coagulant activity, designated CA, expressed in mg/l, is given by the 
formula: 
##EQU1## 
with K and a factors dependent on the milk and on the enzyme under 
consideration (expressed in mgs/l and in s, respectively). 
T: coagulation time (expressed in seconds) 
.alpha.: dilution factor. 
.beta.) Study under fermenter conditions 
The production of endothiapepsin was assessed in a 2-1 fermenter 
(Biolaffite) containing 1.2 l of a culture medium obtained by 
concentration of medium F, sterilised by autoclaving for 45 min at 
120.degree. C. The culture conditions are as follows: agitation at 800 
rpm; aeration: 2 vvm (vvm: volume of air per volume of medium per min). 
The fermenter is inoculated in the proportion of 5% (v/v) with a flask 
preculture as described above (in .alpha.). The temperature is maintained 
at 28.degree. C. Measurement of the coagulant activity is performed after 
approximately 90 h of fermentation according to the assay method described 
above. 
It is verified by determination of the dry weight of the culture that the 
quantity of biomass produced by the overproductive transformants does not 
differ significantly from that produced by the control strain. 
TABLE 8 
______________________________________ 
Composition of medium E 
______________________________________ 
KH.sub.2 PO.sub.4 0.36 g/l 
Na.sub.2 HPO.sub.4.2H.sub.2 O 0.71 g/l 
MgSO.sub.4.7H.sub.2 O 0.50 g/l 
NaCl 0.10 g/l 
Casein hydrolysate 0.05 g/l 
(Difco casamino acids) 
Casein (Hammarsten) 6.0 g/l 
CaCl.sub.2 0.06 g/l 
Saline solution 2 10 ml/l 
(composition specified in Table 2 of 
Example 2) 
Leave stirring for 15 min so as to 
avoid foam formation. 
Add 15 g of agar (Difco Bacto-agar). 
Adjust the pH to 6.2 using 1N HCl or 
1N NaOH. 
Autoclave for 20 min at 120.degree. C. 
______________________________________ 
TABLE 9 
______________________________________ 
Composition of medium F 
______________________________________ 
Cottonseed meal 10 g/l 
Glucose 35 g/l 
Ca(NO.sub.3).sub.2 3.5 g/l 
CaCO.sub.3 0.75 g/l 
Linseed oil 2.5 ml/l 
Sodium oleate 1.5 g/l 
Adjust the pH to 6.20 using 1N HCl or 
1N NaOH. 
Autoclave for 45 min at 120.degree. C. 
______________________________________ 
Analysis of the enzyme secreted by the transformants 
The fermentation must obtained after culturing (either in flasks or in 
fermenters) the overproductive transformants and the untransformed C. 
parasitica control strain SEBR 103 was subjected to a centrifugation so as 
to remove the mycelial mass. After denaturation of the proteins in the 
supernatant in the presence of SDS for 5 min at 100.degree. C., 
electrophoresis was performed on polyacrylamide gel in the presence of 
SDS. After staining with Coomassie blue, a predominant band of molecular 
mass in the region of 36 kDa is observed, corresponding to the molecular 
mass of mature endothiapepsin deduced from its sequence (see FIG. 1) and 
more intensely stained in the case of the overproductive transformants 
than in the case of the control strain; and bands of lesser importance 
which are identical for the overproductive transformants and the control 
strain. 
It was verified, moreover, by an antigen-antibody reaction (Rennetest kit, 
France Biochem) on the culture supernatants of the overproductive 
transformants and of the untransformed control strain that the secreted 
enzyme is identical to that of Cryphonectria parasitica according to the 
identification method described in the Journal Officiel de la Repulique 
Fran.cedilla.aise (Official Journal of the French Republic) of Mar. 20, 
1981. 
In addition, the ratio of coagulant activity to proteolytic activity of the 
secreted enzyme was assessed. The coagulant activity, expressed in g/l, is 
measured according to the method described above in subsection b) 
.alpha.); the proteolytic activity, expressed as glycine milliequivalents 
per litre, is measured using the TNBS reagent, described by R. Fields, 
Biochem. J. (1971) 124: 518-590, by assaying the amino groups which have 
appeared after proteolysis of dimethylcasein. 
This ratio is between 0.045 and 0.050 for the overproductive transformants, 
which is very close to that obtained with the untransformed control 
strain. It may be concluded from these three studies that endothiapepsin 
production is indeed the feature which has been specifically increased in 
the overproductive transformants. 
c) Verification of the integration of the fragment A, B or C by Southern 
blotting. 
The genomic DNA of the selected transformants is prepared according to a 
protocol similar to that described by Raeder and Broda, 1965, Letters in 
Applied Microbiology 1, 17-20, summarised below: 
The mycelium originating from a flask culture performed as described in 
Section 1.1) is lyophilised and filtered off on gauze. After rinsing in 20 
mM EDTA solution, pH 8, the mycelium is again filtered off, then frozen at 
-80.degree. C. and lyophilised. 50 mg of the lyophilisate obtained are 
then ground and thereafter resuspended in 500 .mu.l of the following 
extraction buffer: 200 mM Tris-HCl pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% 
SDS, proteinase K (Sigma) 200 .mu.g/ml. The mixture is incubated for 45 
min at 45.degree. C., and the DNA is then extracted with phenol/chloroform 
and then precipitated with isopropanol. The pellet is then taken up in 100 
.mu.l of TE (10 mM Tris-HCl pH 8, 1 mM EDTA). 
The DNA is then digested with the appropriate endonucleases and the 
fragments obtained are separated by electrophoresis on 0.8% agarose gel. 
The fragments are transferred by capillarity onto a nylon membrane (Hybon 
N.sup.+ -Amersham) according to the method recommended by the 
manufacturer. The filters are then hybridised at 42.degree. C. overnight 
with, successively, the fragment A and the HindIII-HindIII fragment 
containing the mutated gene for .beta.-tubulin of Neurospora of plasmid 
pBT3, these fragments being labelled with horseradish peroxidase 
(Amersham) and revealed with the "Gene detection system" ECL 
chemiluminescent probe kit (RPN 2101, Amersham). The hybridisation signals 
obtained are visualised on a suitable film. 
2) Selection of overproducers from among the 29Pn transformants (containing 
cosmid p8H12 which carries the fragment B--see Section 4) 
164 benomyl-resistant colonies were obtained during the cotransformation 
with cosmid p8H12 and plasmid pBT3. 127 clones were chosen at random from 
among the latter for the selection test on agar medium containing casein, 
which enabled 14 clones to be adopted. From among the latter, 7 chosen at 
random were subjected to the selection test in liquid medium. 3 clones 
were thereby adopted; the clones 29P1, 29P2 and 29P3, possessing after 
flask culture a coagulant activity of 0.9, 1.07 and 0.94 g/l, 
respectively, whereas the untransformed control strain possesses an 
activity of 0.62 g/l (the overproduction factor, namely the ratio of the 
coagulant activity of the overproductive strain to the coagulant activity 
of the control strain is hence 1.45, 1.73 and 1.54, respectively). The 
clones 29P2 and 29P3 were then tested in a 2-1 fermenter, and produced a 
coagulant activity of 1.76 and 2.19 g/l, respectively, the control strain 
producing only 1.20 g/l (the overproduction factor is hence +1.47 and 
+1.83). 
The clone 29P3 and the C. parasitica control strain SEBR 103 were cultured 
in a 20-1 fermenter under less limiting experimental conditions, 
especially as regards agitation, aeration and bulk transfer, and close to 
the conditions used in the industrial process. 
This test enabled a quantity of endothiapepsin equal to approximately twice 
the quantity produced by the control strain to be obtained. 
The 7 clones chosen above at random were analysed by Southern blotting. 
Their genomic DNA and that of the untransformed C. parasitica control 
strain SEBR 103 were digested with the enzyme SmaI, which generates only 
two cleavage sites in cosmid p8H12 and none in the fragment A. After 
hybridisation with the latter, two hybridisation bands were observed with 
the clones 29P1, 29P2 and 29P3 and a single hybridisation band, identical 
in size to one of the two bands mentioned above, with the control strain 
and the other clones not adopted after the selection test in liquid 
medium. The three overproductive clones 29P1, 29P2 and 29P3 have hence 
integrated a copy of cosmid p8H12 at a different locus of their genome, 
since the supernumerary bands observed differ in size. Finally, the 
Southern blot obtained above was hybridised with the HindIII fragment of 
plasmid pBT3 which confers benomyl resistance, and this enabled it to be 
established that the 7 benomyl-resistant clones all received at least one 
copy of plasmid pBT3, since they show several additional hybridisation 
bands compared with the control strain, the latter showing only one band 
corresponding to the endogenous .beta.-tubulin gene (.beta.-tubulin is a 
structural protein present in filamentous fungi, especially Neurospora 
crassa and C. parasitica). 
These results show that the fragment B hence contains the signals necessary 
for the expression (and secretion) of endothiapepsin. It hence contains a 
functional gene for endothiapepsin, that is to say a sequence coding for 
an endothiapepsin precursor, flanked by a functional promoter and a 
functional terminator. This functional promoter hence comprises an 
activator region located upstream of the TATA box localised in the 
fragment A (see Section 1). 
The addition of a further copy of this fragment to the genome of C. 
parasitica strain SEBR 103 by transformation enables transformed strains 
which overproduce endothiapepsin with a factor in the region of 2 to be 
obtained. 
3) Selection of overproducers from among the 30Pn transformants (containing 
plasmid pEp1 which carries the fragment C: see Section 6) 
663 benomyl-resistant colonies were obtained during the cotransformation 
with plasmid pEp1 and plasmid pBT3. 108 clones were chosen at random from 
among the latter for the selection test in agar medium containing casein, 
which enabled 32 clones to be adopted. It will be noted that this test 
enabled a markedly higher level (32/108) of overproducers to be adopted 
from among the 30Pn transformants than that obtained for the 29Pn 
transformants (14/127). This indicates that the frequency of 
cotransformation obtained with the approximately 13.4-kb plasmid pEp1 is 
greater than that obtained with the approximately 40.6-kb cosmid p8H12. 
From among these 32 clones, 12 clones chosen at random were subjected to 
the selection test in liquid medium. 7 clones were thereby adopted: the 
clones 30P.sub.1, 30P.sub.2, 30P.sub.3, 30P.sub.4, 30P.sub.5, 30P.sub.8 
and 30P.sub.7, possessing after flask culture a coagulant activity of 
1.12, 1.06, 0.96, 1.12, 1.05, 0.88 and 0.90 g/l, respectively, whereas the 
untransformed control strain possesses an activity of 0.62 g/l (the 
overproduction factor is hence between +1.42 and +1.81). 
The clones 30P.sub.1, 30P.sub.2 and 30P.sub.5 were then tested in a 2-1 
fermenter, and produced a coagulant activity of 3.05, 3.0 and 3.04 g/l, 
respectively, the control strain producing only 1.20 g/l (the 
overproduction factor is hence +2.54, +2.50 and +2.53, respectively). 
It is probable (see 2 above) that, under the non-limiting experimental 
conditions of the 20-1 fermenter, it would be possible to achieve an 
overproduction factor of approximately 3. 
The 7 clones chosen above at random were analysed by Southern blotting. 
Their genomic DNAs and that of the untransformed C. parasitica control 
strain SEBR 103 were digested with the enzyme SacI, chosen since it 
generates only a single cleavage site in plasmid pEp1, a site localised in 
the fragment C outside the fragment A (see FIG. 4). After hybridisation 
for each of the 7 clones with the fragment A, at least two hybridisation 
bands (2 to 4 depending on the clone) were observed, including the band of 
the endogenous endothiapepsin gene also present for the control strain, 
the profile being different for each of the clones. For the clones 
30P.sub.1, 30P.sub.2 and 30P.sub.5, a hybridisation band identical in size 
to that of the linearised plasmid pEp1 is observed among the bands, which 
indicates the probable integration in tandem of at least two additional 
copies of this plasmid. 
It is, in effect, well known to those skilled in the art that the 
integration in tandem of several copies of a plasmid after transformation 
is a common event in filamentous fungi (Fincham J. R. S., March 
1989,Microbiological Reviews, 148-170), and that digestion of the genomic 
DNA in which the plasmid has thereby been integrated, with an endonuclease 
generating only a single cleavage site in this plasmid, liberates this 
plasmid. 
These results show that the fragment C contains all the signals necessary 
for the expression (and secretion) of endothiapepsin. It hence contains a 
functional gene for endothiapepsin, and hence a complete functional 
promoter. The promoter-activating region is hence located in the fragment 
C. 
The addition of at least two further copies of this fragment to the genome 
of C. parasitica strain SEBR 103 enables transformed strains which 
overproduce endothiapepsin with a factor in the region of 3 to be 
obtained. 
4) Selection of overproducers from among the 31Pn transformants (containing 
plasmid p472 which carries the fragment A: see Section 6) 
840 benomyl-resistant colonies were obtained during the cotransformation 
with plasmid p472 and plasmid pBT3. 108 clones were chosen at random for 
the selection test on agar medium containing casein. In contrast to the 
results obtained above with the 29Pn and 30Pn cotransformants, no 
overproducer could be detected. Nevertheless, 5 clones 30P.sub.1, 
30P.sub.2, 30P.sub.3, 30P.sub.4 and 30P.sub.5, chosen from among those 
giving a hydrolysis halo which was large but not significantly larger than 
that of the untransformed control strain, were subjected to the selection 
test in liquid medium. The abovementioned clones possess after flask 
culture a coagulant activity of 0.38, 0.66, 0.66, 0.56 and 0.65 g/l, 
respectively, whereas the control strain possesses an activity of 0.62 g/l 
(the difference found is hence -38, +6, +6, -22 and +4%). No significant 
overproduction is detected by this test in liquid medium, confirming the 
negative result of the selection test in casein agar medium. 
Analysis by Southern blotting showed that the 5 clones integrated at least 
one copy of plasmid pBT3, and 3 clones out of 5 are genuinely 
cotransformed by plasmid pBT3 and plasmid p472. The addition of a further 
copy of the fragment A contained in plasmid p472 hence does not lead to an 
overproduction of endothiapepsin, which indicates that DNA sequences 
essential for the expression of endothiapepsin are lacking in this 
fragment. The fragment A hence does not contain a functional gene for 
endothiapepsin, which confirms that the fragment A lacks an activating 
region upstream of the promoter necessary for rendering the latter 
functional (see Section 1-7). 
The activating region upstream of the promoter is hence located in the 
fragment C between its 5' end (BamHI site) and the 5' end (HindIII site) 
of the fragment A which it contains. 
A more precise localisation of this activator region may be determined by 
obtaining a series of sub-fragments of the fragment C (prepared, for 
example, by digestion using endonucleases or exonucleases) comprising the 
fragment A flanked at the 5' end by different-sized segments of the 
portion of the fragment C bounded by the 5' end of the fragment A and a 
nucleotide located between the 5' end of the fragment A and the 5' end of 
the fragment C, transformation of Cryphonectria parasitica SEBR 103 with 
these subfragments and selection of the transformants expressing the 
recombinant protease. Examples of such subfragments of the fragment C are 
the fragments D and F, the preparation of which is described in Sections 4 
and 5. 
Section 8 
Method of purification (removal of the selection marker) of a transformant 
over-productive of endothiapepsin, and of amplification of the gene coding 
for endothiapepsin by successive transformations. 
1) Background 
It is known (see, in particular, Fincham J. R. S., March 
1989,Microbiological Reviews, 148-170) that fungal cells comprise several 
nuclei, in general containing the same genetic material. The protoplasts 
obtained after enzymatic digestion of their walls can be anucleate 
(incapable of regenerating), uninucleate or multinucleate. After 
transformation of the latter, it is hence possible to obtain transformed 
cells of the heterokaryon type, containing transformed nuclei, where 
appropriate of different kinds (depending on the mode of integration and 
the nature of the integrated material, which can vary) and untransformed 
nuclei. In the case of C. parasitica, the conidiospores are uninucleate 
(Puhalla J. E. et al., Phytopathology, 1971, 61, 169-173). 
The work described below employs these characteristics of fungal cells to 
construct strains containing only the recombinant DNA of interest and not 
the selection marker. 
2) Purification of the transformant 29P3 (removal of the selection marker) 
for the purpose of testing for benomyl-sensitive overproducers 
A preparation of conidiospores of the initial transformant overproductive 
of endothiapepsin 29P3 [see Section 1.1) and Section 7.2)], sufficiently 
dilute to obtain isolated colonies, was used to inoculate Petri dishes 
containing medium B (see Table 2 above) made into an agar medium by adding 
20 g/l of agar. After incubation for 5 days at 30.degree. C., mycelial 
implants of 50 colonies were subcultured in parallel on the same agar 
medium B and on the latter supplemented with 1 .mu.g/ml of Benlate 
(containing 50% of benomyl). After incubation for 5 days at 30.degree. C., 
6 clones show normal growth on agar medium B and zero growth on agar 
medium B supplemented with Benlate. These 6 benomyl-sensitive clones, each 
derived from the germination of one spore, are of the homokaryon type, 
hence pure. 
The 6 clones and the initial transformant 29P3 were subjected to the test 
of selection of overproduction of endothiapepsin on agar medium containing 
casein [see Section 7.1) a)], which enabled a benomyl-sensitive clone, 
referred to as 29P3 benS, to be selected, which clone shows an 
overproduction not significantly different from that of the initial 
transformant. The clone 29P3 ben.sup.S, the clone 29P3 and the C. 
parasitica control strain SEBR 103 were subjected, after sporulation, to 
the selection test in liquid medium. The clones 29P3 and 29P3 ben.sup.s 
produce a coagulant activity which is identical (taking into account the 
margin of experimental error) but markedly greater than that of the 
control strain. In addition, Southern blotting performed on these two 
clones and the control strain, after digestion of their genomic DNA with 
the endonuclease SmaI, shows that each of the two clones have the same 
hybridisation profile with the fragment A as a probe, which profile 
contains a super-numerary band compared with the control (see Section 
7.2)), indicating that in both clones the integration of the functional 
gene for endothiapepsin is identical, and a different hybridisation 
profile with the HindIII-HindIII fragment of plasmid pBT3 which confers 
resistance to benomyl (see Section 6), the initial transformant 29P3 
showing several hybridisation bands with this fragment and the clone 29P3 
ben.sup.S and also the control strain showing a single hybridisation band 
identical in size (corresponding to the endogenous gene for 
.beta.-tubulin). 
These results show that the purification method made it possible to obtain 
a transformant which was pure (as regards its genotype) and devoid of the 
selection marker (gene conferring benomyl resistance), and that the 
character of overproduction of endothiapepsin is integrated stably in the 
clone 29P3 ben.sup.s since it is derived from the germination of a 
uninucleate conidiospore and all the mycelial cells possess nuclei which 
have integrated cosmid p8H12 in their DNA. 
3) Amplification 
Protoplasts prepared from the clone 29P3 ben.sup.S were transformed with 
plasmid pBT3 according to the protocol described in Section 6. 400 
benomyl-resistant clones were obtained for 1 .mu.g of plasmid DNA. 
It is hence possible to obtain, after cotransformation with a cosmid 
containing the functional gene for endothiapepsin and a plasmid containing 
a selection marker, a transformant which is overproductive of 
endothiapepsin and devoid of the selection marker, capable of being 
transformed again. It is hence possible, by performing several successive 
cycles comprising a step of cotransformation using the above two vectors 
followed by a step of purification enabling the selection marker to be 
removed, to amplify selectively in C. parasitica the gene coding for the 
endothiapepsin precursor. 
Section 9 
Testing for strains deficient in the production of endothiapepsin after 
transformation of C. parasitica SEER 103 
An exhaustive analysis of the benomyl-resistant clones obtained after 
contransformation (see Section 6) of C. parasitica strain SEBR 103 with 
cosmid p8H12 and plasmid pBT3 enabled a clone to be obtained which did not 
produce a halo of hydrolysis on agar medium E containing casein. This 
clone was purified according to the method described in Section 8, and a 
large number of benomyl-sensitive clones which did not produce a halo of 
hydrolysis were obtained. One clone, designated 29P (end.sup.-), was 
chosen at random from among these benomylsensitive clones. After flask 
culture, the clone 29P (end.sup.-) possesses a coagulant activity of less 
than 0.01 g/l, whereas the untransformed Cryphonectria parasitica control 
strain SEBR 103 possesses a coagulant activity of 0.62 g/l (the observed 
fall in production is hence more than 98%). It was, in addition, observed 
that the morphological and physiological characteristics of this clone are 
modified relative to those of C. parasitica strain SEBR 103. 
Southern blot analysis of the genomic DNA of the clone 29P(end.sup.-) and 
of the control strain SEBR 103, after hybridisation with the fragment A on 
the one hand and the fragment containing the benomyl resistance gene of 
plasmid pBT3 on the other hand, showed no differences. 
These results show that the clone 29P(end.sup.-) selected and purified has 
been rendered deficient for the production of endothiapepsin after 
cotransformation of the C. parasitica strain with cosmid p8H12 and plasmid 
pBT3. It is seen to be obvious to those skilled in the art that 
transformants deficient in the production of endothiapepsin could be 
obtained by cotransformation of C. parasitica SEBR 103 with a selection 
marker, such as plasmid pBT3, and a DNA containing the 
preproendothiapepsin gene rendered non-functional, for example by deletion 
of a portion of the coding sequence, followed by selection of the 
benomyl-resistant transformants which do not produce a halo of hydrolysis 
of casein. Such a DNA carrying a non-functional gene may be readily 
obtained by linearisation of the DNA of plasmid pEp1, for example by 
performing an ScaI and SfiI double digestion, then purifying the largest 
fragment by electrophoresis on 0.8% agarose gel and finally treating the 
ends of this fragment with Klenow polymerase in the presence of the four 
deoxyribonucleotide triphosphates dNTP in order to permit a religation of 
the vector and thereby to obtain a plasmid carrying a non-functional gene 
for endothiapepsin. 
Section 10 
Construction of the strain SEBR 3700, deficient in the production of 
endothiapepsin and devoid of a dominant selection marker 
1) Construction of a fragment, referred to as fragment EM, in which the 
sequence coding for endothiapepsin is interrupted by two translation stop 
codons. 
At the beginning of the sequence coding for the endothiapepsin precursor, 
mutations were introduced bringing about a stoppage of translation, as a 
result of which endothiapepsin is no longer produced from the messenger 
RNA carrying these mutations. The fragment A of endothiapepsin comprises 
an approximately 360-bp HindIII-BstEII fragment (see FIG. 2), the BstEII 
site being localised 25 bp downstream of the beginning of the coding 
sequence. The natural sequence in question is as follows (SEQ ID NO:16): 
BsteII 
.vertline. 
.vertline. 
5'-G ATG TCT TCC CCT CTC AAG AAC GCC TTG GTG ACC 
The underlined ATG triplet represents the codon opening the coding frame. 
The BstEII site is indicated by a vertical line. 
The desired mutated sequence (SEQ ID NO:17) is: 
BsteII 
.vertline. 
.vertline. 
5'-G ATG TCT TCC CCT CTC TAA TGA ACG CCT TGG TGA CC 
This sequence differs from the natural sequence by the introduction of 2T, 
one between the 5th and 6th codon, the other within the 6th codon (between 
the 2nd and the 3rd base). These introductions result in the creation of 2 
stop codons stopping the reading frame of the endothiapepsin gene. 
A HindIII-BstEII fragment differing from the wild-type sequence only in 
these two modifications was obtained by the PCR amplification technique 
described in Section 15, using for one of the primers an oligonucleotide 
carrying the desired mutations. 
Oligonucleotide 1 has the following sequence (SEQ ID NO:18): 
HindIII 
.vertline. 
5' - GCT AAA GCT TAT CCG CCG CCG GCG GGG GAA TTC 
This sequence is to be found at the HindIII end of the HindIII-BstEII 
fragment. The HindIII site is designated by a vertical line. 
Oligonucleotide 2 has as its sequence (SEQ ID NO:19): 
BamHIBstEII BamHI BsteII 
.vertline. .vertline. 
5' - CAA TGG ATC CGG TCA CCA AGG CGT TCA TTA GAG AGG GGA AGA 
- CAT C 
This oligonucleotide is complementary to the desired mutated sequence; a 
BamHI site flanking the natural BstEII site has been attached to it. The 
underlined nucleotides correspond to the additions creating the stop 
codons on the complementary strand. 
The DNA used as a template is the DNA of plasmid p472 described in Section 
1. The amplification mixture comprises: 
300 ng (equivalent to 3 .mu.l) of p472 DNA 
100 ng (equivalent to 1 .mu.l) of each oligonucleotide 
5 .mu.l of buffer bipv2 concentrated 10-fold 
40 .mu.l of water 
2 units (equivalent to 0.5 .mu.l) of enzyme: Taq polymerase. 
The buffer bipv2 10-fold concentrated has the following composition: 670 mM 
Tris-HCl pH 8.8; 165 mM ammonium sulphate; 10 mM 2-mercaptoethanol; 
gelatin 2 mg/ml; Triton X-100 1.5%; 67 .mu.M EDTA; 20 mM MgCl.sub.2 ; 2 mM 
DATP; 2 mM dCTP; 2 mM dGTP; 2 mM dTTP. 
Three amplification tests are carried out in parallel. 15 amplification 
cycles are performed, each cycle being divided into 1 min of denaturation 
at 92.degree. C., 1 min of hybridisation at 55.degree. C. and 1 min of 
elongation at 72.degree. C. After PCR amplification, the 3 tubes are 
combined in 1 Eppendorf tube and precipitated with 2 volumes of absolute 
ethanol containing 0.3M ammonium acetate (for 20 min at 0.degree. C.), 
then centrifuged at 10,000 g (for 20 min). The pellet is washed with 70% 
ethanol and then dried under vacuum for 10 min. 
The DNA is taken up in 60 .mu.l of TE solution (10 mM Tris-HCl pH 7.5, 1 mM 
EDTA) and analysed on agarose gel. The band corresponding to the 360-bp 
fragment is eluted from the gel, purified and cloned into the replicative 
form of a phage M13 (Ml3mpl9) between the HindIII and BamHI sites after 
the action of these enzymes. 
The fragment thus cloned was sequenced from the single-stranded fragment, 
and it was verified that the sequence did indeed correspond to the 
fragment mutated at the expected points. 
The approximately 2.1-kb fragment A described in Section 1 is bounded by 2 
HindIII sites and possesses a single BstEII site localised at 360 base 
pairs from one end. The single BstEII site hence bounds two segments of 
the fragment A; the shorter corresponds to the sequence subjected to PCR 
amplification. By preparing the large, BstEII-HindIII segment of the 
fragment A and the mutated, small, HindIII-BstEII segment, and ligating 
the whole in pBR322 cut with HindIII, a fragment A carrying the two 
mutations, referred to as fragment A-M, is thereby reformed. 
The fragment A-M possesses a PstI site which is unique for this fragment. 
This PstI site hence bounds 2 segments of the fragment A-M, including an 
approximately 1.67-kb HindIII-PstI segment which carries the double 
mutation. 
Plasmid pEp3 described in Section 5 is derived from a pUC plasmid into 
which an approximately 3.7-kb SphI fragment, referred to as fragment E, 
has been cloned. This 3.7-kb fragment comprises the fragment A as a 
subfragment. This fragment A was replaced by the fragment A-M in the 
following manner: 
Plasmid pEp3b was digested both with the enzymes ScaI and PstI on the one 
hand and the enzymes ScaI and HindIII on the other hand. From the first 
digestion, the approximately 2.250-kb ScaI-PstI fragment was isolated. 
From the second digestion, the approximately 2.42-kb ScaI-HindIII fragment 
was isolated. Ligation of these 2 fragments with the 1.67-kb HindIII-PstI 
segment carrying the double mutation enables a new plasmid, referred to as 
pEpM3b, which differs from pEP3b only in the double mutation, to be 
obtained. 
The SphI fragment carrying the double mutation, referred to as fragment 
E-M, was prepared from an SphI digestion of pEpM3b followed by a 
separation of the 2 DNA fragments on 0.8% agarose gel and extraction of 
the mutated 3.7-kb fragment in 20 .mu.l of TE solution. 
2) Cotransformation of Cryphonectria parasitica with the fragment EM and 
the SfiI fragment carrying a benomyl resistance gene flanked by telomeric 
sequences. 
The purified fragment EM was used in cotransformation with the DNA of 
plasmid p578.12 digested with SfiI. 
Plasmid p578.12 is a derivative of plasmid pBT3 already-described. The DNA 
of pBT3 was linearised with the endonuclease XhaI and the ends were 
rendered blunt with Klenow DNA polymerase. In it was integrated an XhaI 
fragment, repaired with the polymerase and which comprises the telomeric 
sequences of Tetrahymena originating from plasmid pPAT ura described by 
Perrot, Barreau and Begueret Mol. Cel. Biol. (1987) 7 p. 1725-1730, 
modified so as to replace the BamHI sites by SfiI. Digestion of p578.121 
with SfiI liberates a linear fragment which carries a benomyl resistance 
gene and which terminates at each of its ends with a telomeric sequence. 
Perrot et al. (ref. above) showed that such a fragment could be maintained 
in the state of a linear plasmid (not integrated in the chromosome) in the 
filamentous fungus Podospora anserina. Moreover, Powell and Kistler (J. 
Bacteriology, 1990, vol. 172, pp 3163-3171) showed that repeated sequences 
of the telomeric type permitted the autonomous replication of linear 
plasmids in C. parasitica. This property was turned to good account in 
order to construct appropriate strains by cotransformation with the linear 
fragment. The absence of integration of the benomyl resistance marker in 
the chromosome should enable benomyl-sensitive strains to be recovered 
with a very great frequency from the conidia obtained after sporulation of 
the transformants. 
2 cotransformation experiments were carried out on Cryphonectria parasitica 
strain SEBR 103 with, for each one, approximately 0.5 .mu.g (3 .mu.l) of 
the fragment E-M and 0.5 .mu.g (4 .mu.l) of the SfiI fragment of plasmid 
p578.12. 397 benomyl-resistant colonies were obtained. 395 of these 
colonies were subcultured on medium E (see Section 7) to test for the 
presence of a halo as described in Section 7. 
One colony no longer forming a halo of coagulation of casein was found; 
this colony, referred to as colony AJ7.272, was transferred onto 
sporulation (conidiation) medium according to the method described in 
Section 1. The conidiospores were harvested in bulk and diluted with the 
germination medium. 152 colonies derived from the conidiation were tested 
on casein medium: none of them showed a halo of coagulation. The benomyl 
resistance of these colonies was also tested: 58 of the 152 had lost this 
transformation character. 5 of the 58 were taken; they were referred to as 
colony AJ7272/A, colony AJ7272/B, colony AJ7272/J, colony AJ7272/L and 
colony AJ7272/M. The genomic DNA was extracted from each colony, and the 
hybridisation profiles and that of the strain SEBR 103 were compared by 
the Southern blotting technique, using various restriction enzymes. No 
difference was found between the strain SEBR 103 and the 5 mutants, probed 
with the fragment A (endothiapepsin gene), with pUC18 (bacterial 
sequences) or with the gene for beta-tubulin of Neurospora. With the 
fragment A, the sequences of the endothiapepsin gene are revealed; since 
the hybridisation profile is the same in all the strains, it may be 
concluded that there is no "ectopic" recombination of the mutated fragment 
(that is to say non-homologous recombination) in the mutants. With pUC18, 
no hybridisation is obtained, irrespective of the strain, which indicates 
that no sequence of bacterial origin remains integrated in the mutants. 
With the .beta.-tubulin gene, a sequence is revealed in all the strains; 
this very probably corresponds to the "endogenous" gene for .beta.-tubulin 
of C. parasitica. 
In order to check that the strains derived from the colony AJ7272 were 
indeed mutated in the endothiapepsin structural gene, it was verified that 
their lack could be complemented by transformation. Each of the above 
colonies was used as a receptor for a cotransformation involving the DNAs 
of plasmid pBT3 and of plasmid pEP2. 
The following table shows that the majority of benomyl-resistant 
transformants obtained in this manner again produce endothiapepsin. 
______________________________________ 
Benomyl-resistant 
colonies Colonies productive 
Strain (test on casein) of protease 
______________________________________ 
AJ7.272/A 2 2 
AJ7.272/B 8 7 
AJ7.272/J 13 11 
AJ7.272/L 17 16 
AJ7.272/M 10 10 
______________________________________ 
Control: transformation of the segregants with plasmid pBT3 alone gives n 
transformant productive of protease. 
Analysis of the culture supernatants by acrylamide gel electrophoresis 
confirmed that the mutated strains did not produce a detectable amount of 
endothiapepsin before transformation. 
A strain AJ7.272 which, after sporulation, gave rise to 5 colonies which 
differ from the strain SEBR 103 only in one or more mutations affecting 
the endothiapepsin structural gene was hence constructed. One of these 
colonies was adopted. It is referred to as SEBR 3700. It is deficient in 
the production of endothiapepsin and devoid of a dominant selection 
marker. 
Section 11 
Verification of the functionality of the fragments D and F by 
complementation of the strain SEER 3700 deficient in the production of 
endothiapepsin. 
It was demonstrated in Section 7, subsection 3, that the fragment C 
contained in plasmid pEp1 carries all the signals necessary for the 
expression of endothiapepsin. Using the strain SEBR 3700 deficient for the 
production of endothiapepsin, obtained in Section 10, the functionality of 
the fragments D and F contained in plasmids pEp2 and pEp4 was verified. 
1) Transformation of protoplasts of the strain SEBR 3700 with plasmids pEp2 
and pBT3 
Plasmid pEp2 carries both the benomyl resistance gene and the fragment D, 
and can hence be used directly for transformation. Plasmid pBT3 which does 
not carry a gene coding for endothiapepsin is used as a negative control. 
The strain SEBR 3700 was transformed according to a protocol identical to 
that described for the strain SEBR 103 (see Section 6) with approximately 
1 .mu.g of plasmid pEp2 and 1 .mu.g of plasmid pBT3. 
2) Cotransformation of protoplasts of the strain SEBR 3700 with plasmids 
pEp1 and PBT3 and plasmids pEp4 and pBT3. 
Plasmids pEp1 and pEp4 do not carry a dominant selection marker. Plasmid 
pEp1 is used as a positive control-of complementation of the strain SEBR 
3700. 
The strain SEBR 3700 was cotransformed with the following mixtures of 
plasmids: 4 .mu.g of pEp1 and 1 .mu.g of pBT3, 4 .mu.g of pEp4 and 1 .mu.g 
of pBT3. 
3) Detection of transformants producing endothiapepsin 
Mycelial implants of several transformants selected for each of the 
transformations and cotransformations described above were subcultured on 
medium E, an agar medium containing casein. After incubation, the colonies 
showing a halo of precipitation characteristic of the secretion of 
endothiapepsin were identified. The results obtained are as follows: 
transformants obtained with plasmid pBT3: no clone gave a halo of 
precipitation 
transformants obtained either with plasmids pEp1 and pBT3, or with plasmids 
pEp4 and pBT3: a proportion of approximately 30% of the clones gave halos 
of precipitation 
transformants obtained with plasmid pEp2: 92% of the clones tested gave 
halos of precipitation. 
These results show that plasmids pEp1, pEp2 and pEp4 complement the strain 
SEBR 3700 deficient in the production of endothiapepsin, and hence that 
the fragments C, D and F contained in these plasmids all carry a 
functional promoter of endothiapepsin. However, the relative strength of 
the promoter present in each of the fragments cannot be deduced from this 
qualitative test. 
It may be deduced from these results that the SphI-HindIII segment of the 
fragment F, the sequence of which has been determined in Section 5, 
possesses signals involved in the activation of the promoter of the gene 
coding for preproendothiapepsin. 
Section 12 
Selection of transformants overproductive of endothiapepsin and devoid of a 
dominant selection marker. 
1) Selection protocol 
The different steps of the protocol are shown in the table below. Its 
principle is as follows: 
C. parasitica SEBR 103 protoplasts are cotransformed, according to a 
protocol identical to that described in Section 6, with a mixture composed 
of 0.5 to 2 .mu.g of fragment C, D or F, previously purified on agarose 
gel after digestion of plasmids pEp1 or pEp2 and extracted according to 
the instructions of the Biorad Gene-clean kit, and 0.5 .mu.g of the 
selection plasmid pBT3 or pBT6, either in circular form or in linear form. 
After regeneration of the protoplasts on medium C, the transformants 
obtained, which are designated initial transformants, are simultaneously 
subcultured on medium B made into an agar medium by adding 20 g/l of agar 
(non-selective medium) and on medium E, an agar medium containing casein, 
where appropriate supplemented with 5 .mu.g/ml of pepstatin. The clones 
possessing a ratio of the diameter of the halo of precipitation to the 
colony diameter significantly higher than that of the untransformed 
control strain are designated overproductive initial transformants and are 
set up for sporulation. A preparation of conidiospores of these strains is 
made according to the method used in Section 1.1, and a dilution of each 
conidiospore suspension is plated out on medium G so as to obtain isolated 
colonies. Mycelial implants originating from about fifty colonies are 
subcultured for each overproductive initial transformant, simultaneously 
on medium B made into an agar medium by adding 20 g/l of agar, where 
appropriate supplemented with 0.5 mg/l of Benlate (benomyl sensitivity 
test) and on medium E (test on casein medium). At this stage, the clones 
which are sensitive to benomyl and overproductive of endothiapepsin are 
designated overproductive benomyl(s) segregants and are set up for 
sporulation. A conidiospore preparation is then made in order to verify 
the overproduction of endothiapepsin after culturing in flasks, to verify 
the integration of one or more copies of the fragment C, D or F by 
hybridisation of the genomic DNA with a probe consisting of the fragment 
A, and to check the absence of heterologous DNA using a probe consisting 
of all or part of the selection plasmid. The strain satisfying these three 
criteria positively is designated overproductive appropriate strain. A 
further cycle of cotransformation/selection may then be performed from 
this overproductive appropriate strain in order to amplify again the 
fragment C, D or F. 
The advantage of this process, in addition to that obtained by 
amplification of the fragment of interest, is to construct transformants 
devoid of a dominant selection marker, which are more acceptable from the 
standpoint of statutory regulations. 
Protocol for the selection of overproductive transformants devoid of a 
dominant selection marker 
##STR15## 
2) Selection of overproductive transformants devoid of a dominant 
selection marker after cotransformation of SEBR 103 with the fragment C, D 
or F and plasmid pBT3 
The following table summarises the results obtained 
______________________________________ 
Cotransformation 
Fragment F Fragment C Fragment D 
______________________________________ 
Quantity 0.5 .mu.g 2 .mu.g 0.5 .mu.g 
Selection plasmid circular circular linear 
Initial trans- 4 137 106 
formants 
Overproductive 1 26 33 
initial trans- 
formants 
Benomyl(s) 1 4 * 8 ** 
segregants 
Overproductive 0 2 7 
benomyl(s) 
segregants 
overproductive 0 1 4 
appropriate 
strains 
______________________________________ 
No overproductive appropriate strain could be obtained with the fragment 
C, which is not surprising in view of the low number of initial 
transformants. 
* selection performed on the progeny of 12 overproductive initial 
transformants 
** selection performed on the progeny of 21 overproductive initial 
transformants 
The overproductive appropriate strain obtained with the fragment D was 
tested in a 2-litre fermenter and produced a coagulant activity of 1.9 
g/l, the control strain producing only a coagulant activity of 1.2 g/l 
(the overproduction factor is hence +1.58). Analysis of the genomic DNA by 
Southern blotting of the strain SEBR 3574 showed that it had integrated at 
least 3 additional copies of the fragment D, including 2 copies in tandem. 
Among the four overproductive appropriate strains obtained with the 
fragment F, the strain SEBR 3912 was tested in a 2-litre fermenter and 
produced a coagulant activity of 2.4 g/l (the overproduction factor 
relative to the control strain is hence +2). Analysis of the genomic DNA 
by Southern blotting showed that it had integrated at least 5 additional 
copies of the fragment F, in tandem according to a head-to-tail 
orientation. 
In the overproductive strains obtained, no integration of the selection 
plasmid was detected. These strains may hence be subjected to a further 
cycle of cotransformation/selection. Furthermore, if the over-production 
factor observed for these strains is compared with the presumed number of 
copies integrated, it is found that the ratio obtained is in the region of 
0.2, whereas it is approximately 1 for the 30Pn transformants which have 
integrated two additional copies of plasmid pEp1 containing the fragment 
C. This hence suggests that, upstream of the BglII site located at the 5' 
end of the fragment D up to the BamHI site, there are regulatory sequences 
which are important for a strong expression of the endothiapepsin gene. 
Section 13 
Construction of plasmid pEMR713, a vector for the expression of 
preproendothiapepsin in C. parasitica which comprises the promoter region 
of the gene coding for glyceraldehyde-3-phosphate dehydrogenase of 
Aspergillus nidulans 
Plasmid pAN52 (Punt et al., 1987, Gene, 56, 117-124) carries, in addition 
to the gene coding for ampicillin resistance and the origin of replication 
of pUC18, the promoter region of the gene coding for glyceraldehyde-3 
-phosphate dehydrogenase (gpd) of Aspergillus nidulans [Punt et al., gene, 
93, (1990) 101-109] and the terminator region of the trpC gene of 
Aspergillus nidulans [Mullaney et al., Mol. Gen. Genet. (1985) 189; 
37-45]. The promoter region and the terminator region are separated by a 
DNA sequence which comprises nucleotide sequences recognised by the 
restriction enzymes NcoI and MluI, which sequences are unique in plasmid 
pAN52. The sequence of the NcoI site, namely CCATGG, is especially useful 
inasmuch as it comprises the ATG codon which codes for a methionine which 
is the initiation codon for the majority of proteins. 
The aim of this experiment is to express the gene coding for endothiapepsin 
using the expression signals described above. Integration of the gene 
coding for endothiapepsin is carried out by the PCR (polymerase chain 
reaction) technique, which is described in detail in Section 15 below. 
1--Description of the primers used for the PCR Gene amplification by PCR 
permits modification of the sequence to be amplified. This property is 
used in order to introduce an NcoI site at the 5' end of the prepro 
sequence of the gene coding for endothiapepsin, and an MluI site at the 
non-coding 3' end of the gene coding for endothiapepsin. The sequences of 
the two primers are hence as follows: 
- 5' primer carrying the NcoI site SEQ ID NO:20: 
- 5'-ACG-TCC-ATG-GCT-TCC-CCT-CTC-AAG-AAC-GCC-3' 
This primer consists mainly of: 
a) The sequence recognised by the restriction enzyme NcoI: CCATGG 
b) The modified sequence of the 5' end of the signal peptide of 
endothiapepsin. 
The modification consists in the change of the first codon after the 
methionine; in effect, the TCT codon coding for a serine is replaced by 
the GCT codon coding for an alanine. This modification has no effect on 
the efficacy of the signal peptide. 
- 3' primer carrying the MluI site SEQ ID NO:21: 
- 5' -ACG-TAC-GCG-TCC-ACG-CCT-ACC-CAA-CAA-GAC-3' 
This primer consists mainly of: 
a) The sequence recognised by the restriction enzyme MluI: ACGCGT 
b) The sequence of the non-coding 3' end of endothiapepsin, which is the 
sequence complementary to the sequence located between nucleotides 1921 
and 1940 of FIG. 2. 
2--Production of the amplified fragment containing the gene coding for 
modified endothiapepsin Plasmid p472, the production of which is described 
in Section 1, is used as a template. 
a) The PCR reaction 
100 ng of plasmid p472, previously purified on a P10 column, are mixed with 
100 ng of the 5' primer, 100 ng of the 3' primer, 2 mM MgCl.sub.2, 0.2 mM 
dNTP and 5 .mu.l of reaction mixture concentrated 10-fold (final quantity: 
67 mM Tris-HCl pH 8.8, 16.6 mM (NH.sub.4).sub.2 SO.sub.4, 1 mM 
.beta.-mercaptoethanol, 6.7 mM EDTA, 0.15% Triton X-100, 200 g/ml of 
gelatin). 
The volume of the mixture is then brought to 50 .mu.l by adding water. 
The reaction mixture thereby obtained is incubated for 4 min at 94.degree. 
C. and then brought to a temperature of 50.degree. C., which is maintained 
for 4 min. 
0.5 .mu.l, equivalent to 2.5 units, of Taq polymerase (Boehringer Mannheim 
Ref. 1146-165) is then added. The reaction mixture is then covered with 
paraffin in order to prevent evaporation of the aqueous solution. 
The amplification is carried out during 18 reaction cylces, the steps of 
which are as follows: 
2 min at 92.degree. C..fwdarw.denaturation 
2 min at 50.degree. C..fwdarw.hybridisation 
2 min at 72.degree. C..fwdarw.polymerisation. 
After the 18 cycles, the enzymatic reaction is stopped by adding 20 mM EDTA 
solution. 
The DNA fragment thus amplified, which possesses the expected size of 
approximately 1620 bp, is then isolated and purified on 1% agarose gel, 
dialysed on a P10 column (Pharmacia) and then hydrolysed simultaneously 
with the enzymes NcoI and MluI. After hydrolysis, the fragment is purified 
on a P10 column. 
b) Production of plasmid pEMR713 
The DNA of plasmid pAN52 is hydrolysed with the restriction enzymes NcoI 
and MluI. The fragment carrying the promoter region of the gpd gene, the 
origin of replication of E. coli, the gene coding for ampicillin 
resistance and the trpC terminator is purified. 100 ng of this fragment 
are ligated in the presence of DNA ligase to 100 ng of the amplified 
fragment carrying the endothiapepsin gene (see subsection 2 above). The 
ligation mixture is then used to transform the strain RRI. The resulting 
plasmid is pEMR713 in which the gene coding for modified endothiapepsin is 
placed under the control of the promoter region of gpd and the terminator 
region of trpC. 
Preparation of protoplasts 
see Section 6 
Cotransformation of protoplasts of the strain SEBR 3700 with plasmid 
DEMR713 and plasmid pBT3 
see Section 6 
Approximately 2,000 transformants obtained are capable of growing on agar 
medium B containing 0.5 mg/l of Benlate, which indicates that all these 
colonies carry at least one plasmid pBT3. 
Selection of transformed strains productive of endothiapepsin 
A) General method 
a) Selection on agar medium containing casein 
Mycelial implants of the 2,000 benomyl-resistant colonies are subcultured 
on an agar medium containing casein, referred to as medium E and whose 
composition is specified in TABLE 8 above . On this medium, the 
Cryphonectria parasitica colonies which produce the protease give rise to 
a halo of precipitation whose area is proportional to the quantity of 
endothiapepsin secreted. 
The productive strains are adopted on the basis of the presence of the halo 
of precipitation. A preparation of conidiospores of these productive 
strains is made according to the method used in Section 1.1). In addition, 
it was verified by adding 5 .mu.g/ml of pepstastin, a substance 
specifically inhibiting aspartic proteases, to medium E that the increase 
in the halos observed in the overprodutive strains was reduced. This 
result shows that the observed effect is indeed due to an overproduction 
of an aspartic protease. 
3 transformants capable of producing a halo of coagulation were isolated. 
Control experiments show that the size of the halo of the recombinant 
clones transformed with plasmids pBT3 and pEMR713, referred to as clone 1, 
clone 2 and clone 3, is comparable to that obtained for the strain SEBR 
103. 
b) Selection in liquid medium by a study in flasks 
To confirm this result, tests of production in flasks were carried out in 
the following manner: inoculation of 250-ml flasks containing 40 ml of 
medium F (see Section 7). The flasks are then incubated at 28.degree. C. 
on an eccentric rotary agitator adjusted to 220 rpm for 48 h. For each 
strain, culturing was carried out in 3 different flasks and the mean of 
the results of assay of coagulant activity for the 3 flasks was 
calculated. The control consists of untransformed Cryphonectria parasitica 
strain SEBR 103. The assay of coagulant activity is carried out according 
to the official method of determination of the enzyme content of coagulant 
solutions, published in the Journal Officiel de la Republique 
Fran.cedilla.aise (Official Journal of the French Republic) of Mar. 20, 
1981 (section C), summarised in Section 7. 
c) Analysis of the enzyme secreted by the clones 1, 2 and 3 
The fermentation must obtained after culturing the productive transformants 
and that of the untransformed C. parasitica control strain SEBR 3700 were 
subjected to a centrifugation so as to remove the mycelial mass. After 
denaturation of the proteins in the supernatant in the presence of SDS for 
5 min at 100.degree. C., an electrophoresis was performed on 
polyacrylamide gel in the presence of SDS. After staining with Coomassie 
blue, a predominant band of molecular mass in the region of 36 kDa is 
observed, corresponding to the molecular mass of mature endothiapepsin, 
deduced from its sequence (see FIG. 1), of the same intensity in the case 
of the productive transformants as in the case of the control strain, and 
bands of lesser importance which are identical for the overproductive 
transformants and the control strain. 
It was verified, moreover, by an antigen-antibody reaction (Rennetest kit, 
France Biochem) on the culture supernatants of the overproductive 
transformants and of the untransformed control strain that the secreted 
enzyme is identical to that of Cryphonectria parasitica according to the 
identification method described in the Journal Officiel de la Republique 
Fran.cedilla.aise (Official Journal of the French Republic) of 20th Mar. 
1981. 
In addition, the ratio of coagulant activity to proteolytic activity of the 
secreted enzyme was assessed. The coagulant activity, expressed in g/l, is 
measured using the TNBS reagent, described by R. Fields, Biochem. J. 
(1971) 124: 581-590, by assaying the amino groups which have appeared 
after proteolysis of dimethylcasein. 
This ratio is between 0.040 and 0.045 for the overproductive transformants, 
which is very close to that obtained with the untransformed control 
strain. It may be concluded from these three studies that endothiapepsin 
has indeed been specifically produced by the clones 1, 2 and 3. 
This experiment shows that it is possible to express in C. parasitica the 
gene coding for endothiapepsin, using a promoter region and a heterologous 
terminator region (not belonging to C. parasitica). 
Section 15 
Amplification by the PCR technique of the complementary DNA coding for the 
endothiapepsin precursor. 
1) Isolation of the messenger RNAs of C. parasitica 
C. parasitica strain SEBR 103 was cultured under conditions of production 
of endothiapepsin. The mycelium was recovered by filtration on gauze, 
washed with water and frozen in liquid nitrogen. 
15 g of frozen mycelium (wet weight) are suspended in 45 ml of lysis buffer 
and then taken up in the same volume of beads (0.45 .mu.m in diameter). 
The lysis buffer consists of 4M guanidine thiocyanate, 10 mM Tris-HCl pH 
7.6, 10 mM EDTA and 50 mg/l .beta.-mercaptoethanol. The mycelial 
suspension is ground for 5 min. 
The ground preparation is recovered and the beads removed after settling 
has taken place. Approximately 45 ml of supernatant are withdrawn, lithium 
chloride is added to a final concentration of 3M and the preparation is 
stored at 0.degree. C. 
After two days, the above solution is centrifuged for 60 min at 10,000 rpm. 
The supernatant is withdrawn and the pellet is taken up in 40 ml of 3M 
LiCl. The suspension obtained is recentrifuged at 10,000 rpm for 1 h 30 
min. Proteinase K (SIGMA) 40 .mu.g/ml, SDS (0.1% w/v) and 20 mM EDTA are 
added. The mixture is incubated at 37.degree. C. for 3 h. Precipitation is 
performed with 2 volumes of ethanol and the precipitate is then washed 
with 70% ethanol. The pellet is taken up in 0.5 ml of TE buffer (10 mM 
Tris-HCl, 1 mM EDTA pH 7.5), the mixture is extracted twice with 
chloroform and the product is precipitated with ethanol. The RNA is stored 
at -80.degree. C. in ethanol. 
2) Purification of the poly(A).sup.+ fraction of the RNA 
Approximately 1 mg of RNA is precipitated for 20 min at 4.degree. C. 
(15,000 rpm), then washed with 70% ethanol and then dried. The pellet is 
taken up in 1 ml of TE buffer and suspended by vortexing. Type 3 
oligo(dT)-cellulose (marketed by Collaborative Research Inc, Biomedicals 
Product Division) is prepared according to the manufacturer's 
recommendations. The RNA is applied to the oligo(dT), agitated gently to 
resuspend the beads and then heated for 1 min to 65.degree. C. 
The suspension is adjusted to 0.5 M NaCl and then agitated gently for 10 
min. The suspension is then centrifuged for 1 min at 1,000 rpm, the 
supernatant is removed and the pellet is washed twice with 1 ml of TE 
buffer containing 0.5M NaCl. The supernatants are removed. Elution of the 
polyadenylated fraction of the RNA (consisting of messenger RNAs) is 
obtained by suspending the beads in 1 ml of TE buffer and then heating 
this suspension to 60.degree. C. for 1 min, followed by agitation for 10 
min on a rocking-stage mixer. The mixture is then centrifuged for 1 min at 
1,000 rpm, permitting recovery, on the one hand of the supernatant 
containing free mRNAs in solution, and on the other hand of the pellet of 
cellulose beads. All the above operations (beginning with elution) are 
repeated. The supernatants thereby obtained are combined, the excess beads 
are removed by centrifugation and the supernatant is precipitated with 
ethanol containing NaCl according to the usual techniques (Maniatis: op. 
cit.). 
3) Description of the polymerase chain reaction (PCR) technique 
The polymerase chain reaction (PCR) technique is a method, well known to 
those skilled in the art, which enables both strands of a previously 
denatured DNA sequence to be copied simultaneously using two 
oligonucleotides as primers (see, in particular, the work by H.A. Erlich: 
"PCR Technology: Principles and Applications for DNA amplification" 
published in 1989 by Macmillan Publishers Ltd publications, United 
Kingdom, and that of M. A. INNIS et al. "PCR Protocols" published in 1990 
by Academic Press Inc. San Diego, Calif. 92101, USA). The principle of 
this technique is summarised below. 
A large number of cycles, each of which consists of three steps, produce 
amplification of the DNA strands of interest; these three steps are: 
a) denaturation of the template 
b) hybridisation of the primers with the template 
c) extension of the primers. 
After a few hours of cycles, hundreds of thousands of copies of the 
original template have been produced using a heat-stable DNA polymerase of 
Thermus acuaticus, commonly referred to as Taq polymerase. 
The PCR technique is based on the repetition of three steps. 
a) Denaturation of the template 
The double-stranded DNA is denatured to single-stranded DNA by incubation 
at high temperature (from 92.degree. C. to 96.degree. C.) for 
approximately 2 min. 
b) Hybridisation of the primers 
These primers are a pair of synthetic oligonucleotides which hybridise with 
the ends of the region to be amplified. The two primers hybridise with the 
opposite strands. The primers are added in excess so that formation of the 
primer-template complex is favoured. 
c) Extension of the primers 
The step during which Taq polymerase effects extension of the 
primer-template complex from 5' to 3' is performed at 72.degree. C. 
In the PCR technique, the product of interest appears in the third cycle 
and it is then amplified significantly. As the cycles proceed, the 
amplification product rapidly becomes the major template with which the 
primers hybridise. 
4) Description of the primers used 
Two synthetic oligonucleotides were prepared from the sequence of the 
fragment A (see FIG. 2). 
The first oligonucleotide, referred to as primer 1 and whose sequence is as 
follows (SEQ ID NO:22): 
##STR16## 
possesses two distinct regions: region 1, which carries a cloning site 
AAGCTT corresponding to the recognition site of the endonuclease HindIII, 
and region 2, which is a region intended for hybridisation with the 
non-coding region of the coding strand of the fragment A, located on the 
3' side of the sequence coding for preproendothiapepsin (see FIG. 
2--position 1870-1881). 
The second oligonucleotide, referred to as primer 2 and whose sequence is 
as follows (SEQ ID NO:23): 
##STR17## 
also consists of two distinct regions: region 1, which carries a cloning 
site CATATG corresponding to the recognition site of the endonuclease NdeI 
and in which is included the sequence of the initiation codon ATG, and 
region 2, which carries a nucleotide sequence identical to that coding for 
the first five amino acids of preproendothiapepsin which follow the 
initial methionine. This region is intended for hybridisation with the 
non-coding strand of the fragment A. 
5) Production of the amplified fragment representing the complementary DNA 
of endothiapepsin 
A pool of messenger RNA known to contain the messenger RNA coding for 
endothiapepsin is used as a template; an enzymatic reaction using reverse 
transcriptase is performed on the messenger RNA before amplification. 
a) Demonstration of the presence of the messenger RNA coding for 
endothiapepsin in the total RNA preparation. 
.alpha.) Northern blotting 
The Northern technique is used (Maniatis). It consists essentially in 
separating approximately 10 .mu.g of total RNA by electrophoresis on 1.0% 
agarose gel under denaturing conditions (20 mM MOPS pH 7, 5 mM sodium 
acetate, 1 mM EDTA, 6.6% formaldehyde). The RNA thus separated is 
transferred onto a nitrocellulose sheet (Maniatis op. cit.). Two different 
nitrocellulose filters are thereby prepared, one of which is hybridised 
with radiolabelled probe 1 and the other with radiolabelled probe 2 [see 
Section 1.2) and 1.3) for the preparation of the probes and their 
labelling with .sup.32 P]. 
.beta.) Hybridisation with radiolabelled probe 1 and radiolabelled probe 2 
The hybridisation conditions are the same as those described in Section 
1.5). After hybridisation, each of the filters is washed individually in a 
solution containing 0.5.times.SSC at 42.degree. C. The filters are then 
exposed to a photographic film (Kodak XAR5) overnight. 
Analysis of the films shows that an RNA population responds specifically to 
both probes, thereby indicating that the messenger RNA coding for 
endothiapepsin is present in the preparation. 
b) The reaction using reverse transcriptase 
The reaction of reverse transcriptase with 1 .mu.g of messenger RNA is 
performed in the presence of 10 mM dithiothreitol DTT, RNasin (RNase 
inhibitor, Genofit) 0.0040 U/.mu.l, a mixture of the four 
deoxyribonucleotide triphosphates dNTP at a concentration of 10 mM, buffer 
of composition 50 mM Tris-HCl pH 8.3, 20 mM KCl and 10 mM MgCl.sub.2, 0.7 
unit of reverse transcriptase (Stratagene) and 0.1 ng of primer 1 as well 
as 0.1 ng of primer 2 for a final volume of 10 .mu.l. After incubation for 
half an hour at 46.degree. C., the reaction is stopped by adding 20 mM 
EDTA and the mixture is then incubated for 5 min at 65.degree. C. 
c) The PCR reaction 
The mixture described above is subjected to chromatography on a P10 
polyacrylamide gel column in order to remove the small molecules 
(nucleotides, EDTA, and the like). The solution then obtained is incubated 
for 2 min at 92.degree. C. in order to denature the template composed of a 
strand of complementary DNA and a strand of messenger RNA. 100 ng of 
primer 1, 100 ng of primer 2, 2 mM MgCl.sub.2, 0.2 mM dNTP and 5 .mu.l of 
reaction mixture concentrated 10-fold (final quantity: 67 mM Tris-HCl pH 
8.8, 16.6 mM (NH.sub.4).sub.2 SO.sub.4, 1 mM .beta.-mercaptoethanol, 6.7 
mM EDTA, 0.15% Triton X-100, 200 g/ml of gelatin) are then added to the 
tube. 
The volume of the mixture is then brought to 50 .mu.l by adding water. 
The reaction mixture thereby obtained is incubated for 4 min at 94.degree. 
C. and then taken to a temperature of 41.degree. C., which is maintained 
for 4 min. The temperature of 41.degree. C. corresponds to a value 5 
degrees lower than the temperature of half-denaturation of the 
oligonucleotide, calculated with an empirical formula well known to those 
skilled in the art. 
0.5 .mu.l, equivalent to 2.5 units, of Taq polymerase (Boehringer Mannheim 
ref. 1146-165) is then added. The reaction mixture is then covered with 
paraffin in order to prevent evaporation of the aqueous solution. 
The amplification is carried out during 30 reaction cycles, the steps of 
one cycle being as follows: 
2 min at 92.degree. C..fwdarw.denaturation 
2 min at 41.degree. C..fwdarw.hybridisation 
2 min at 72.degree. C..fwdarw.polymerisation. 
After the 30 cycles, the enzymatic reaction is stopped by adding 20 mM 
EDTA. 
The DNA fragment thus amplified, which possesses the expected size of 
approximately 1,300 bp, is then isolated and purified on 1% agarose gel, 
dialysed on a P10 column and then hydrolysed simultaneously with the 
enzymes NdeI and HindIII according to the usual techniques well known to 
those skilled in the art (Maniatis, op. cit.) in order to form the NdeI 
and HindIII cohesive ends. After hydrolysis, the fragment is purified on a 
P10 column. 
Section 16 
Construction of plasmid p572, a vector for the cloning and expression in E. 
coli of the complementary DNA coding for the endothiapepsin precursor. 
Determination of the sequence of this complementary DNA and expression of 
the latter. 
1) Construction of plasmid p572 
Plasmid p572 was prepared from plasmid p466, a vector for the cloning and 
expression of the complementary DNA of urate oxidase of Aspergillus flavus 
in E. coli, described in Patent Application PCT-FR-90/00,532, which 
comprises a fragment of plasmid pBR327 including the origin of replication 
and the ampicillin resistance gene, a synthetic promoter of E. coli (R. 
RODRIGUEZ and M. CHAMBERLIN "Promoters-Structure and function" (1982) 
Preager), a Shine-Dalgarno sequence followed by a polylinker possessing 
single NdeI and HindIII sites, a transcription terminator (derived from 
phage fd) and the lac i gene. 
a) Construction of plasmid p466 
Plasmid p466, an expression vector in E. coli, was prepared. It comprises a 
fragment of pBR327 including the origin of replication and the ampicillin 
resistance gene; it also comprises a synthetic promoter of E. coli (R. 
RODRIGUEZ and M. CHAMBERLIN "Promoters-Structure and function" (1982) 
Preager), a Shine-Dalgarno sequence followed by a polylinker possessing 
single NdeI and KpnI sites, a transcription terminator (derived from phage 
fd) and the lac i gene. 
This plasmid was constructed from a plasmid for the expression of hGH in E. 
coli (p462) by substitution of a fragment carrying the hGH gene by the 
cDNA of urate oxidase. 
The construction of plasmid p466 will now be described in greater detail in 
the account below, in which reference will be made to FIGS. 6, 7, 8, 9 and 
10. 
1) Construction of plasmid p373,2 
The strategy employed makes use of fragments obtained from pre-existing 
plasmids available to the public and fragments prepared synthetically 
according to techniques now in common use. The cloning techniques employed 
are those described by T. MANIATIS, E. F. FRITSCH and J. SAMBROOK, Cold 
Spring Harbor Laboratory (1982). Oligonucleotide synthesis is carried out 
using a Biosearch 4600 DNA synthesiser. 
Plasmid p163,1 (FIG. 6), described in Patent Application EP-A-0,245,138 and 
deposited with the CNCM under reference I-530 on 17th Feb. 1986, was 
subjected to a digestion with the enzymes PvuI and BamhI. This plasmid 
contains the gene coding for hGH. The PvuI-BamHI fragment--hereinafter 
fragment 1--containing the site of action of the restriction enzyme XhoI, 
shown in FIG. 6, was purified. 
Similarly, plasmid pBR327, well known to those sklilled in the art (see 
SOBERON, X et al., Gene, 9 (1980) 287-305), was subjected to a digestion 
with the enzymes PvuI and BamHI. The PvuI-BamHI fragment--herein-after 
fragment 2--containing the origin of replication was purified. 
The fragment 3, which is a synthetic BamHI(1)-BamHI(2) fragment containing 
the lac i gene and its promoter and whose sequence is as follows (SEQ ID 
NO:24), on which sequence the two ends of the strand are identified by the 
numbers 1 and 2 in order to specify the orientation of the fragment in the 
plasmids described in FIGS. 7 and 8, was then prepared. 
__________________________________________________________________________ 
FRAGMENT 3 (SEQ ID NO: 24) 
__________________________________________________________________________ 
BamHI(1) 
5' GATCC GCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT 
- GAGCTAACTT ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG 
- GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA 
- GGCGGTTTGC GTATTGGGCG CCAGGGTGGT TTTTCTTTTC ACCAGTGAGA 
- CGGGCAACAG CTGATTGCCC TTCACCGCCT GGCCCTGAGA GAGTTGCAGC 
- AAGCGGTCCA CGCTGGTTTG CCCCACCACC CGAAAATCCT GTTTGATGGT 
- GGTTAACGGC GGGATATAAC ATGAGCTGTC TTCGGTATCG TCGTATCCCA 
- CTACCGAGAT ATCCGCACCA ACGCGCAGCC CGGACTCGGT AATGGCGCGC 
- ATTGCGCCCA GCGCCATCTG ATCGTTGGCA ACCAGCATCG CAGTGGGAAC 
- GATGCCCTCA TTCAGCATTT GCATGGTTTG TTGAAAACCG GACATGGCAC 
- TCCAGTCGCC TTCCCGTTCC GCTATCGGCT GAATTTGATT GCGAGTGAGA 
- TATTTATGCC AGCCAGCCAG ACGCAGACGC GCCGAGACAG AACTTAATGG 
- GCCCGCTAAC AGCGCGATTT GCTGGTGACC CAATGCGACC AGATGCTCCA 
- CGCCCAGTCG CGTACCGTCT TCATGGGAGA AAATAATACT GTTGATGGGT 
- GTCTGGTCAG AGACATCAAG AAATAACGCC GGAACATTAG TGCAGGCAGC 
- TTCCACAGCA ATGGCATCCT GGTCATCCAG CGGATAGTTA ATGATCAGCC 
- CACTGACGCG TTGCGCGAGA AGATTGTGCA CCGCCGCTTT ACAGGCTTCG 
- ACGCCGCTTC GTTCTACCAT CGACACCACC ACGCTGGCAC CCAGTTGATC 
- GGCGCGAGAT TTAATCGCCG CGACAATTTG CGACGGCGCG TGCAGGGCCA 
- GACTGGAGGT GGCAACGCCA ATCAGCAACG ACTGTTTGCC CGCCAGTTGT 
- TGTGCCACGC GGTTGGGAAT GTAATTCAGC TCCGCCATCG CCGCTTCCAC 
- TTTTTCCCGC GTTTTCGCAG AAACGTGGCT GGCCTGGTTC ACCACGCGGG 
- AAACGGTCTG ATAACAGACA CCGGCATACT CTGCGACATC GTATAACGTT 
- ACTGGTTTCA CATTCACCAC CCTGAATTGA CTCTCTTCCG GGCGCTATCA 
- TGCCATACCG CGAAAGGTTT TGCGCCATTC GATGGTGTCC G 3' 
- BamHI(2) 
__________________________________________________________________________ 
The fragments 1, 2 and 3 were then ligated so as to obtain plasmid p160, 
shown in FIG. 7. 
This plasmid was subjected to a partial digestion with the restriction 
enzymes HincII and PstI. The large HincII-PstI fragment, containing the 
origin of replication and shown in FIG. 7, was then ligated to the 
fragment 4 (SEQ ID NO:25), shown below, which is a synthetic DNA fragment 
carrying a sequence coding for the first 44 amino acids of a natural 
precursor of hGH and, upstream of this sequence, regulation signals (SEQ 
ID NO:26). 
__________________________________________________________________________ 
FRAGMENT 4 (SEQ ID NO: 25) 
__________________________________________________________________________ 
ClaI 
.tangle-soliddn. 
5' TCGAGCTGACTGACCTGTTGCTTATATTACATCGA 
----------------------------------- 
AGCTCGACTGACTGGACAACGAATATAATGTAGCT 
.tangle-solidup. 
NdeI 
.tangle-soliddn. 
TAGCGTATAATGTGTGGAATTGTGAGCGATAACAATTTCACACAGTTTAACTTTAAGAAGGAGATATACAT 
ATCGATATTACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCAAATTGAAATTCTTCCTCTATATGTA 
- ATG GCT ACC GGA TCC CGG ACT AGT CTG CTC CTG GCT TTT GGC CTG CTC TGC 
CTG 
TAC CGA TGG CCT AGG GCC TGA TCA GAC GAG GAC CGA AAA CCG GAC GAC ACG GAC 
.tangle-solidup. 
M A T G S R T S L L L A F G L L C L 
-26 
- XbaI 
.tangle-soliddn. 
CCC TGG CTT CAA GAG GGC AGT GCC TTC CCA ACC ATT CCC TTA TCT AGA CTT TTT 
----------------------------------------------------- 
GGG ACC GAA GTT CTC CCG TCA CGG AAG GGT TGG TAA GGG AAT AGA TCT GAA AAA 
.tangle-sol 
idup. 
P W L Q E G S A F P T I P L S R L F 
-1 1 
- GAC AAC GCT ATG CTC CGC GCC CAT CGT CTG CAC CAG CTG GCC TTT GAC ACC 
TAC 
CTG TTG CGA TAC GAG GCG CGG GTA GCA GAC GTG GTC GAC CGG AAA CTG TGG ATC 
D N A M L R A H R L H Q L A F L T Y 
PstI 
CAG GAG TTT GAA GAA GCC TAT ATC CCA AAG GAA CAG AAG TAT TCA TTC CTG CA 
GTC CTC AAA CTT CTT CGG ATA TAG GGT TTC CTT GTC TTC ATA AGT AAG G 
Q E F E E A Y I P K E Q K Y S F 
44 
__________________________________________________________________________ 
In this fragment, the amino acids are designated by letters according to 
the following code: 
______________________________________ 
A = Alanine M = Methionine 
C = Cysteine N = Asparagine 
D = Aspartic acid P = Proline 
E = Glutamic acid Q = Glutamine 
F = Phenylalanine R = Arginine 
G = Glycine S = Serine 
H = Histidine T = Threonine 
I = Isoleucine V = Valine 
K = Lysine W = Tryptophan 
L = Leucine Y = Tyrosine 
______________________________________ 
In this fragment, the sequences -35 (TTGCTT) and -10 (TATAAT) of the 
promoter sequence and the Shine-Dalgarno sequence which is well known to 
those skilled in the art are successively underlined. 
Plasmid p380,1 was thereby obtained. 
Plasmid p380,1 was then subjected to a digestion with the restriction 
enzymes ClaI and NdeI so as to remove from it the small ClaI-NdeI fragment 
of the above fragment 4 and to substitute for it the ClaI-NdeI fragment 
below (SEQ ID NO:27): 
ClaI 
- 5' CGATAGCGTATAATGTGTGGAATTGTGAGCGGATAACA 
- TATCGCATATTACACACCTTAACACTCGCCTATTGT 
- NdeI 
- ATTTCACACAGTTTTTCGCGAAGAAGGAGATATACA 
- TAAAGTGTGTCAAAAAGCGCTTCTTCCTCTATATGTAT 5' 
2) Construction of plasmid p466 
Plasmid p373,2 was subjected to a double digestion with the enzymes BglII 
and HindIII. The large fragment derived from this digestion was purified 
and ligated with a synthetic DNA fragment whose sequence (SEQ ID NO:28), 
given below, is intended for re-forming the end of the hGH gene followed 
at the 3' end by KpnI and SnaBI cloning sites. 
B 
g 
l 
I 
I 
GATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGAT 
----+----------+----------+---- 
-------+---------+ 
AAGTTCGTCTGGATGTCGTTCAAGCTGTGTTTGAGTGTGTTGCTA 
GACGCACTACTCAAGAACTACG 
GGCTGCTCTACTGCTTCAGGAAGGACATGGA 
CAAGGTC 
----------+----------+----------+----------+----------+--------- 
+ 
CTGCGTGATGAGTTCTTGATGCCCGACGAGATGACGAAGTCCTTCCTGTACCTGTTCCAG 
F 
s 
p 
I 
GAGACATTCCTGCGCATCGTGC 
AGTGCCGCTCTGTGGAGGGCAGCTGTGGCTT 
CTAGTAA 
----------+----------+----------+---------+-----------+--------- 
+ 
CTCTGTAAGGACGCGTAGCACGTCACGGCGAGACACCTCCCGTCGACACCGAAGATCATT 
H 
i 
S n 
K n d 
p a I 
n B I 
I I I 
GGTACCCTGCCCTACGTACCA 
----------+----------+----- 
CCATGGGACGGGATGCATGGTTCGA 
This fragment comprises the BglII and HindIII cohesive ends. The new 
plasmid thereby formed, p462 (see FIG. 9), thus comprises a KpnI site and 
an NdeI site which will be used for cloning the fragment carrying the cDNA 
of urate oxidase into the expression vector. 
The hybrid plasmid derived from pTZ19R carrying the approximately 1.2-kb 
cDNA (clone 9C) of urate oxidase comprises a single KpnI site. This site 
is localised a few base pairs downstream of the cloning site of the cDNA. 
Moreover, the cDNA of urate oxidase contains an AccI site situated in 
proximity to the 5' end. 
The AccI-KpnI fragment comprising the larger part of this cDNA was hence 
isolated and purified. Moreover, two complementary oligonucleotides, whose 
sequence (SEQ ID NOS:29 and 30), given below: 
5'-TATGTCTGCGGTAAAAGCAGCGCGCTACGGCAAGGACAATGTTCGCGT 
- ACAGACGCCATTTTCGTCGCGCGATGCCGTTCCTGTTACAAGCGCAGA-5' 
is intended for re-forming the 5' end of the cDNA, were synthesised. This 
synthetic fragment thereby obtained possesses an NdeI end and an AccII 
end. The fragment and the synthetic sequence were ligated with the 
expression vector cut with KpnI and with NdeI. This three-fragment 
ligation enables the vector, designated p466, for the expression of urate 
oxidase for E. coli to be obtained (see FIG. 10). This plasmid was 
subjected to a series of enzymatic hydrolyses with restriction enzymes, 
which enabled the presence of the expected restriction sites to be 
verified, especially those carried by the gene coding for urate oxidase. 
Plasmid p466 hence contains by construction a gene coding for urate 
oxidase, of the sequence below (SEQ ID NO:31): 
ATGTCTGCGG TAAAAGCAGC GCGCTACGGC AAGGACAATG TTCGCGTCTA 
- CAAGGTTCAC AAGGACGAGA AGACCGGTGT CCAGACGGTG TACGAGATGA 
- CCGTCTGTGT GCTTCTGGAG GGTGAGATTG AGACCTCTTA CACCAAGGCC 
- GACAACAGCG TCATTGTCGC AACCGACTCC ATTAAGAACA CCATTTACAT 
- CACCGCCAAG CAGAACCCCG TTACTCCTCC CGAGCTGTTC GGCTCCATCC 
- TGGGCACACA CTTCATTGAG AAGTACAACC ACATCCATGC CGCTCACGTC 
- AACATTGTCT GCCACCGCTG GACCCGGATG GACATTGACG GCAAGCCACA 
- CCCTCACTCC TTCATCCGCG ACAGCGAGGA GAAGCGGAAT GTGCAGGTGG 
- ACGTGGTCGA GGGCAAGGGC ATCGATATCA AGTCGTCTCT GTCCGGCCTG 
- ACCGTGCTGA AGAGCACCAA CTCGCAGTTC TGGGGCTTCC TGCGTGACGA 
- GTACACCACA CTTAAGGAGA CCTGGGACCG TATCCTGAGC ACCGACGTCG 
- ATGCCACTTG GCAGTGGAAG AATTTCAGTG GACTCCAGGA GGTCCGCTCG 
- CACGTGCCTA AGTTCGATGC TACCTGGGCC ACTGCTCGCG AGGTCACTCT 
- GAAGACTTTT GCTGAAGATA ACAGTGCCAG CGTGCAGGCC ACTATGTACA 
- AGATGGCAGA GCAAATCCTG GCGCGCCAGC AGCTGATCGA GACTGTCGAG 
- TACTCGTTGC CTAACAAGCA CTATTTCGAA ATCGACCTGA GCTGGCACAA 
- GGGCCTCCAA AACACCGGCA AGAACGCCGA GGTCTTCGCT CCTCAGTCGG 
- ACCCCAACGG TCTGATCAAG TGTACCGTCG GCCGGTCCTC TCTGAAGTCT 
- AAATTG. 
(The nucleotides different from the nucleotides of the cDNA isolated from 
A. flavus are underlined in the above sequence. These differences were 
introduced into the synthetic AccI-KpnI fragment so as to have, downstream 
of the ATG, a nucleotide sequence conforming more closely to those usually 
encountered in a prokaryotic gene). 
Plasmid p466 was hydrolysed with the enzymes NdeI and HindIII, and the 
fragment carrying the lac i gene, the origin of replication and the gene 
coding for ampicillin resistance was purified according to the techniques 
known to those skilled in the art (Maniatis, op. cit.). 
This fragment was ligated to the amplified complementary DNA fragment 
previously hydrolysed with the endonucleases NdeI and HindIII. The product 
of this ligation was used for transformation in E. coli strain K12 RRI. 
(Gibco BRL-ref.: 520-8261 SA). A transformant, referred to as clone 512, 
which contains the plasmid referred to as p572, was adopted. 
2) Determination of the sequence of the complementary DNA 
Plasmid p572 was hydrolysed, on the one hand with the endonucleases ClaI 
and KpnI, and on the other hand with the enzymes KpnI and HindIII. (The 
endonuclease KpnI cuts the coding sequence of the fragment A). The 
ClaI-KpnI fragment carrying the 5' end of the DNA coding for the protein 
and the KpnI-HindIII fragment carrying the 3' end of the DNA coding for 
the protein were cloned into phage M13mp19 (Pharmacia) and sequenced by 
the cyclone technique ("Cyclone I Biosystem" of IBI). 
The nucleotide sequence of the complementary DNA thereby obtained is shown 
in FIG. 11. It is observed that the coding sequence of the complementary 
DNA is exactly identical to that of the genomic DNA, the only difference 
being that the latter is interrupted by three introns, which have been 
correctly localised (see Section 1.7)). 
3) Expression of the complementary DNA of preproendothiapepsin 
E. coli strain K12 RRI (Gibco BRL Ref.: 520-8261A) was transformed for 
ampicillin resistance with plasmid p572 carrying the complementary DNA of 
preproendothiapepsin, whose sequence was determined in 2), and with a 
negative control plasmid pBR322. 
Ampicillin-resistant colonies were obtained in both cases. 
1 colony of each type was cultured in LB liquid medium (of composition 
specified in Table 4, but without agar) supplemented with 100 .mu.g/ml of 
ampicillin. After stirring overnight at 37.degree. C., both cultures were 
diluted 100-fold in liquid LB medium supplemented with 100 .mu.g/ml of 
ampicillin. After 1 h of culture, IPTG 
(isopropyl-.beta.-D-thiogalactoside) was added to a concentration of 1 mM 
for 3 h. 
Immunodetection of preproendothiapepsin by Western blotting 
a) Procedure 
An aliquot fraction corresponding to 0.2 ml with an OD=1 is withdrawn from 
the culture medium obtained after 3 h of induction with IPTG. The aliquot 
is centrifuged and the supernatant is removed. The pellet is then 
subjected to Western blotting, a technique well known to those skilled in 
the art, which comprises the following steps: 
Solubilisation of the pellet by boiling for 10 min in a buffer, designated 
loading buffer, consisting of 0.125M Tris-HCl pH 6.8, 4% SDS, 0.002% 
bromophenol blue, 20% glycerol and 10% .beta.-mercaptoethanol (according 
to the protocol described by LAEMMLI (U. K. LAEMMLI, Nature, 227 (1970), 
680-685)), 
Electrophoretic separation of the different proteins contained in the 
solubilisate according to the protocol described by LAEMMLI (U. K. 
LAEMMLI, Nature, 227, (1970), 680-685), 
Transfer of the said proteins contained in the gel onto a nitrocellulose 
filter (according to the technique of H. TOWBIN et al. Proc. Natl. Acad. 
Sci. USA 76 (1979) 4350-4354), 
Immunodetection, carried out according to the technique of BURNETTE (W. W. 
BURNETTE Ana. Biochem. 112 (1981) 195-203); this involves successively: 
Rinsing the nitrocellulose filter for 10 min with a buffer of composition 
10 mM Tris-HCl, 170 mM NaCl, 1 mM KCl. 
Bringing the nitrocellulose filter into contact for 30 min at 37.degree. C. 
with buffer A supplemented with bovine serum albumin in the proportion of 
3 g per 100 ml. 
Bringing the nitrocellulose filter into contact for 1 h at 37.degree. C. 
with the polyclonal antibodies of the Rennetest France Biochem kit, 
according to the method of identification of endothiapepsin described in 
the Journal Officiel de la Republique Fran.cedilla.aise (Official Journal 
of the French Republic) of Mar. 20, 1981. 
Rinsing the nitrocellulose filter with buffer A supplemented with 3 g/100 
ml of bovine serum albumin. 
Bringing the nitrocellulose filter into contact for 1 h at 37.degree. C. 
with a solution of iodine-125-labelled protein G having an activity of 0.1 
microcurie/ml. 
Rinsing the filter with buffer A. 
Drying the filter between two absorbent sheets. 
Bringing the filter into contact with a radiographic film. 
Developing the film. 
b) Results 
It is found that the strain transformed with plasmid p572 overproduces a 
protein of apparent molecular weight approximately 43 kDa corresponding to 
the expected molecular mass of preproendothiapepsin, which is recognised 
by antibodies directed towards endothiapepsin and which is absent in the 
control strain. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 33 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 330 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: endothiapepsin 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Ser Thr Gly Ser Ala Thr Thr Thr - # Pro Ile Asp Ser Leu Asp 
Asp Ala 
1 - # 5 - # 10 - # 
15 
- - Tyr Ile Thr Pro Val Gln Ile Gly - # Thr Pro Ala Gln Thr Leu 
Asn Leu 
20 - # 25 - # 30 
- - Asp Phe Asp Thr Gly Ser Ser Asp - # Leu Trp Val Phe Ser Ser 
Glu Thr 
35 - # 40 - # 45 
- - Thr Ala Ser Glu Val Asp Gly Gln - # Thr Ile Tyr Thr Pro Ser 
Lys Ser 
50 - # 55 - # 60 
- - Thr Thr Ala Lys Leu Leu Ser Gly - # Ala Thr Trp Ser Ile Ser 
Tyr Gly 
65 - # 70 - # 75 - # 
80 
- - Asp Gly Ser Ser Ser Ser Gly Asp - # Val Tyr Thr Asp Thr Val 
Ser Val 
- # 85 - # 90 - # 
95 
- - Gly Gly Leu Thr Val Thr Gly Gln - # Ala Val Glu Ser Ala Lys 
Lys Val 
100 - # 105 - # 110 
- - Ser Ser Ser Phe Thr Glu Asp Ser - # Thr Ile Asp Gly Leu Leu 
Gly Leu 
115 - # 120 - # 125 
- - Ala Phe Ser Thr Leu Asn Thr Val - # Ser Pro Thr Gln Gln Lys 
Thr Phe 
130 - # 135 - # 140 
- - Phe Asp Asn Ala Lys Ala Ser Leu - # Asp Ser Pro Val Phe Thr 
Ala Asp 
145 - # 150 - # 155 - # 
160 
- - Leu Gly Tyr His Ala Pro Gly Thr - # Tyr Asn Phe Gly Phe Ile 
Asp Thr 
- # 165 - # 170 - # 
175 
- - Thr Ala Tyr Thr Gly Ser Ile Thr - # Tyr Thr Ala Val Ser Thr 
Lys Gln 
180 - # 185 - # 190 
- - Gly Phe Trp Glu Trp Thr Ser Thr - # Gly Tyr Ala Val Gly Ser 
Gly Thr 
195 - # 200 - # 205 
- - Phe Lys Ser Thr Ser Ile Asp Gly - # Ile Ala Asp Thr Gly Thr 
Thr Leu 
210 - # 215 - # 220 
- - Leu Tyr Leu Pro Ala Thr Val Val - # Ser Ala Tyr Trp Ala Gln 
Val Ser 
225 - # 230 - # 235 - # 
240 
- - Gly Ala Lys Ser Ser Ser Ser Val - # Gly Gly Tyr Val Phe Pro 
Cys Ser 
- # 245 - # 250 - # 
255 
- - Ala Thr Leu Pro Ser Phe Thr Phe - # Gly Val Gly Ser Ala Arg 
Ile Val 
260 - # 265 - # 270 
- - Ile Pro Gly Asp Tyr Ile Asp Phe - # Gly Pro Ile Ser Thr Gly 
Ser Ser 
275 - # 280 - # 285 
- - Ser Cys Phe Gly Gly Ile Gln Ser - # Ser Ala Gly Ile Gly Ile 
Asn Ile 
290 - # 295 - # 300 
- - Phe Gly Asp Val Ala Leu Lys Ala - # Ala Phe Val Val Phe Asn 
Gly Ala 
305 - # 310 - # 315 - # 
320 
- - Thr Thr Pro Thr Leu Gly Phe Ala - # Ser Lys 
- # 325 - # 330 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 419 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: preproendothiap - #epsin 
- - (ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 90..419 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Ser Ser Pro Leu Lys Asn Ala - # Leu Val Thr Ala Met Leu 
Ala Gly 
- # -85 - # -80 - # 
-75 
- - Gly Ala Leu Ser Ser Pro Thr Lys - # Gln His Val Gly Ile Pro 
Val Asn 
-70 - # -65 - # -60 
- - Ala Ser Pro Glu Val Gly Pro Gly - # Lys Tyr Ser Phe Lys Gln 
Val Arg 
-55 - # -50 - # -45 
- - Asn Pro Asn Tyr Lys Phe Asn Gly - # Pro Leu Ser Val Lys Lys 
Thr Tyr 
-40 - # -35 - # -30 
- - Leu Lys Tyr Gly Val Pro Ile Pro - # Ala Trp Leu Glu Asp Ala 
Val Gln 
-25 - # -20 - # -15 - # 
-10 
- - Asn Ser Thr Ser Gly Leu Ala Glu - # Arg Ser Thr Gly Ser Ala 
Thr Thr 
- # -5 - # 1 - #5 
- - Thr Pro Ile Asp Ser Leu Asp Asp - # Ala Tyr Ile Thr Pro Val 
Gln Ile 
10 - # 15 - # 20 
- - Gly Thr Pro Ala Gln Thr Leu Asn - # Leu Asp Phe Asp Thr Gly 
Ser Ser 
25 - # 30 - # 35 
- - Asp Leu Trp Val Phe Ser Ser Glu - # Thr Thr Ala Ser Glu Val 
Asp Gly 
40 - # 45 - # 50 - # 
55 
- - Gln Thr Ile Tyr Thr Pro Ser Lys - # Ser Thr Thr Ala Lys Leu 
Leu Ser 
- # 60 - # 65 - # 
70 
- - Gly Ala Thr Trp Ser Ile Ser Tyr - # Gly Asp Gly Ser Ser Ser 
Ser Gly 
75 - # 80 - # 85 
- - Asp Val Tyr Thr Asp Thr Val Ser - # Val Gly Gly Leu Thr Val 
Thr Gly 
90 - # 95 - # 100 
- - Gln Ala Val Glu Ser Ala Lys Lys - # Val Ser Ser Ser Phe Thr 
Glu Asp 
105 - # 110 - # 115 
- - Ser Thr Ile Asp Gly Leu Leu Gly - # Leu Ala Phe Ser Thr Leu 
Asn Thr 
120 - # 125 - # 130 - # 
135 
- - Val Ser Pro Thr Gln Gln Lys Thr - # Phe Phe Asp Asn Ala Lys 
Ala Ser 
- # 140 - # 145 - # 
150 
- - Leu Asp Ser Pro Val Phe Thr Ala - # Asp Leu Gly Tyr His Ala 
Pro Gly 
155 - # 160 - # 165 
- - Thr Tyr Asn Phe Gly Phe Ile Asp - # Thr Thr Ala Tyr Thr Gly 
Ser Ile 
170 - # 175 - # 180 
- - Thr Tyr Thr Ala Val Ser Thr Lys - # Gln Gly Phe Trp Glu Trp 
Thr Ser 
185 - # 190 - # 195 
- - Thr Gly Tyr Ala Val Gly Ser Gly - # Thr Phe Lys Ser Thr Ser 
Ile Asp 
200 - # 205 - # 210 - # 
215 
- - Gly Ile Ala Asp Thr Gly Thr Thr - # Leu Leu Tyr Leu Pro Ala 
Thr Val 
- # 220 - # 225 - # 
230 
- - Val Ser Ala Tyr Trp Ala Gln Val - # Ser Gly Ala Lys Ser Ser 
Ser Ser 
235 - # 240 - # 245 
- - Val Gly Gly Tyr Val Phe Pro Cys - # Ser Ala Thr Leu Pro Ser 
Phe Thr 
250 - # 255 - # 260 
- - Phe Gly Val Gly Ser Ala Arg Ile - # Val Ile Pro Gly Asp Tyr 
Ile Asp 
265 - # 270 - # 275 
- - Phe Gly Pro Ile Ser Thr Gly Ser - # Ser Ser Cys Phe Gly Gly 
Ile Gln 
280 - # 285 - # 290 - # 
295 
- - Ser Ser Ala Gly Ile Gly Ile Asn - # Ile Phe Gly Asp Val Ala 
Leu Lys 
- # 300 - # 305 - # 
310 
- - Ala Ala Phe Val Val Phe Asn Gly - # Ala Thr Thr Pro Thr Leu 
Gly Phe 
315 - # 320 - # 325 
- - Ala Ser Lys 
330 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1252 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: DNA coding - #for preproendothiapepsin having 
SEQ ID - #NO:2 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - ATGTCTTCCC CTCTCAAGAA CGCCTTGGTG ACCGCCATGT TGGCTGGTGG TG - 
#CTCTCAGC 60 
- - TCGCCTACAA AGCAACACGT TGGAATTCCC GTCAACGCCT CTCCTGAAGT TG - 
#GCCCCGGA 120 
- - AAGTACTCGT TCAAGCAAGT CCGGAACCCC AACTACAAGT TCAACGGGCC TC - 
#TGTCGGTC 180 
- - AAGAAGACGT ACCTCAAGTA CGGCGTGCCG ATCCCAGCCT GGCTGGAGGA TG - 
#CTGTCCAG 240 
- - AACTCTACCT CGGGCCTGGC TGAGCGCTCG ACCGGTTCTG CGACCACAAC TC - 
#CCATCGAC 300 
- - AGCCTCGATG ATGCTTACAT CACTCCGGTT CAGATCGGCA CCCCTGCGCA GA - 
#CTCTGAAC 360 
- - CTGGACTTTG ACACTGGATC TTCGGATCTG TGGGTCTTCA GCAGCGAGAC TA - 
#CAGCCAGC 420 
- - GAGGTCGATG GGCAGACCAT CTACACCCCC AGCAAGAGCA CCACCGCCAA GC - 
#TGCTGTCG 480 
- - GCGCTACCTG GTCCATCTCC TACGGAGACG GTAGCTCTTC CAGCGGCGAT GT - 
#CTACACTG 540 
- - ACACCGTCTC GGTTGGAGGC CTTACCGTGA CGGGCCAGGC TGTCGAGTCG GC - 
#CAAGAAGG 600 
- - TTTCTTCCAG CTTCACCGAG GACTCGACCA TTGACGGTCT CCTGGGCCTG GC - 
#CTTCAGCA 660 
- - CCCTGAACAC TGTGTCGCCT ACCCAGCAAA AGACTTTCTT CGACAATGCG AA - 
#GGCGTCCT 720 
- - TGGACTCGCC TGTGTTCACG GCTGATCTTG GCTACCATGC CCCTGGTACC TA - 
#CAACTTCG 780 
- - GCTTCATCGA TACCACTGCC TACACGGGCT CCATCACCTA CACCGCTGTC TC - 
#GACCAAGC 840 
- - AAGGGTTCTG GGAGTGGACT TCGACCGGCT ACGCCGTCGG CTCCGGCACC TT - 
#CAAGTCGA 900 
- - CTTCCATCGA CGGCATCGCT GACACTGGCA CGACCCTCCT GTACCTCCCT GC - 
#CACCGTCG 960 
- - TGTCGGCCTA CTGGGCCCAG GTCTCGGGCG CCAAGTCCAG CTCTTCCGTC GG - 
#CGGCTACG 1020 
- - TCTTCCCCTG CAGCGCGACC CTGCCTTCCT TCACCTTCGG CGTTGGCTCA GC - 
#TCGCATTG 1080 
- - TGATTCCTGG CGACTACATT GATTTCGGCC CCATCTCCAC TGGAAGCTCG TC - 
#TTGCTTTG 1140 
- - GCGGCATCCA GTCCAGCGCT GGTATCGGCA TCAACATCTT CGGTGATGTC GC - 
#TCTGAAGG 1200 
- - CTTTGTCGTC TTCAACGGGG CTACAACTCC CACTCTTGGC TTTGCTTCCA AG - # 
1252 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1533 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: DNA sequence - # coding for endothiapepsin 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - ATGTCTTCCC CTCTCAAGAA CGCCTTGGTG ACCGCCATGT TGGCTGGTGG TG - 
#CTCTCAGC 60 
- - TCGCCTACAA AGCAACACGT TGGAATTCCC GTCAACGCCT CTCCTGAAGT TG - 
#GCCCCGGA 120 
- - AAGTACTCGT TCAAGCAAGG TGAGTAGAGC TGCTTCTGTG TGTTGCAACA GA - 
#AGACCAAC 180 
- - GCAAAAAGAA GAGGTCAAGG CAAGACGGAT ATTTTACTGA CAATTATACT TT - 
#TGAAGTCC 240 
- - GGAACCCCAA CTACAAGTTC AACGGGCCTC TGTCGGTCAA GAAGACGTAC CT - 
#CAAGTACG 300 
- - GCGTGCCGAT CCCAGCCTGG CTGGAGGATG CTGTCCAGAA CTCTACCTCG GG - 
#CCTGGCTG 360 
- - AGCGCTCGAC CGGTTCTGCG ACCACAACTC CCATCGACAG CCTCGATGAT GC - 
#TTACATCA 420 
- - CTCCGGTTCA GATCGGCACC CCTGCGCAGA CTCTGAACCT GGACTTTGAC AC - 
#TGGATCTT 480 
- - CGGATCTGTG GGTCTTCAGC AGCGAGACTA CAGCCAGCGA GGTTGGTCAA CC - 
#CTCGCCCG 540 
- - CATTTTATTG CATACATTTT TAGTTTTTTT GGTAATCAGA ATACTAACAT TG - 
#GGAATTTC 600 
- - CCAACTGTAG GTCGATGGGC AGACCATCTA CACCCCCAGC AAGAGCACCA CC - 
#GCCAAGCT 660 
- - GCTGTCGGGC GCTACCTGGT CCATCTCCTA CGGAGACGGT AGCTCTTCCA GC - 
#GGCGATGT 720 
- - CTACACTGAC ACCGTCTCGG TTGGAGGCCT TACCGTGACG GGCCAGGCTG TC - 
#GAGTCGGC 780 
- - CAAGAAGGTT TCTTCCAGCT TCACCGAGGA CTCGACCATT GACGGTCTCC TG - 
#GGCCTGGC 840 
- - CTTCAGCACC CTGAACACTG TGTCGCCTAC CCAGCAAAAG ACTTTCTTCG AC - 
#AATGCGAA 900 
- - GGCGTCCTTG GACTCGCCTG TGTTCACGGC TGATCTTGGC TACCATGCCC GT - 
#GAGTGACC 960 
- - CCTCTTGATA CATATACTTT TTGATGAATC TTGTTGGAGA AGCATTCCCC AC - 
#TAATATGG 1020 
- - AAATTGTTTG TATCTACAGC TGGTACCTAC AACTTCGGCT TCATCGATAC CA - 
#CTGCCTAC 1080 
- - ACGGGCTCCA TCACCTACAC CGCTGTCTCG ACCAAGCAAG GGTTCTGGGA GT - 
#GGACTTCG 1140 
- - ACCGGCTACG CCGTCGGCTC CGGCACCTTC AAGTCGACTT CCATCGACGG CA - 
#TCGCTGAC 1200 
- - ACTGGCACGA CCCTCCTGTA CCTCCCTGCC ACCGTCGTGT CGGCCTACTG GG - 
#CCCAGGTC 1260 
- - TCGGGCGCCA AGTCCAGCTC TTCCGTCGGC GGCTACGTCT TCCCCTGCAG CG - 
#CGACCCTG 1320 
- - CCTTCCTTCA CCTTCGGCGT TGGCTCAGCT CGCATTGTGA TTCCTGGCGA CT - 
#ACATTGAT 1380 
- - TTCGGCCCCA TCTCCACTGG AAGCTCGTCT TGCTTTGGCG GCATCCAGTC CA - 
#GCGCTGGT 1440 
- - ATCGGCATCA ACATCTTCGG TGATGTCGCT CTGAAGGCCG CCTTTGTCGT CT - 
#TCAACGGG 1500 
- - GCTACAACTC CCACTCTTGG CTTTGCTTCC AAG - # - 
# 1533 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 328 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (ix) FEATURE: 
(A) NAME/KEY: TATA.sub.-- - #signal 
(B) LOCATION: 1..328 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - AAGCTTATCC GCCGCCGGCG GGGGAATTCT ATTGAACTTG TTCGAATCAT TG - 
#GTCCGTGG 60 
- - TCTTTTCGTC CATGCGGGCT CCGCTGGCGG ATGAATGACC TTCTGGCTTC TA - 
#GCCTGGCG 120 
- - AAGCGATGTT ACTCTGTTGT CTATACTATA CGATATGGTC AAGAGAGCAC AT - 
#GTGCCGCC 180 
- - AGATGAAGAC ATGTATATAA AAGGAGTGGC CTCGACGGTT GCTCAACCAT CT - 
#TCTGTCTG 240 
- - TCCCAACGCC ATCGACTCTT CAACTTCTCC TTCGTGTTCC ACCACCATCA CC - 
#TTGCTCCA 300 
- - GACTTAGGAC TTTCAGCAAC CTTCAAAG - # - # 
328 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 608 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: segment X - #of fragment C having an activator 
region 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - GCATGCTTGG CTCTTTAACG TCCTGCCCAT TCAGGGCCTT CAGCCGGCAC TG - 
#GTCCTTCA 60 
- - TCAAGGGGGA CCTCATGACC ATGAACTAAT CTGTGATATC TGATATATTC TA - 
#GAAGGCTT 120 
- - GGCTCCTCAA AGTTTCCAGC TAATGAATCA GCGGCCCGCC GCCCTTAAAC CG - 
#CATCAGGC 180 
- - AAGTCGTTTG GTGTTGCCAG GCGATGGCGA CAGGAGAGTG GTGTTGATGG GA - 
#CAAGGGGA 240 
- - GGGAGGCTTA GCCGACTTCA TCCATAGCAC CCACCTGCTT GGCGCCGATA AG - 
#TCTGACGA 300 
- - TCCGCTTGAG CTGCAAAACG GCTCCTTGAC CTTTGTTTGG TCGACCGAGG GA - 
#AATAGTCT 360 
- - CTTTTTGCGT GATCGTGCGC GCTTCGTATA GCAATAGCAG CCAGCACCAG CA - 
#GGACGGGC 420 
- - CGTTGTCACG GTCACATCGT TCGCAACATG CCGAGCGTAG GGATGAACGA AT - 
#GACTCGAG 480 
- - CCTTGCCTGA CAGTCTGGCA ATCAATCTAT GGTCACGCAC GATCACAAGC CA - 
#ATCGCTGT 540 
- - GACTGCGTTA CTAGCCCAAT AATCCCTTGT TCGATCAGAG TGTTCTACAG AC - 
#TTCAAGTG 600 
- - AGGTTCAC - # - # 
- # 608 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: peptide 1 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - Val Asp Gly Gln Thr 
1 - # 5 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: peptide 2 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - Gly Phe Trp Glu Trp Thr 
1 - # 5 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # / probe 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - GTNGAYGGNC ARACN - # - # 
- # 15 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotid - #/ probe 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - GGNTTYTGGG ARTGGACN - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - ATGTCTTCCC CTCTCAAGAA CGCCTTGGTG ACCGCCATGT TGGCTGGTGG TG - 
#CTCTCAGC 60 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: peptide sign - #al 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - Met Ser Ser Pro Leu Lys Asn Ala - # Leu Val Thr Ala Met Leu 
Ala Gly 
1 - # 5 - # 10 - # 
15 
- - Gly Ala Leu Ser 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 207 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: pro nucleoti - #de sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - TCGCCTACAA AGCAACACGT TGGAATTCCC GTCAACGCCT CTCCTGAAGT TG - 
#GCCCCGGA 60 
- - AAGTACTCGT TCAAGCAAGT CCGGAACCCC AACTACAAGT TCAACGGGCC TC - 
#TGTCGGTC 120 
- - AAGAAGACGT ACCTCAAGTA CGGCGTGCCG ATCCCAGCCT GGCTGGAGGA TG - 
#CTGTCCAG 180 
- - AACTCTACCT CGGGCCTGGC TGAGCGC - # - # 
207 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 69 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: pro peptide - #sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - Ser Pro Thr Lys Gln His Val Gly - # Ile Pro Val Asn Ala Ser 
Pro Glu 
1 - # 5 - # 10 - # 
15 
- - Val Gly Pro Gly Lys Tyr Ser Phe - # Lys Gln Val Arg Asn Pro 
Asn Tyr 
20 - # 25 - # 30 
- - Lys Phe Asn Gly Pro Leu Ser Val - # Lys Lys Thr Tyr Leu Lys 
Tyr Gly 
35 - # 40 - # 45 
- - Val Pro Ile Pro Ala Trp Leu Glu - # Asp Ala Val Gln Asn Ser 
Thr Ser 
50 - # 55 - # 60 
- - Gly Leu Ala Glu Arg 
65 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 89 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: prepro pepti - #de sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - Met Ser Ser Pro Leu Lys Asn Ala - # Leu Val Thr Ala Met Leu 
Ala Gly 
1 - # 5 - # 10 - # 
15 
- - Gly Ala Leu Ser Ser Pro Thr Lys - # Gln His Val Gly Ile Pro 
Val Asn 
20 - # 25 - # 30 
- - Ala Ser Pro Glu Val Gly Pro Gly - # Lys Tyr Ser Phe Lys Gln 
Val Arg 
35 - # 40 - # 45 
- - Asn Pro Asn Tyr Lys Phe Asn Gly - # Pro Leu Ser Val Lys Lys 
Thr Tyr 
50 - # 55 - # 60 
- - Leu Lys Tyr Gly Val Pro Ile Pro - # Ala Trp Leu Glu Asp Ala 
Val Gln 
65 - # 70 - # 75 - # 
80 
- - Asn Ser Thr Ser Gly Leu Ala Glu - # Arg 
- # 85 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Start of - #natural seq. coding for precursor of 
endothiapeps - #in 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - GATGTCTTCC CCTCTCAAGA ACGCCTTGGT GACC - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Fragment EM - #in which the sequence coding for 
endothiapeps - #in 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- - GATGTCTTCC CCTCTCTAAT GAACGCCTTG GTGACC - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- - GCTAAAGCTT ATCCGCCGCC GGCGGGGGAA TTC - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- - CAATGGATCC GGTCACCAAG GCGTTCATTA GAGAGGGGAA GACATC - # 
46 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # /primer 5' carrying site NcoI 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- - ACGTCCATGG CTTCCCCTCT CAAGAACGCC - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # / primer 3' carrying site 
MluI 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- - ACGTACGCGT CCACGCCTAC CCAACAAGAC - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # / primer 1 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- - AGAAAGCTTG GAGGAGCGAG GGCCC - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # / primer 2 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- - GCAGAATTCA CATATGTCTT CCCCTCTCAA G - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1236 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Fragment 3:s - #ynthetic fragment 
BamHI(1)-Bam - #HI(2) containing 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- - GATCCGCGGA AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AA - 
#CTTACATT 60 
- - AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AG - 
#CTGCATTA 120 
- - ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCCAGG GT - 
#GGTTTTTC 180 
- - TTTTCACCAG TGAGACGGGC AACAGCTGAT TGCCCTTCAC CGCCTGGCCC TG - 
#AGAGAGTT 240 
- - GCAGCAAGCG GTCCACGCTG GTTTGCCCCA CCACCCGAAA ATCCTGTTTG AT - 
#GGTGGTTA 300 
- - ACGGCGGGAT ATAACATGAG CTGTCTTCGG TATCGTCGTA TCCCACTACC GA - 
#GATATCCG 360 
- - CACCAACGCG CAGCCCGGAC TCGGTAATGG CGCGCATTGC GCCCAGCGCC AT - 
#CTGATCGT 420 
- - TGGCAACCAG CATCGCAGTG GGAACGATGC CCTCATTCAG CATTTGCATG GT - 
#TTGTTGAA 480 
- - AACCGGACAT GGCACTCCAG TCGCCTTCCC GTTCCGCTAT CGGCTGAATT TG - 
#ATTGCGAG 540 
- - TGAGATATTT ATGCCAGCCA GCCAGACGCA GACGCGCCGA GACAGAACTT AA - 
#TGGGCCCG 600 
- - CTAACAGCGC GATTTGCTGG TGACCCAATG CGACCAGATG CTCCACGCCC AG - 
#TCGCGTAC 660 
- - CGTCTTCATG GGAGAAAATA ATACTGTTGA TGGGTGTCTG GTCAGAGACA TC - 
#AAGAAATA 720 
- - ACGCCGGAAC ATTAGTGCAG GCAGCTTCCA CAGCAATGGC ATCCTGGTCA TC - 
#CAGCGGAT 780 
- - AGTTAATGAT CAGCCCACTG ACGCGTTGCG CGAGAAGATT GTGCACCGCC GC - 
#TTTACAGG 840 
- - CTTCGACGCC GCTTCGTTCT ACCATCGACA CCACCACGCT GGCACCCAGT TG - 
#ATCGGCGC 900 
- - GAGATTTAAT CGCCGCGACA ATTTGCGACG GCGCGTGCAG GGCCAGACTG GA - 
#GGTGGCAA 960 
- - CGCCAATCAG CAACGACTGT TTGCCCGCCA GTTGTTGTGC CACGCGGTTG GG - 
#AATGTAAT 1020 
- - TCAGCTCCGC CATCGCCGCT TCCACTTTTT CCCGCGTTTT CGCAGAAACG TG - 
#GCTGGCCT 1080 
- - GGTTCACCAC GCGGGAAACG GTCTGATAAC AGACACCGGC ATACTCTGCG AC - 
#ATCGTATA 1140 
- - ACGTTACTGG TTTCACATTC ACCACCCTGA ATTGACTCTC TTCCGGGCGC TA - 
#TCATGCCA 1200 
- - TACCGCGAAA GGTTTTGCGC CATTCGATGG TGTCCG - # 
- # 1236 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 321 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (ix) FEATURE: 
(A) NAME/KEY: -35.sub.-- - #signal 
(B) LOCATION: 18..23 
- - (ix) FEATURE: 
(A) NAME/KEY: -10.sub.-- - #signal 
(B) LOCATION: 41..46 
- - (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: 107..184 
- - (ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- - #peptide 
(B) LOCATION: 185..316 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 107..316 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- - TCGAGCTGAC TGACCTGTTG CTTATATTAC ATCGATAGCG TATAATGTGT GG - 
#AATTGTGA 60 
- - GCGATAACAA TTTCACACAG TTTAACTTTA AGAAGGAGAT ATACAT ATG - #GCT ACC 
115 
- # - # Met Ala - #Thr 
- # - # -26 -25 
- - GGA TCC CGG ACT AGT CTG CTC CTG GCT TTT GG - #C CTG CTC TGC CTG CCC 
163 
Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gl - #y Leu Leu Cys Leu Pro 
-20 - # -15 - # -10 
- - TGG CTT CAA GAG GGC AGT GCC TTC CCA ACC AT - #T CCC TTA TCT AGA CTT 
211 
Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Il - #e Pro Leu Ser Arg Leu 
-5 - # 1 - # 5 
- - TTT GAC AAC GCT ATG CTC CGC GCC CAT CGT CT - #G CAC CAG CTG GCC TTT 
259 
Phe Asp Asn Ala Met Leu Arg Ala His Arg Le - #u His Gln Leu Ala Phe 
10 - # 15 - # 20 - # 25 
- - GAC ACC TAC CAG GAG TTT GAA GAA GCC TAT AT - #C CCA AAG GAA CAG AAG 
307 
Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Il - #e Pro Lys Glu Gln Lys 
30 - # 35 - # 40 
- - TAT TCA TTC CTGCA - # - # 
- # 321 
Tyr Ser Phe 
- - - - (2) INFORMATION FOR SEQ ID NO:26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- - Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Le - #u Ala Phe Gly Leu Leu 
26 -25 - # -20 - # -15 
- - Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ph - #e Pro Thr Ile Pro Leu 
10 - #-5 - # 1 5 
- - Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Al - #a His Arg Leu His Gln 
10 - # 15 - # 20 
- - Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Gl - #u Ala Tyr Ile Pro Lys 
25 - # 30 - # 35 
- - Glu Gln Lys Tyr Ser Phe 
40 
- - - - (2) INFORMATION FOR SEQ ID NO:27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 74 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- - CGATAGCGTA TAATGTGTGG AATTGTGAGC GGATAACAAT TTCACACAGT TT - 
#TTCGCGAA 60 
- - GAAGGAGATA TACA - # - # 
- # 74 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 190 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- - GATCTTCAAG CAGACCTACA GCAAGTTCGA CACAAACTCA CACAACGATG AC - 
#GCACTACT 60 
- - CAAGAACTAC GGGCTGCTCT ACTGCTTCAG GAAGGACATG GACAAGGTCG AG - 
#ACATTCCT 120 
- - GCGCATCGTG CAGTGCCGCT CTGTGGAGGG CAGCTGTGGC TTCTAGTAAG GT - 
#ACCCTGCC 180 
- - CTACGTACCA - # - # 
- # 190 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # complementary to SEQ ID NO:30 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- - TATGTCTGCG GTAAAAGCAG CGCGCTACGG CAAGGACAAT GTTCGCGT - # 
48 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: oligonucleotide - # complementary to SEQ ID NO:29 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- - AGACGCGAAC ATTGTCCTTG CCGTAGCGCG CTGCTTTTAC CGCAGACA - # 
48 
- - - - (2) INFORMATION FOR SEQ ID NO:31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 906 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- - ATGTCTGCGG TAAAAGCAGC GCGCTACGGC AAGGACAATG TTCGCGTCTA CA - 
#AGGTTCAC 60 
- - AAGGACGAGA AGACCGGTGT CCAGACGGTG TACGAGATGA CCGTCTGTGT GC - 
#TTCTGGAG 120 
- - GGTGAGATTG AGACCTCTTA CACCAAGGCC GACAACAGCG TCATTGTCGC AA - 
#CCGACTCC 180 
- - ATTAAGAACA CCATTTACAT CACCGCCAAG CAGAACCCCG TTACTCCTCC CG - 
#AGCTGTTC 240 
- - GGCTCCATCC TGGGCACACA CTTCATTGAG AAGTACAACC ACATCCATGC CG - 
#CTCACGTC 300 
- - AACATTGTCT GCCACCGCTG GACCCGGATG GACATTGACG GCAAGCCACA CC - 
#CTCACTCC 360 
- - TTCATCCGCG ACAGCGAGGA GAAGCGGAAT GTGCAGGTGG ACGTGGTCGA GG - 
#GCAAGGGC 420 
- - ATCGATATCA AGTCGTCTCT GTCCGGCCTG ACCGTGCTGA AGAGCACCAA CT - 
#CGCAGTTC 480 
- - TGGGGCTTCC TGCGTGACGA GTACACCACA CTTAAGGAGA CCTGGGACCG TA - 
#TCCTGAGC 540 
- - ACCGACGTCG ATGCCACTTG GCAGTGGAAG AATTTCAGTG GACTCCAGGA GG - 
#TCCGCTCG 600 
- - CACGTGCCTA AGTTCGATGC TACCTGGGCC ACTGCTCGCG AGGTCACTCT GA - 
#AGACTTTT 660 
- - GCTGAAGATA ACAGTGCCAG CGTGCAGGCC ACTATGTACA AGATGGCAGA GC - 
#AAATCCTG 720 
- - GCGCGCCAGC AGCTGATCGA GACTGTCGAG TACTCGTTGC CTAACAAGCA CT - 
#ATTTCGAA 780 
- - ATCGACCTGA GCTGGCACAA GGGCCTCCAA AACACCGGCA AGAACGCCGA GG - 
#TCTTCGCT 840 
- - CCTCAGTCGG ACCCCAACGG TCTGATCAAG TGTACCGTCG GCCGGTCCTC TC - 
#TGAAGTCT 900 
- - AAATTG - # - # - 
# 906 
- - - - (2) INFORMATION FOR SEQ ID NO:32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2140 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: base pairs - #402-405 : site BstIII 
- - (ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 468..565 
(D) OTHER INFORMATION: - #/standard.sub.-- name= "intron 1" 
- - (ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 850..938 
(D) OTHER INFORMATION: - #/standard.sub.-- name= "intron 2" 
- - (ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 1279..1367 
(D) OTHER INFORMATION: - #/standard.sub.-- name= "intron 3" 
- - (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: join(329..46 - #7, 566..693) 
- - (ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- - #peptide 
(B) LOCATION: join(694..84 - #9, 939..1278, 1368..1861) 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(329..46 - #7, 566..849, 939..1278, 1368..186 
1) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- - AAGCTTATCC GCCGCCGGCG GGGGAATTCT ATTGAACTTG TTCGAATCAT TG - 
#GTCCGTGG 60 
- - TCTTTTCGTC CATGCGGGCT CCGCTGGCGG ATGAATGACC TTCTGGCTTC TA - 
#GCCTGGCG 120 
- - AAGCGATGTT ACTCTGTTGT CTATACTATA CGATATGGTC AAGAGAGCAC AT - 
#GTGCCGCC 180 
- - AGATGAAGAC ATGTATATAA AAGGAGTGGC CTCGACGGTT GCTCAACCAT CT - 
#TCTGTCTG 240 
- - TCCCAACGCC ATCGACTCTT CAACTTCTCC TTCGTGTTCC ACCACCATCA CC - 
#TTGCTCCA 300 
- - GACTTAGGAC TTTCAGCAAC CTTCAAAG ATG TCT TCC CCT CTC - #AAG AAC GCC 
352 
- # Met Ser Ser - #Pro Leu Lys Asn Ala 
- # -89 - # -85 
- - TTG GTG ACC GCC ATG TTG GCT GGT GGT GCT CT - #C AGC TCG CCT ACA AAG 
400 
Leu Val Thr Ala Met Leu Ala Gly Gly Ala Le - #u Ser Ser Pro Thr Lys 
-80 - # -75 - # -70 
- - CAA CAC GTT GGA ATT CCC GTC AAC GCC TCT CC - #T GAA GTT GGC CCC GGA 
448 
Gln His Val Gly Ile Pro Val Asn Ala Ser Pr - #o Glu Val Gly Pro Gly 
65 - - #60 - - #55 - - 
#50 
- - AAG TAC TCG TTC AAG CAA G GTGAGTAGAG CTGCTTC - #TGT GTGTTGCAAC 
497 
Lys Tyr Ser Phe Lys Gln 
-45 
- - AGAAGACCAA CGCAAAAAGA AGAGGTCAAG GCAAGACGGA TATTTTACTG AC - 
#AATTATAC 557 
- - TTTTGAAG TC CGG AAC CCC AAC TAC AAG TTC A - #AC GGG CCT CTG TCG 
GTC 606 
Val Arg Asn Pro Asn T - #yr Lys Phe Asn Gly Pro Leu Ser Val 
-42 - #-40 - #-35 - 
#-30 
- - AAG AAG ACG TAC CTC AAG TAC GGC GTG CCG AT - #C CCA GCC TGG CTG 
GAG 654 
Lys Lys Thr Tyr Leu Lys Tyr Gly Val Pro Il - #e Pro Ala Trp Leu Glu 
-25 - # -20 - # -15 
- - GAT GCT GTC CAG AAC TCT ACC TCG GGC CTG GC - #T GAG CGC TCG ACC GGT 
702 
Asp Ala Val Gln Asn Ser Thr Ser Gly Leu Al - #a Glu Arg Ser Thr Gly 
-10 - # -5 - # 1 
- - TCT GCG ACC ACA ACT CCC ATC GAC AGC CTC GA - #T GAT GCT TAC ATC ACT 
750 
Ser Ala Thr Thr Thr Pro Ile Asp Ser Leu As - #p Asp Ala Tyr Ile Thr 
5 - # 10 - # 15 
- - CCG GTT CAG ATC GGC ACC CCT GCG CAG ACT CT - #G AAC CTG GAC TTT GAC 
798 
Pro Val Gln Ile Gly Thr Pro Ala Gln Thr Le - #u Asn Leu Asp Phe Asp 
20 - # 25 - # 30 - # 35 
- - ACT GGA TCT TCG GAT CTG TGG GTC TTC AGC AG - #C GAG ACT ACA GCC AGC 
846 
Thr Gly Ser Ser Asp Leu Trp Val Phe Ser Se - #r Glu Thr Thr Ala Ser 
40 - # 45 - # 50 
- - GAG GTTGGTCAAC CCTCGCCCGC ATTTTATTGC ATACATTTTT AGTTTTTTT - #G 
899 
Glu 
- - GTAATCAGAA TACTAACATT GGGAATTTCC CAACTGTAG GTC GAT GGG - # CAG ACC 
953 
- # - # Val Asp Gly Gln Thr 
- # - # 55 
- - ATC TAC ACC CCC AGC AAG AGC ACC ACC GCC AA - #G CTG CTG TCG GGC GCT 
1001 
Ile Tyr Thr Pro Ser Lys Ser Thr Thr Ala Ly - #s Leu Leu Ser Gly Ala 
60 - # 65 - # 70 
- - ACC TGG TCC ATC TCC TAC GGA GAC GGT AGC TC - #T TCC AGC GGC GAT GTC 
1049 
Thr Trp Ser Ile Ser Tyr Gly Asp Gly Ser Se - #r Ser Ser Gly Asp Val 
75 - # 80 - # 85 
- - TAC ACT GAC ACC GTC TCG GTT GGA GGC CTT AC - #C GTG ACG GGC CAG GCT 
1097 
Tyr Thr Asp Thr Val Ser Val Gly Gly Leu Th - #r Val Thr Gly Gln Ala 
90 - # 95 - #100 - #105 
- - GTC GAG TCG GCC AAG AAG GTT TCT TCC AGC TT - #C ACC GAG GAC TCG ACC 
1145 
Val Glu Ser Ala Lys Lys Val Ser Ser Ser Ph - #e Thr Glu Asp Ser Thr 
110 - # 115 - # 120 
- - ATT GAC GGT CTC CTG GGC CTG GCC TTC AGC AC - #C CTG AAC ACT GTG TCG 
1193 
Ile Asp Gly Leu Leu Gly Leu Ala Phe Ser Th - #r Leu Asn Thr Val Ser 
125 - # 130 - # 135 
- - CCT ACC CAG CAA AAG ACT TTC TTC GAC AAT GC - #G AAG GCG TCC TTG GAC 
1241 
Pro Thr Gln Gln Lys Thr Phe Phe Asp Asn Al - #a Lys Ala Ser Leu Asp 
140 - # 145 - # 150 
- - TCG CCT GTG TTC ACG GCT GAT CTT GGC TAC CA - #T GCC C GTGAGTGACC 
1288 
Ser Pro Val Phe Thr Ala Asp Leu Gly Tyr Hi - #s Ala 
155 - # 160 - # 165 
- - CCTCTTGATA CATATACTTT TTGATGAATC TTGTTGGAGA AGCATTCCCC AC - 
#TAATATGG 1348 
- - AAATTGTTTG TATCTACAG CT GGT ACC TAC AAC TTC - #GGC TTC ATC GAT 
ACC 1399 
- # Pro Gly Thr Tyr Asn Phe Gly Phe Ile - # Asp Thr 
- # - # 170 - # 175 
- - ACT GCC TAC ACG GGC TCC ATC ACC TAC ACC GC - #T GTC TCG ACC AAG CAA 
1447 
Thr Ala Tyr Thr Gly Ser Ile Thr Tyr Thr Al - #a Val Ser Thr Lys Gln 
180 - # 185 - # 190 
- - GGG TTC TGG GAG TGG ACT TCG ACC GGC TAC GC - #C GTC GGC TCC GGC ACC 
1495 
Gly Phe Trp Glu Trp Thr Ser Thr Gly Tyr Al - #a Val Gly Ser Gly Thr 
195 - # 200 - # 205 
- - TTC AAG TCG ACT TCC ATC GAC GGC ATC GCT GA - #C ACT GGC ACG ACC CTC 
1543 
Phe Lys Ser Thr Ser Ile Asp Gly Ile Ala As - #p Thr Gly Thr Thr Leu 
210 - # 215 - # 220 
- - CTG TAC CTC CCT GCC ACC GTC GTG TCG GCC TA - #C TGG GCC CAG GTC TCG 
1591 
Leu Tyr Leu Pro Ala Thr Val Val Ser Ala Ty - #r Trp Ala Gln Val Ser 
225 2 - #30 2 - #35 2 - 
#40 
- - GGC GCC AAG TCC AGC TCT TCC GTC GGC GGC TA - #C GTC TTC CCC TGC 
AGC 1639 
Gly Ala Lys Ser Ser Ser Ser Val Gly Gly Ty - #r Val Phe Pro Cys Ser 
245 - # 250 - # 255 
- - GCG ACC CTG CCT TCC TTC ACC TTC GGC GTT GG - #C TCA GCT CGC ATT GTG 
1687 
Ala Thr Leu Pro Ser Phe Thr Phe Gly Val Gl - #y Ser Ala Arg Ile Val 
260 - # 265 - # 270 
- - ATT CCT GGC GAC TAC ATT GAT TTC GGC CCC AT - #C TCC ACT GGA AGC TCG 
1735 
Ile Pro Gly Asp Tyr Ile Asp Phe Gly Pro Il - #e Ser Thr Gly Ser Ser 
275 - # 280 - # 285 
- - TCT TGC TTT GGC GGC ATC CAG TCC AGC GCT GG - #T ATC GGC ATC AAC ATC 
1783 
Ser Cys Phe Gly Gly Ile Gln Ser Ser Ala Gl - #y Ile Gly Ile Asn Ile 
290 - # 295 - # 300 
- - TTC GGT GAT GTC GCT CTG AAG GCC GCC TTT GT - #C GTC TTC AAC GGG GCT 
1831 
Phe Gly Asp Val Ala Leu Lys Ala Ala Phe Va - #l Val Phe Asn Gly Ala 
305 3 - #10 3 - #15 3 - 
#20 
- - ACA ACT CCC ACT CTT GGC TTT GCT TCC AAG TA - #AATTAAGG GCCCTCGCTC 
1881 
Thr Thr Pro Thr Leu Gly Phe Ala Ser Lys 
325 - # 330 
- - CTCCATAGCT GCGATAAATG AGGCAGGCTC AAGTGGAAAG TCTTGTTGGG TA - 
#GGCGTGGA 1941 
- - TACGTATTGT CTACTTAATT AATTAATGCC AAAGCAGACC TGAAGATAGC TT - 
#TAGTAATT 2001 
- - AATTCAATAA GCACATGGAG ATCCTTCGGA TCAATATGCT AACTCGGTCT TC - 
#ATCTCTAA 2061 
- - ACGAATGTGT TGTTGCTTGA GTTTCAGATG AATTTCCTGC TGTGATATCC CT - 
#CTAAGGTG 2121 
- - TAGTATGGAC AGTAAGCTT - # - # 
214 - #0 
- - - - (2) INFORMATION FOR SEQ ID NO:33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 419 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
- - Met Ser Ser Pro Leu Lys Asn Ala Leu Val Th - #r Ala Met Leu Ala Gly 
89 -85 - # -80 - # -75 
- - Gly Ala Leu Ser Ser Pro Thr Lys Gln His Va - #l Gly Ile Pro Val Asn 
-70 - # -65 - # -60 
- - Ala Ser Pro Glu Val Gly Pro Gly Lys Tyr Se - #r Phe Lys Gln Val Arg 
-55 - # -50 - # -45 -42 
- - Asn Pro Asn Tyr Lys Phe Asn Gly Pro Leu Se - #r Val Lys Lys Thr Tyr 
-40 - # -35 - # -30 
- - Leu Lys Tyr Gly Val Pro Ile Pro Ala Trp Le - #u Glu Asp Ala Val Gln 
25 - - #20 - - #15 - - 
#10 
- - Asn Ser Thr Ser Gly Leu Ala Glu Arg Ser Th - #r Gly Ser Ala Thr 
Thr 
-5 - # 1 - # 5 
- - Thr Pro Ile Asp Ser Leu Asp Asp Ala Tyr Il - #e Thr Pro Val Gln 
Ile 
10 - # 15 - # 20 
- - Gly Thr Pro Ala Gln Thr Leu Asn Leu Asp Ph - #e Asp Thr Gly Ser 
Ser 
25 - # 30 - # 35 
- - Asp Leu Trp Val Phe Ser Ser Glu Thr Thr Al - #a Ser Glu Val Asp 
Gly 
40 - #45 - #50 - # 
55 
- - Gln Thr Ile Tyr Thr Pro Ser Lys Ser Thr Th - #r Ala Lys Leu Leu 
Ser 
60 - # 65 - # 70 
- - Gly Ala Thr Trp Ser Ile Ser Tyr Gly Asp Gl - #y Ser Ser Ser Ser Gly 
75 - # 80 - # 85 
- - Asp Val Tyr Thr Asp Thr Val Ser Val Gly Gl - #y Leu Thr Val Thr Gly 
90 - # 95 - # 100 
- - Gln Ala Val Glu Ser Ala Lys Lys Val Ser Se - #r Ser Phe Thr Glu Asp 
105 - # 110 - # 115 
- - Ser Thr Ile Asp Gly Leu Leu Gly Leu Ala Ph - #e Ser Thr Leu Asn Thr 
120 1 - #25 1 - #30 1 - 
#35 
- - Val Ser Pro Thr Gln Gln Lys Thr Phe Phe As - #p Asn Ala Lys Ala 
Ser 
140 - # 145 - # 150 
- - Leu Asp Ser Pro Val Phe Thr Ala Asp Leu Gl - #y Tyr His Ala Pro Gly 
155 - # 160 - # 165 
- - Thr Tyr Asn Phe Gly Phe Ile Asp Thr Thr Al - #a Tyr Thr Gly Ser Ile 
170 1 - #75 1 - #80 
- - Thr Tyr Thr Ala Val Ser Thr Lys Gln Gly Ph - #e Trp Glu Trp Thr Ser 
185 - # 190 - # 195 
- - Thr Gly Tyr Ala Val Gly Ser Gly Thr Phe - #Lys Ser Thr Ser Ile Asp 
200 2 - #05 2 - #10 2 - 
#15 
- - Gly Ile Ala Asp Thr Gly Thr Thr Leu Leu Ty - #r Leu Pro Ala Thr 
Val 
220 - # 225 - # 230 
- - Val Ser Ala Tyr Trp Ala Gln Val Ser Gly Al - #a Lys Ser Ser Ser Ser 
235 - # 240 - # 245 
- - Val Gly Gly Tyr Val Phe Pro Cys Ser Ala Th - #r Leu Pro Ser Phe Thr 
250 - # 255 - # 260 
- - Phe Gly Val Gly Ser Ala Arg Ile Val Ile Pr - #o Gly Asp Tyr Ile Asp 
265 - # 270 - # 275 
- - Phe Gly Pro Ile Ser Thr Gly Ser Ser Ser Cy - #s Phe Gly Gly Ile Gln 
280 2 - #85 2 - #90 2 - 
#95 
- - Ser Ser Ala Gly Ile Gly Ile Asn Ile Phe Gl - #y Asp Val Ala Leu 
Lys 
300 - # 305 - # 310 
- - Ala Ala Phe Val Val Phe Asn Gly Ala Thr Th - #r Pro Thr Leu Gly Phe 
315 - # 320 - # 325 
- - Ala Ser Lys 
330 
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