Abstract:
An isolated DNA having the promoter sequence of the hex gene of  P. chrysogenum  or a DNA fragment that is hybridizable to the complement of the promoter sequence under stringent conditions and is capable of directing expression of DNA downstream of the fragment in  P. chrysogenum . Also a process for promoting expression of a coding sequence of interest in a microorganism using the isolated DNA and a process to block expression of a gene of interest in a microorganism using the isolated DNA are disclosed.

Description:
This application is a divisional of application Ser. No. 09/171,337 filed on May 14, 1999, now U.S. Pat. No. 6,300,095, which is International Application PCT/ES98/00056 filed on Mar. 5, 1998 and which designated the U.S., claims the benefit thereof and incorporates the same by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to the technical field of the expression of the gdh and hex genes of  Penicillium chrysogenum  and of the act gene also of  P. chrysogenum  and of  Acremonium chrysogenum . From analysis of the nucleotide sequence of said genes the existence of a promoter region which includes the translation initiation site, and which can be used to construct powerful expression and secretion vectors that are useful both for  P. chrysogenum  and for  A. chrysogenum  and related species, is deduced. In addition, these promoters can be used to block gene expression by means of antisense constructs. The expression of other genes in filamentous fungi can be directed under the control of the aforesaid promoters, with the production of antibiotics and/or proteins inherent therein being increased. 
     PRIOR ART 
       P. chrysogenum  and  A. chrysogenum  are filamentous fungi which are of industrial interest because of their ability to produce penicillin and cephalosporin, respectively. During the last decade there has been considerable development of genetic manipulation techniques applicable in both microorganisms. The techniques for genetic manipulation of  P. chrysogenum  and  A. chrysogenum  include the transformation of protoplasts with vectors which use the phleomycin resistance gene (hereinafter called ble R  gene) (Kolar, M. et al. (1988), Gene 62, 127-134) as a selection marker, as well as the expression of additional intact copies of genes of interest and the replacement of the promoter of the gene in question by another promoter which is able to improve its expression. The expression of homologous genes in fungi such as  P. chrysoqenum  or  A. chrysogenum  can be negatively regulated, whereas in the case of heterologous genes it is possible that their promoter may not be efficiently recognized by the said fungi. With the aim of avoiding these problems, genes were identified and cloned which are expressed constitutively and in which the said expression preferably does not show negative catabolic regulation, called hereinafter strong promoters. In general it is considered that the high-expression genes have signals in the promoter region which facilitate high transcription levels and which play a fundamental rôle in functions implicated in primary cellular metabolism. These genes include: the genes which code for NADP-dependent glutamate dehydrogenase (EC.1.4.1.4) (hereinafter called gdh gene), β- N -acetylhexosaminidase (EC.3.2.1.52) (hereinafter called hex gene) and γ-actin (hereinafter called act gene). 
     There are earlier references to the gdh, hex and act genes from microorganisms other than those which are used in the present invention. The most relevant bibliography includes: (I) the nucleotide sequence of the gdh gene of the fungus  Neurospora crassa  (Kinnaird, J. H. and Fincham, J. R. S. (1983), Gene 26, 253-260) as well as the regulation of the expression of the gdhA gene of  Aspergillus nidulans  (Hawkins, A. R. et al. (1989), Mol. Gen. Genet. 418, 105-111), (II) the cloning and expression of the hex1 gene of  Candida albicans  (Cannon, R. D. et al. (1994), J. Bacteriol. 2640-2647) and (III) the characterization of the act gene of  A. nidulans  (Fidel, S. et al. (1988), Gene 70, 283-293). The expression of heterologous genes in  P. chrysogenum  using the promoters of the pcbC or penDE genes was described by Cantwell, C. A. et al. in 1992 (Proc. R. Soc. London Ser. B 248, 283-289). In addition, the expression of heterologous genes in  A. chrysogenum  using the promoters of the β-isopropyl malate dehydrogenase gene (Japanese Patent Laid Open Publication No. 80295/1989) and glyceraldehyde 3-phosphate dehydrogenase gene (European Patent Application 0376226A1/1989) has also been described. 
     The inactivation of gene expression in industrial strains is sometimes necessary for the elimination of undesirable enzyme activities. Owing to the fact that the level of ploidy of many industrial strains makes it difficult in most cases to block expression by direct gene disruption, it is necessary to use systems for inactivation of expression which are independent of the level of ploidy. The development of antisense constructs expressed under the control of strong promoters makes interruption of gene expression possible. Constructs of this type are especially useful in industrial strains owing to the fact that their levels of ploidy (Künkel et al. (1992) Appl. Microbiol. Biotech. 36, 499-502) make it difficult to obtain complete gene inactivation. The use of antisense constructs for blocking enzyme activities has been described in yeasts (Atkins, D. et al. (1994), Biol. Chem. H-S 375, 721-729) and plants (Hamada, T. (1996), Transgenic Research 5, 115-121; John, M. E. (1996) Plant Mol. Biol. 30, 297-306). The hex promoter has the special feature of coding for an extracellular enzyme, which allows it to be used for the expression of extracellular proteins. 
     There are no citations in the prior art, however, which describe either the gene sequences of the filamentous fungi used in the present invention or those of the enzymes synthesized by the expression thereof. Nor is there any description in said Prior Art of the use of the strong promoters of the genes of the fungi described in the present invention for the expression, secretion or inactivation of gene expression. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The use of strong promoters to overexpress certain genes can lead to improvement in the production of penicillin or cephalosporin, and also to the synthesis of new antibiotics derived from the latter. 
     This invention describes a new process for obtaining strains of  P. chrysogenum  and  A. chrysogenum  with the ability to express homologous or heterologous genes under the control of strong promoters. The characterization and subsequent use of the promoters corresponding to the genes which code for NADP-dependent glutamate dehydrogenase (EC.1.4.1.4)—gdh gene—of  P. chrysogenum , β- N -acetylhexosaminidase (EC.3.2.1.52)—hex gene—of  P. chrysogenum  and γ-actin—act gene—of  P. chrysogenum  and  A. chrysogenum  are described. The use of said promoters to overexpress genes related to the biosynthesis of penicillin and/or cephalosporin in the above-mentioned strains is one of the aims of the present invention. These promoters can also be used to block gene expression by means of antisense constructs. 
     The present invention is based on  P. chrysogenum  and  A. chrysogenum  as nucleic acid donors. Once the genomic DNA had been purified, DNA libraries of both microorganisms were constructed as described in Examples 1 and 4, and they were screened with: (I) synthetic oligonucleotides corresponding to the gdh gene of  N. crassa  in order to clone the homologous gene of  P. chrysogenum , (II) combinations of oligonucleotides synthesized on the basis of the amino terminal sequence of the enzyme β- N -acetylhexosaminidase in order to clone the hex gene of  P. chrysogenum  and (III) a fragment of the act gene of  A. nidulans  in order to clone the homologous genes of  P. chrysogenum  and  A. chrysogenum . The clones purified by virtue of their ability to generate positive hybridization with the corresponding probe were subsequently analysed, the presence of the genes sought being determined. 
     The gdh gene of  P. chrysogenum  was identified in a 7.2 kb EcoRI fragment and in two BamHI fragments of 2.9 and 1.5 kb respectively. The restriction map of the DNA region which includes it is shown in FIG.  1 . The 2,816 nucleotide sequence (SEQ ID NO:1) was then determined, which includes an open reading frame (ORF) with a very marked preferential codon usage pattern, the ATG translation initiation codon of which was found in position 922 and the TAA translation termination codon in position 2,522. The presence of 2 introns of 159 bp and 56 bp was also determined between positions 971-1130 and 1262-1318 respectively. Said ORF codes for a protein of 49,837 Da, with an isoelectric point of 6.18, the 461 amino acid sequence of which (SEQ ID NO:5) has 72.4% identity with the amino acid sequence of the NADP-dependent glutamate dehydrogenase enzyme of  N. crassa . In the promoter region there are found pyrimidine-rich zones similar to those which appear in highly expressed genes, as well as two presumed TATA boxes (this box is found in certain promoters of fungi 30 to 50 bp upstream from the site of transcription initiation) (Davis, M. A. and Hynes, M. J. (1991), More Gene Manipulations in Fungi, Academic Press,. San Diego, Calif.) and a CCAAT box (which is found in about 30% of promoters of eukaryotic genes 50 to 200 bp upstream from the site of transcription initiation) (Bucher, P. (1990) J. Mol. Biol. 212: 563-578). This promoter was then used to express in  P. chrysogenum  and  A. chrysogenum  the  E. coli  gene which codes for β-galactosidase (hereinafter called lacZ gene) and the ble R  gene of  S. hindustanus . The plasmids pSKGSu and pALfleo7 (FIG. 5) were constructed for this purpose, as described in Example 1. From-the results obtained it is deduced that the gdh promoter (hereinafter called Pgdh) is able to control the expression of the heterologous lacZ and ble R  genes both in  P. chrysogenum  and  A. chrysogenum  and also in  E. coli.    
     The development of antisense constructs expressed under the control of strong promoters makes the interruption of gene expression possible. The plasmid pALP888 (FIG. 5) was constructed for this purpose, as described in Section 1.3 of Example 1. The results obtained confirm the possibility of totally or partially blocking undesirable enzyme activities in  P. chrysogenum  by the use of antisense constructs using Pgdh. 
     The hex gene of  P. chrysogenum  was identified in a 3.2 kb SacI fragment and in a 2.1 kb SalI fragment. The restriction map of the DNA region which includes the hex gene is shown in FIG.  2 . The 5,240 nucleotide sequence (SEQ ID NO:2) was then determined, confirming the existence of two ORFs with a very marked preferential codon usage pattern, one of which matched the hex gene. The ATG translation initiation codon of the hex gene was found in position 1,324 and the TGA termination codon in position 3,112. Said ORF has no introns and codes for a protein of 66,545 Da, with an isoelectric point of 5.34, the 596 amino acid sequence of which (SEQ ID NO:6) has 49.0% identity with the amino acid sequence of the β- N -acetylhexosaminidase enzyme of  Candida albicans . In addition, the deduced amino acid sequence includes the polypeptides determined chemically from the purified enzyme in positions 19-40 and 99-120. In the promoter region there are found two pyrimidine-rich zones, a presumed TATA box and the CAAT box. This promoter was then used to express the ble R  gene of  S. hindustanus  in  P. chrysogenum . The plasmid pALP480 (FIG. 6) was constructed for this purpose, as described in Example 2. From the results obtained it is deduced that the hex promoter (hereinafter called Phex) is able to control the expression of the heterologous ble R  gene in  P. chrysogenum . In addition, the fact that the enzyme β- N -acetylhexosaminidase is a protein abundantly secreted by  P. chrysogenum  to the culture medium makes it possible to use the hex gene for the expression and secretion of homologous or heterologous proteins in  P. chrysogenum  or related filamentous fungi. The genes to be expressed can be fused in a reading frame with the promoter region, including the secretion signal sequence of the hex gene, or else they can be fused in a reading frame with the complete hex gene. 
     The act gene of  P. chrysogenum  (hereinafter called actPc) was identified in a 5.2 kb BamHI fragment, a 4.9 kb EcoRI fragment and a 5.9 kb HindIII fragment. The restriction map of the DNA region which includes the actPc gene is shown in FIG.  3 . Once the 2,994 nucleotide sequence (SEQ ID NO:3) had been determined, the existence of an ORF with a very marked preferential codon usage pattern was confirmed. The ATG translation initiation codon was found in position 494 and the TAA termination codon in position 2,250. Said ORF has 5 introns and codes for a protein of 41,760 Da, with an isoelectric point of 5.51, the 375 amino acid sequence of which (SEQ ID NO:7) has 98.1% identity with the amino acid sequence of the γ-actin protein of  A. nidulans . In the promoter region there are found two pyrimidine-rich zones, a presumed TATA box and four CAAT boxes. This promoter was then used to express the ble R  gene of  S. hindustanus  in  P. chrysogenum . The plasmid pALPfleo1 (FIG. 6) was constructed for this purpose, as described in Example 3. From the results obtained it is deduced that the act promoter of  P. chrysogenum  (hereinafter called PactPc) is able to control the expression of the heterologous ble R  gene in  P. chrysogenum.    
     The act gene of  A. chrysogenum  (hereinafter called actAc) was identified in SalI fragments of 2.4 and 1.1 kb, a 3.9 kb SmaI fragment and an 8.7 kb HindIII fragment. The restriction map of the DNA region which includes the actAc gene is shown in FIG.  4 . The 3,240 nucleotide sequence determined (SEQ ID NO:4) confirmed the existence of an ORF with a very marked preferential codon usage pattern. The ATG translation initiation codon was found in position 787 and the TAA termination codon in position 2,478. Said ORF has 5 introns and codes for a protein of 41,612 Da, with an isoelectric point of 5.51, the 375 amino acid sequence of which (SEQ ID NO:8) has 98.4% and 98.1% identity with the amino acid sequences corresponding to the γ-actin proteins of  A. nidulans  and  P. chrysogenum , respectively. In the promoter region there are found pyrimidine-rich zones and a CAAT box, the existence of a TATA box not being observed. This promoter was then used to express the ble R  gene of  S. hindustanus  in  A. chrysogenum . The plasmid pALCfleo1 (FIG. 6) was constructed for this purpose, as described in Example 4. From the results obtained it is deduced that the act promoter of  A. chrysogenum  (hereinafter called PactAc) is able to control the expression of the heterologous ble R  gene in  A. chrysogenum.    
     In all cases, the expression of the heterologous gene in  P. chrysogenum  or  A. chrysogenum  under the control of the fungal promoter was achieved by fusing the said gene in the correct reading frame. Although the lacZ and ble R  genes were expressed by way of example, it would be possible in the same way to express genes which code for enzymes involved in the biosynthesis of penicillin: pcbAB (α-aminoadipyl-cysteinyl-valine synthetase), pcbC (isopenicillin N synthase), penDE (acyl-CoA:6-APA acyltransferase), pcl (phenylacetyl-CoA ligase), etc.; or of cephalosporin: pcbAB (α-aminoadipyl-cysteinyl-valine synthetase), pcbC (isopenicillin N synthase), cefD (isopenicillin N isomerase), cefEF (deacetoxycephalosporin C synthase/hydroxylase), cefG (deacetylcephalosporin C acetyltransferase), etc. The gene to be expressed may have been obtained by different methods: isolated from chromosome DNA, cDNA synthesized from mRNA, synthesized chemically, etc. The fundamental processes for correct promoter-gene fusion are described in Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA, and Ausubel et al. (1987), Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, USA. 
       P. chrysogenum  and  A. chrysogenum  were used as host strains, but any related strain or mutant strain derived from them can be used. The process employed for the production of protoplasts and transformation of  P. chrysogenum  was based on that described by Cantoral et al. in 1987 (Biotechnology 5: 494-497) and Díez et al. in 1987 (Curr. Genet. 12: 277-282) and is described in Example 1. The production of protoplasts and transformation of  A. chrysogenum  are described in Example 4. In both cases use was made of the antibiotic phleomycin as selection marker and the plasmids pALfleo7, pALP480, pALPfleo1 or pALCfleo1, which are carriers of the ble R  gene expressed under the control of Pgdh, Phex, PactPc and PactAc, respectively. It would be possible, however, to use any marker which can selectively separate the transformant strains from the others, which are not. 
     The transformant may be grown in culture media containing carbon and nitrogen sources which can be assimilated. Examples of carbon sources are glucose, sucrose, lactose, starch, glycerine, organic acids, alcohols, fatty acids, etc., used alone or in combination. Examples of nitrogen sources would be peptone, malt extract, yeast extract, corn steep liquor, gluten, urea, ammonium salts, nitrates, NZ-amine, ammonium sulphate, etc., used alone or in combination. Inorganic salts which can be used as components of the culture medium include phosphates (for example potassium phosphate), sulphates (for example sodium sulphate), chlorides (for example magnesium chloride), etc., and iron, magnesium, calcium, manganese, cobalt, etc., can be used as ions. The cultural conditions such as incubation temperature, pH of the culture medium, aeration, incubation time, etc., must be selected and adjusted in accordance with the strain used. In general terms, however, fermentation is carried out for a period of 4 to 14 days under aerobic conditions at a temperature between 20° C. and 30° C. and a pH between 5 and 9. 
     In summary, the present invention includes: (I) DNA fragments which contain the promoters of the gdh, hex and act genes of  P. chrysogenum  and of the act gene of  A. chrysogenum , (II) plasmids which incorporate the aforesaid promoters together with their translation initiation site, (III) plasmids in which a homologous or heterologous structural gene or an antisense DNA fragment is situated, under the control of the said promoters, (IV)  P. chrysogenum  or  A. chrysogenum  strains transformed with said plasmids, (VI) transformant strains able to express the structural gene or the antisense DNA situated in the plasmid under the control of the promoter and (VII) transformant strains able to secrete homologous or heterologous extracellular proteins under the control of the Phex. 
    
    
     The following examples describe the present invention in detail, without limiting its scope. 
     EXAMPLE 1 
     1.1. Cloning and Characterization of the gdh Gene of  P. chrysogenum.    
     With the aim of cloning the gdh gene of  P. chrysogenum , a DNA library was constructed in the phage vector λGEM12, using established procedures (Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA). To this end, the total DNA of the fungus (purified by the method described by Barredo et al. (1994) Spanish Patent P9400931) was partially digested with Sau3AI and the fragments of about 20 kb were purified in a sucrose gradient (10-40%). These fragments were ligated with the arms of the vector, which had previously been digested with BamHI and purified, and the ligation mixture was then packaged in vitro using the Gigapack II Gold (Stratagene) system in accordance with the manufacturer&#39;s instructions. The packaging reaction, resuspended in 500 μl of SM, was used to make infections of  E. coli  LE392, in order to titrate the number of phages present, and of  E. coli  NM539, with the aim of determining the percentage of recombinant phages.  E. coli  NM539 is a lysogenic strain of the phage P2 and only produces lysis plaques when the phage which infects it lacks the dispensable central region. The phage titre was found to be 132 pfu/μl (a total of 66,000 pfu) in  E. coli  LE392 and 113 pfu/μl (a total of 56,500 pfu) in  E. coli  NM539. This meant that about 85% of the phages were carrying an exogenous DNA insert. The number of recombinant phages needed to make up a complete DNA library was calculated with the equation: N=ln(1−p)/ln(1−f), where “p” is the desired probability, “f” is the proportion of the genome of the selected organism which is contained in a recombinant, and “N” is the number of recombinants needed. Assuming that the genome of  P. chrysogenum  is contained in about 30,000 kb (Fierro et al. (1993),  Mol. Gen. Genet.  241: 573-578) and that the average of the packaged inserts was 18 kb (in spite of the fact that sizes around 20 kb had been selected), a  P. chrysogenum  DNA library had been obtained with 99.999% probability with the number of recombinant phages obtained. After this series of theoretical verifications had been carried out,  E. coli  NM539 was infected and the complete DNA library was spread on 5 Petri dishes of 150 mm diameter (about 11,300 pfu/Petri dish), collected in 50 ml of SM plus 2.5 ml of chloroform, and kept at 4° C. In this way a sufficient and representative volume of recombinant phages (5,300 pfu/μl) ready to be plated out at any time was available. 
     About 60,000 pfu were spread on 3 Petri dishes of 150 mm diameter and then transferred to nitrocellulose filters (BA85, 0.45 μm, Schleicher &amp; Schuell). Said filters were hybridized using standard protocols (Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA) with synthetic oligonucleotides corresponding to the gdh gene of  N. crassa . A total of 10 positive clones were purified via a second and third hybridization cycle and their DNA was then digested with a series of restriction endonucleases and analysed by the Southern blot technique. In this way the gdh gene was identified in a 7.2 kb EcoRI fragment and in two BamHI fragments of 2.9 and 1.5 kb, respectively. After the corresponding subcloning in the plasmids pBluescript I KS(+) (Stratagene) and pUC13 had been carried out, the plasmids pALP784 and pALP785, which match both orientations of a 2.9 kb Sau3AI-XbaI fragment that includes the gdh gene, were constructed. The restriction map of the DNA region which includes said gene is shown in FIG.  1 . 
     With the aim of determining the nucleotide sequence of the gdh gene, a series of clones were constructed from the plasmids pALP784 and pALP785 by the “Erase a base” method (Promega) and then sequenced by the dideoxynucleotide method using the “Sequenase” test kit (USB), in both cases in accordance with the manufacturer&#39;s instructions. The 2,816 nucleotide sequence obtained (SEQ ID NO:1) was analysed with the Geneplot program (DNASTAR), confirming the existence of an ORF with a very marked preferential codon usage pattern. The ATG translation initiation codon was found in position 922 and the TAA termination codon in position 2,522. The presence of 2 introns of 159 bp and 56 bp was also determined between positions 971-1130 and 1262-1318, respectively. Said ORF codes for a protein of 49,837 Da, with an isoelectric point of 6.18, the 461 amino acid sequence of which (SEQ ID NO:5) has 72.4% identity with the amino acid sequence of the NADP-dependent glutamate dehydrogenase enzyme of  N. crassa.    
     In the promoter region there are found various pyrimidine-rich zones, although that located between positions 766-796 is the most extensive one. These zones are found in highly expressed genes and are located immediately upstream from the site of transcription initiation. In addition there are two presumed TATA boxes (the consensus sequence of which in fungi is TATAAA) in positions 752 (TATATAATT) and 852 (TATAATTT). These TATA boxes are found in fungi 30 to 50 bp upstream from the site of transcription initiation, so it is most likely that the authentic TATA box is the one situated in position 752, i.e. 42 bp upstream from the site of transcription initiation. The sequence CCAAT is found in the promoter region of about 30% of known eukaryotic genes, situated between 50 and 200 bp upstream from the site of transcription initiation. The CCAAT box is in position 691 in the promoter region of the gdh gene, i.e. about 105 bp upstream from the presumable site of transcription initiation. 
     1.2. Expression of Control Genes in  P. chrysogenum  and  A. chrysogenum  Under the gdh Promoter 
     The process of transformation and selection of  P. chrysogenum  and  A. chrysogenum  transformants was carried out as described below, depending on their resistance to the antibiotic phleomycin. For this purpose it was necessary to construct the plasmid pALfleo7, which has a size of 5.4 kb and carries the the ble R  gene of  S. hindustanus  expressed under the control of Pgdh as marker in fungi, the chloramphenicol resistance gene as marker in  E. coli  and the polylinker of the plasmid pBC KS (+) (Stratagene). 
     The procedure used for the production of protoplasts and transformation of  P. chrysogenum  was that described by Cantoral et al. in 1987 (Biotechnology 5: 494-497) and Díez et al. in 1987 (Curr. Genet. 12: 277-282), with slight modifications. First of all,  P. chrysogenum  was grown in the PM defined medium (Anné, J., (1997), Agricultura 25) with addition of 10% yeast extract for 18-21 hours at 25° C., and the mycelium was recovered by filtration through a nylon filter and washed with 3-5 volumes of 0.9% NaCl. After drying it between filter paper, it was resuspended (100 mg/ml) in protoplasts buffer. When the micellar suspension was considered to be homogeneous, a volume of a 4 mg/ml Caylasa solution (Cayla) in protoplasts buffer was added to it and it was incubated for 3 hours at 25° C. with agitation at 100 r.p.m. The appearance of protoplasts was observed microscopically. When most of them had been released, they were separated from the mycelium by filtration through a 30 μm pore nylon filter. The protoplasts suspension was washed 3 times with 0.7 M KCl, centrifuging at 400×g for 3 minutes between washings. The precipitated protoplasts were resuspended in 10 ml of KCM solution and after estimation of their concentration by counting in a Thoma chamber they were adjusted to 1-5×10 8  protoplasts/ml with KCM. Next, 100 μl of this solution were carefully mixed with 1-10 μg of DNA plus 10 μl of PCM, and the mixture was incubated in a chilled water bath for 20 minutes. 500 ml of PCM were then added and the mixture was kept at ambient temperature for 20 minutes, after which 600 μl of KCM were added. Transformants were selected on the basis of the ability given by the phleomycin resistance gene present in the plasmids pALfleo7, pALP480 and pALPfleo1 to grow in 30 μg/ml of phleomycin. For this purpose, 200 μl of the transformation reaction were mixed with 5 ml of Czapeck&#39;s medium with the addition of sorbitol (1 M) and phleomycin (30 μg/ml), and it was then spread on a Petri dish with 5 ml of the same medium. The plates were incubated at 25° C. until the appearance of transformants was seen (4-8 days). 
     The procedure used for the production of protoplasts and transformation of  A. chrysogenum  was that described by Gutiérrez et al. (1991), Mol. Gen. Genet. 225: 56-64. First of all, the strain of  A. chrysogenum  was grown in the MMC defined medium for 20-24 hours at 28° C., and the mycelium was recovered by filtration through a nylon filter and washed with 3-5 volumes of 0.9% NaCl. After drying it between filter paper, it was resuspended (50 mg/ml) in protoplasts buffer. When the micellar suspension was considered to be homogeneous, DTT at a final concentration of 10 mM was added to it and it was incubated at 28° C. and 150 r.p.m. for 1 hour. It was then centrifuged at 12,000×g for 15 minutes and the precipitate was resuspended in 20 ml of protoplasts buffer. Next, a volume of a 4 mg/ml Caylasa solution (Cayla) in protoplasts buffer was added and it was incubated for 3 hours at 25° C. with agitation at 100 r.p.m. The appearance of protoplasts was observed microscopically. When most of them had been released, they were separated from the mycelium by filtration through a 25 μm pore nylon filter. The protoplasts suspension was washed 3 times with 0.7 M KCl, centrifuging at 1,000×g for 3 minutes between washings. The precipitated protoplasts were resuspended in 10 ml of NCM buffer and after estimation of their concentration by counting in a Thoma chamber they were adjusted to 1-5×10 8  protoplasts/ml. Next, 100 μl of this solution were carefully mixed with 1-10 μg of DNA, and the mixture was kept in a chilled water bath for 20 minutes, after which 1 ml of CCM was added, followed by incubation at ambient temperature for a further 20 minutes. The mixture was centrifuged at 1,000×g for 5 minutes and the sediment was resuspended in 800 μl of NCM buffer. Transformants were selected on the basis of the ability given by the phleomycin resistance gene present in the plasmids pALfleo7 and pALCfleo1 to grow in 10 μg/ml of phleomycin. For this purpose, 200 μl of the transformation reaction were mixed with 5 ml of the TSA medium with addition of sucrose (0.3 M) and phleomycin (10 μg/ml), and it was then spread on a Petri dish with 5 ml of the same medium. The plates were incubated at 28° C. until the appearance of transformants was seen (5-8 days). 
     In the transformants obtained, an analysis was made of (I) the presence of DNA corresponding to the plasmid used in the transformation, (II) the existence of a transcript corresponding to the control gene and (III) the enzymatic activity corresponding to the gene expressed. Total DNA was obtained in accordance with the conditions described by Barredo et al. in 1994 (Spanish Patent P9400931), and it was then analysed by the Southern blot technique, using the procedure described by Sambrook et al. in 1989 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA). Total RNA was purified by the method described by Ausubel et al. in 1987 (Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, USA). The RNA obtained was kept precipitated in ethanol at −20° C. In order to use it, it was recovered by centrifugation at 4° C. and 10,000×g for 20 minutes. The separation of the RNA molecules on the basis of their molecular size was carried out by agarose-formaldehyde electrophoresis. The RNA was then transferred to a nitrocellulose filter and hybridized with the desired probe, all this being done by the method described by Sambrook et al. in 1989 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA). The appearance of hybridization bands revealed the existence of transcripts and thus the ability to express a bacterial gene in the host fungus:  P. chrysogenum  or  A. chrysogenum . The β-galactosidase enzyme activity was assessed in the transformants by the method described by Sambrook et al. in 1989 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA). The expression of the phleomycin resistance gene was assessed on the basis of the level of resistance given to  P. chrysogenum  or  A. chrysogenum  in Czapeck&#39;s solid medium after incubation at 25° C. for 7 days. 
     1.2.1. Expression of the lacZ Gene of  E. coli  in  P. chrysogenum  and  E. coli  Under the Pgdh 
     The lacZ gene of  E. coli  was fused translationally with the Pgdh with the aim of expressing it in  P. chrysogenum . To this end the lacZ gene was subcloned between the EcoRI and SalI sites of the plasmid pML1 (Carramolino et al. 1989, Gene 77: 31-38), generating the plasmid pMLac. The Pgdh was then introduced between the EcoRI and SmaI sites of pMLac, giving rise to the plasmid pSKG (FIG.  5 ). Finally, the sulphonamide resistance gene (Carramolino et al. 1989, Gene 77: 31-38) was introduced at the EcoRI site of pSKG, giving rise to the plasmid pSKGSu (FIG.  5 ). In the  P. chrysogenum  transformants with the plasmid pSKGSu selected for their sulphonamide resistance, analyses were made for the presence of the plasmid by the Southern blot technique and the existence of a transcript corresponding to the lacZ gene by the Northern blot technique. The β-galactosidase enzyme activity was then measured in the transformants which were positive in the two preceding analyses. The transformants efficiently expressed the lacZ gene of  E. coli , and it was observed that β-galactosidase activity levels were higher in those which contained a copy of the plasmid integrated into their genome than in single-copy transformants which expressed the lacZ gene under the control of the tryptophan C gene promoter (trpC). 
     The plasmid pSKG was introduced into  E. coli  DH5α (ΔlacZ) with the aim of finding out whether the Pgdh of  P. chrysogenum  was also able to direct the expression of the lacZ gene in  E. coli . The transformants obtained had the ability to generate blue-coloured colonies after 10 days of incubation at 25° C. in LB medium to which isopropyl-β- D -galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β- D -galactoside (X-gal) had been added. This result confirmed that the Pgdh-lacZ construct expresses the β-galactosidase enzyme activity in  E. coli , although less efficiently than the endogenous lacz gene of  E. coli.    
     1.2.2. Expression of the ble R  Gene of  S. hindustanus  in  P. chrysogenum  and  A. chrysogenum  Under the Pgdh 
     The ble R  gene without its promoter region was obtained from the plasmid pUT737 (Mullaney et al. (1985), Mol. Gen. Genet. 199: 37-45) as a 1,100 bp NcoI-ApaI fragment. This fragment was then subcloned in the plasmid pUT713 previously digested with NcoI-ApaI, giving the plasmid pALfleo5. The Pgdh was recovered from pALP25 as a 726 bp EcoRI-BamHI fragment, which was then subcloned in pALfleo5 (previously digested with EcoRI-BamHI) to generate pALfleo6. This last plasmid has a size of 4.2 kb, the ble R  gene expressed under the control of the Pgdh and the ampicillin resistance gene as marker in  E. coli . With the aim of replacing the latter marker with the chloramphenicol resistance gene, a 1,900 bp EcoRI-NotI fragment which included the Pgdh, ble R  and the terminator of the trpC gene (TtrpC) was purified from pALfleo6 and ligated to the plasmid pBC KS (+) (Stratagene) digested with EcoRI-NotI. This resulted in the plasmid pALfleo7 (FIG.  5 ), which has a size of 5.4 kb and carries the ble R  gene of  S. hindustanus  under the Pgdh as selection marker in fungi, the chloramphenicol resistance gene as marker in  E. coli  and the polylinker of the plasmid pBC KS (+). The sequencing of the fusion region between Pgdh and ble R  confirmed the arrangement of the latter gene in the correct reading frame. 
     Transformations of  P. chrysogenum  and  A. chrysogenum  were carried out with the plasmid pALfleo7, the transformants being selected on the basis of their resistance to 30 μg/ml and 10 μg/ml of phleomycin, respectively. The maximum level of phleomycin resistance of the transformants was then established in a solid medium, some being obtained that were capable of growing in the presence of more than 100 μg/ml of phleomycin. In the transformants selected for their phleomycin resistance, analyses were made for the presence of the plasmid by the Southern blot technique and the existence of a transcript corresponding to the ble R  gene by the Northern blot technique, positive results being obtained in both cases. These results confirmed the possibility of expressing heterologous genes in  P. chrysogenum  and  A. chrysogenum  under the control of the Pgdh. 
     The plasmid pALfleo7 was introduced into  E. coli  with the aim of finding out whether the Pgdh of  P. chrysogenum  was also able to direct the expression of the ble R  gene in  E. coli . The transformants obtained had the ability to grow in LB with 0.2 μg/ml of phleomycin, the minimum inhibitory concentration of the phleomycin being less than 0.025 μg/ml for  E. coli . This result confirmed that the Pgdh was expressed in  E. coli , although less efficiently than in  P. chrysogenum . A transformant of  E. coli  DH5α with the plasmid pALfleo7 has been deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT4849. Other plasmids such as pALP784 and pALP785 can be obtained from the deposited plasmid simply by selecting the 2.9 kb Sau3AI-XbaI fragment by hybridization with the promoter of the gdh gene included in pALfleo7, and subcloning it in pBluescript I KS(+) or pUC13, respectively. 
     1.3. Antisense Expression in  P. chrysogenum  and  A. chrysogenum  Under the gdh Promoter 
     The inactivation of gene expression in industrial strains is sometimes necessary for the elimination of undesirable enzyme activities. Owing to the fact that the level of ploidy of many industrial strains makes it difficult in most cases to block expression by direct gene disruption, it is necessary to use systems for inactivation of expression which are independent of the level of ploidy. The development of antisense constructs expressed under the control of strong promoters makes interruption of gene expression possible. 
     By way of example, the use of the Pgdh to inactivate the expression of the gene which codes for phenylacetate 2-hydroxylase (pahA) in  P. chrysogenum  is described below. First of all the plasmid pALP873, which carries the Pgdh and the TtrpC fused via a single BamHI site, was constructed. The plasmid pALP873 was digested with BamHI, its ends were filled in with the Klenow fragment of DNA polymerase I and it was ligated with a 1,053 bp cDNA fragment inside the pahA gene obtained from the plasmid pALP555 by EcoRV digestion. The resultant plasmid, called pALP874, was selected because it carried the antisense pahA gene fragment relative to the Pgdh. From this plasmid a 2.5 kb EcoRI-XbaI fragment, carrying the antisense cassette, which was filled in with Klenow and subcloned at the EcoRV site of the plasmid pALfleo7, giving rise to the plasmid pALP888, was purified. This last plasmid is characterized by having a size of 7.9 kb and carrying (I) the antisense cassette of the pahA gene under the control of the Pgdh, (II) the ble R  gene as selection marker in fungi, (III) the chloramphenicol resistance gene as marker in  E. coli  and (IV) the polylinker of the plasmid pBC KS (+). 
     Transformations of  P. chrysogenum  were performed with the plasmid pALP888, the transformants being selected on the basis of their resistance to 30 μg/ml of phleomycin. Of the transformants selected, about 20% showed reduced ability to oxidize phenylacetic acid, with some of them lacking detectable levels of said activity. In these transformants, analyses were made for the presence of the plasmid by the Southern blot technique and the existence of an antisense transcript corresponding to the pahA gene by the Northern blot technique, using an oligonucleotide corresponding to the coding strand as a probe. In both cases positive results were obtained, confirming the possibility of totally or partially blocking undesirable enzyme activities in  P. chrysogenum  by the use of antisense constructs. These results can be extrapolated to related filamentous fungi and to any enzyme activity, using any of the promoters described in the present patent (Pgdh, Phex, PactPc and PactAc) or any available promoter. 
     EXAMPLE 2 
     2.1. Cloning and Characterization of the hex Gene of  P. chrysogenum    
     The presence of a major protein which after purification and characterization was found to be the enzyme β- N -acetylhexosaminidase was determined in the  P. chrysogenum  mycelium obtained from industrial fermentation under conditions of penicillin G production. The amino acid sequence of the amino terminal end of the purified protein was determined by Edman&#39;s degradation method, two different sequences being obtained: 
     (A) Ala-Pro-Ser-Gly-Ile-His-Asn-Val-Asp-Val-(His)-Val-Val-(Asp)-Asn-(Asp)-Ala-(Asp)-Leu-Gln-Tyr-(Gly)(SEQ ID NO:9) 
     (B) Val-Gln-Val-Asn-Pro-Leu-Pro-Ala-Pro-(Arg)-(Arg)-Ile-(Thr)-???-(Gly)-(Ser)-(Ser)-(Gly)-(Pro)-(Ile/Thr)-???-(Val)(SEQ ID NO:10) 
     On the basis of these sequences, and assuming the codon usage trend which exists in a series of  P. chrysogenum  genes, the following combinations of synthetic oligonucleotides were designed: 
     
       
         5′ TCGACGACGTGSACGTCSACGTTGTGGATGCC 3′ (SEQ ID NO: 11)  (I) 
       
     
     
       
         5′ CCGTAYTGSAGGTCRGCGTCGTTGTCGACGAC 3′ (SEQ ID NO: 12)  (II) 
       
     
     
       
         5′ GGGGCVGGSAGVGGGTTGACYTG 3′ (SEQ ID NO: 13)  (III) 
       
     
     The hex gene of  P. chrysogenum  was cloned using the DNA library and the procedures described in Example 1. A total of 11 positive clones were purified and their DNA was then digested with a series of restriction endonucleases and analysed by the Southern blot technique. In this way the hex gene was identified in a 3.2 kb SacI fragment and in a 2.1 kb SalI fragment. Subcloning of the SalI fragment in the plasmid pBC KS(+) (Stratagene) in both orientations generated the plasmids pALP295 and pALP303. The restriction map of the DNA region which includes the hex gene is shown in FIG.  2 . 
     In order to determine the nucleotide sequence of the hex gene, use was made of the above-mentioned plasmids pALP295 and pALP303, as well as pALP319 and pALP461 (both orientations of a 2.8 kb BamHI fragment), pALP388 and pALP389 (both orientations of a 2.4 kb SalI fragment) and pALP377 and pALP378 (both orientations of a 1.2 kb PstI fragment) (FIG.  2 ). A series of clones were constructed from the said plasmids by the “Erase a base” method (Promega) and then sequenced by the dideoxynucleotide method using the “Sequenase” test kit (USB), in both cases in accordance with the manufacturer&#39;s instructions. The 5,240 nucleotide sequence obtained (SEQ ID NO:2) was analysed with the Geneplot program (DNASTAR), confirming the existence of two ORFs with a very marked preferential codon usage pattern. The ATG translation initiation codon of the hex gene was found in position 1,324 and the TGA termination codon in position 3,112. The said ORF lacks introns and codes for a protein of 66,545 Da, with an isoelectric point of 5.34, the 596 amino acid sequence of which (SEQ ID NO:6) has 49.0% identity with the amino acid sequence of the β- N -acetylhexosaminidase enzyme of  Candida albicans . In addition, in positions 19-40 and 99-120 the deduced amino acid sequence includes the amino acid sequences determined chemically from the purified enzyme. A protease recognition site (Lys-Arg) appears in the positions immediately adjacent to the amino acid sequence (A) described above (amino acids 97-98). 
     In the promoter region there are found two pyrimidine-rich zones between positions 1,106-1,128 and 1,182-1,200, a presumed TATA box in position 1,258 (ATAAATA) and a CAAT box in position 1,163. 
     2.2. Expression of the ble R  gene of  S. hindustanus  in  P. chrysogenum  Under the Phex 
     The processes of (I) transformation and selection of  P. chrysogenum  transformants, (II) analysis of DNA, (III) analysis of RNA and (IV) enzyme measurements were carried out as described in Section 1.2 of Example 1. 
     In order to express the ble R  gene under the Phex, first of all an NcoI site was constructed above the ATG codon which codes for the initiator methionine of the hex gene. This was carried out by PCR using the following oligonucleotides as primers: 
     
       
         5′ CTCCATGGTGATAAGGTGAGTGACGATG 3(SEQ ID NO:14) 
       
     
     
       
         5′ GTAAAACGACGGCCAGTG 3′ (Primer −20)  (SEQ ID NO:15) 
       
     
     The DNA fragment obtained by PCR was subcloned in both orientations in the SmaI site of the plasmid pBC KS (+) (Stratagene), giving rise to pALP427 and pALP428. The inserts of both plasmids were sequenced using the test kits “Erase a base” (Promega) and “Sequenase” (USB), in both cases in accordance with the manufacturer&#39;s instructions. In this way it was shown that the Phex obtained lacked mutations and included the NcoI site above the ATG which codes for the initiator methionine of the protein. 
     pALP427 was the plasmid chosen for carrying out the subcloning of the ble R  gene. The ble R  gene without its promoter region was obtained from the plasmid pUT737 (Mullaney et al. (1985), Mol. Gen. Genet. 199: 37-45) as a 1,100 bp NcoI-ApaI fragment. This fragment was then subcloned in the plasmid pALP427 (carrying the Phex) previously digested with NcoI-ApaI, giving the plasmid pALP480 (FIG.  6 ). This last plasmid has a size of 5.4 kb, the ble R  gene expressed under the control of the Phex, the terminator of the trpC gene under the ble R  gene, the chloramphenicol resistance gene as marker in  E. coli  and the polylinker of the plasmid pBC KS (+). The sequencing of the fusion region between Phex and ble R  confirmed the arrangement of the latter gene in the correct reading frame. 
     Transformations of  P. chrysogenum  were performed with the plasmid pALP480, the transformants being selected on the basis of their resistance to 30 μg/ml of phleomycin. The maximum level of phleomycin resistance of the transformants was then established in a solid medium, some being obtained that were capable of growing in the presence of more than 100 μg/ml of phleomycin. In the transformants selected for their phleomycin resistance, analyses were made for the presence of the plasmid by the Southern blot technique and the existence of a transcript corresponding to the ble R  gene by the Northern blot technique, positive results being obtained in both cases. These results confirmed the possibility of expressing heterologous genes in  P. chrysogenum  under the control of the Phex. A transformant of  E. coli  DH5α with the plasmid pALP480 has been deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT4852. The plasmids pALP295, pALP319, pALP377 and pALP388 can be obtained from the deposited plasmid simply by selecting the DNA fragments 2.1 kb SalI, 2.8 kb BamHI, 1.2 kb PstI and 2.4 kb SalI, respectively, by hybridization with the promoter of the hex gene included in pALP480, and then subcloning them in pBluescript I KS(+). 
     2.3. Extracellular Production of Proteins in  P. chrysogenum  Using the hex Gene 
     The enzyme β- N -acetylhexosaminidase is a protein which is abundantly secreted by  P. chrysogenum  to the culture medium in industrial fermenters under conditions of penicillin G production. The ability of this enzyme to be secreted makes it possible to use the hex gene for the expression and secretion of homologous or heterologous proteins in  P. chrysogenum  or related filamentous fungi. 
     The enzyme has a secretion signal sequence made up of the following amino acids: Met-Lys-Phe-Ala-Ser-Val-Leu-Asn-Val-Leu-Gly-Ala-Leu-Thr-Ala-Ala-Ser-Ala (amino acids 1 to 18 of SEQ ID NO: 6). In general, signal peptides have three conserved structural domains (Takizawa, N. et al. (1994) Recombinant microbes for industrial and agricultural applications, Murooka, Y. and Imanaka, T. (eds), Marcel Dekker, Inc. New York) (I) a positively charged amino terminal region called “n”, which usually has from 1‥5 residues and is needed for the efficient translocation of the protein across the membrane (Met-Lys), (II) a hydrophobic region called “h”, made up of 7 to 15 residues (Phe-Ala-Ser-Val-Leu-Asn-Val-Leu) (amino acids 3 to 10 of SEQ ID NO: 6) and (III) a polar region at the carboxyl end, called “c”, made up of 3 to 7 residues (Gly-Ala-Leu-Thr-Ala-Ala-Ser-Ala) (amino acids 11 to 18 of SEQ ID NO: 6). 
     There are two possibilities when it comes to expressing and secreting proteins using the hex gene: (I) fusing the promoter region, including the secretion signal sequence, to the coding region of the gene to be expressed, in a reading frame, and (II) fusing the complete hex gene to the coding region of the gene to be expressed, in a reading frame. Using standard techniques of molecular biology (Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA; Ausubel et al. (1987), Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, USA), any person skilled in the art would be able to use the promoter, including the secretion sequence of the hex gene, or else the complete gene, for the expression and secretion of proteins of interest in  P. chrysogenum  or related filamentous fungi. 
     EXAMPLE 3 
     3.1. Cloning and Characterization of the act Gene of  P. chrysogenum    
     The act gene of  P. chrysogenum  was cloned using the DNA library and the procedures described in Example 1. In this case the hybridization was performed with an 888 bp NcoI-ClaI fragment originating from the act gene of  A. nidulans  (Fidel et al. (1988), Gene 70: 283-293). A total of 10 positive clones were purified and their DNA was then digested with a series of restriction endonucleases and analysed by the Southern blot technique. In this way the act gene was identified in a 5.2 kb BamHI fragment, a 4.9 kb EcoRI fragment and a 5.9 kb HindIII fragment. The HindIII fragment was subcloned in both orientations in the plasmid pBluescript I KS(+) (Stratagene), generating the plasmids pALP298 and pALP299. The subcloning of the EcoRI fragment in the plasmid pBluescript I KS(+) (Stratagene) in both orientations generated the plasmids pALP315 and pALP316. The restriction map of the DNA region which includes the act gene is shown in FIG.  3 . 
     In order to determine the nucleotide sequence of the act gene, use was made of the above-mentioned plasmids pALP315 and pALP316. A series of clones were constructed from the said plasmids by the “Erase a base” method (Promega) and then sequenced by the dideoxynucleotide method using the “Sequenase” test kit (USB), in both cases in accordance with the manufacturer&#39;s instructions. The 2,994 nucleotide sequence obtained (SEQ ID NO:3) was analysed with the Geneplot program (DNASTAR), confirming the existence of an ORF with a very marked preferential codon usage pattern. The ATG translation initiation codon of the act gene was found in position 494 and the TAA termination codon in position 2,250. Said ORF has 5 introns in positions 501-616, 649-845, 905-1046, 1078-1180 and 1953-2021 and codes for a protein of 41,760 Da, with an isoelectric point of 5.51, the 375 amino acid sequence of which (SEQ ID NO:7) has 98.1% identity with the amino acid sequence of the γ-actin protein of  A. nidulans . In the promoter region there are found two extensive pyrimidine-rich zones between positions 356-404 and 418-469, a presumed TATA box in position 259 (TATAAAAAT) and four CAAT boxes in positions 174, 217, 230 and 337. 
     3.2. Expression of the ble R  Gene in  P. chrysogenum  Under the PactPc 
     In order to express the ble R  gene under the PactPc, first of all an NcoI site was constructed above the ATG codon which codes for the initiator methionine of the hex gene. This was carried out by PCR using the following oligonucleotides as primers: 
     
       
         5′ CTCCATGGTGACTGATTAAACAAGGGAC 3′  (SEQ ID NO:19) 
       
     
     
       
         5′ GTAAAACGACGGCCAGTG 3′ (Primer −20)  (SEQ ID NO:20) 
       
     
     The DNA fragment obtained by PCR was subcloned in both orientations in the SmaI site of the plasmid pBC KS (+) (Stratagene), giving rise to pALPact1 and pALPact2. The inserts of both plasmids were sequenced using the test kits “Erase a base” (Promega) and “Sequenase” (USB), in both cases in accordance with the manufacturer&#39;s instructions. In this way it was shown that the PactPc obtained lacked mutations and included the NcoI site above the ATG. which codes for the initiator methionine of the protein. 
     pALPact1 was the plasmid chosen for carrying out the subcloning of the ble R  gene. The ble R  gene without its promoter region was obtained from the plasmid pUT737 (Mullaney et al. (1985), Mol. Gen. Genet. 199: 37-45) as a 1,100 bp NcoI-ApaI fragment. This fragment was then subcloned in the plasmid pALPact1 (carrying the PactPc) previously digested with NcoI-ApaI, giving the plasmid pALPfleo1 (FIG.  6 ). This last plasmid has the ble R  gene expressed under the control of the PactPc, the terminator of the trpC gene under the ble R  gene, the chloramphenicol resistance gene as marker in  E. coli  and the polylinker of the plasmid pBC KS (+). The sequencing of the fusion region between PactPc and ble R  confirmed the arrangement of the latter gene in the correct reading frame. 
     Transformations of  P. chrysogenum  were performed with the plasmid pALPfleo1, the transformants being selected on the basis of their resistance to 30 μg/ml of phleomycin. The maximum level of phleomycin resistance of the transformants was then established in a solid medium, some being obtained that were capable of growing in the presence of more than 100 μg/ml of phleomycin. In the transformants selected for their phleomycin resistance, analyses were made for the presence of the plasmid by the Southern blot technique and the existence of a transcript corresponding to the ble R  gene by the Northern blot technique, positive results being obtained in both cases. These results confirmed the possibility of expressing heterologous genes in  P. chrysogenum  under the control of the PactPc. A transformant of  E. coli  DH5α with the plasmid pALP315, which carries the act gene, has been deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT4851. The plasmid pALP316 can be obtained from the deposited plasmid pALP315 simply by subcloning the pALP315 insert in the EcoRI site of pBluescript I KS(+) in the opposite orientation. 
     EXAMPLE 4 
     4.1. Cloning and Characterization of the act Gene of  A. chrysogenum    
     With the aim of cloning the gdh gene of  A. chrysogenum , a DNA library was constructed in the phage vector λGEM12, as described in Section 1.1 of Example 1. The phage titre obtained was 50 pfu/μl (a total of 25,000 pfu) in  E. coli  LE392 and 41 pfu/μl (a total of 20,500 pfu) in  E. coli  NM539. This meant that about 82% of the phages were carrying an exogenous DNA fragment and that an  A. chrysogenum  DNA library had been obtained with 99.999% probability. After this series of theoretical verifications had been carried out,  E. coli  NM539 was infected and the complete DNA library was spread on 3 Petri dishes of 150 mm diameter (about 7,000 pfu/Petri dish), collected in 50 ml of SM plus 2.5 ml of chloroform, and kept at 4° C. In this way a sufficient and representative volume of recombinant phages (2,100 pfu/μl) ready to be plated out at any time was available. 
     About 20,000 pfu were spread on 2 Petri dishes of 150 mm diameter and then transferred to nitrocellulose filters (BA85, 0.45 μm, Schleicher &amp; Schuell) said filters were hybridized using standard protocols (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA) with an 888 bp NcoI-ClaI fragment corresponding to the act gene of  A. nidulans . A total of 5 positive clones were purified and their DNA was then digested with a series of restriction endonucleases and analysed by the Southern blot technique. In this way the act gene was identified in an 8.7 kb HindIII fragment. This fragment was subcloned in both orientations in the plasmid pBluescript I KS(+) (Stratagene), generating the plasmids pALC52 and pALC53. The restriction map of the DNA region which includes the act gene is shown in FIG.  4 . 
     The above-mentioned plasmids pALC52 and pALC53 were used to determine the nucleotide sequence of the act gene. A series of clones were constructed from the said plasmids by the “Erase a base” method (Promega) and then sequenced by the dideoxynucleotide method using the “Sequenase” test kit (USB), in both cases in accordance with the manufacturer&#39;s instructions. The 3,240 nucleotide sequence obtained (SEQ ID NO:4) was analysed with the Geneplot program (DNASTAR), confirming the existence of an ORF with a very marked preferential codon usage pattern. The ATG translation initiation codon of the act gene was found in position 787 and the TAA termination codon in position 2,478. Said ORF has 5 introns in positions 794-920, 952-1,123, 1,180-1,289, 1,321-1,410 and 2,183-2,249 and codes for a protein of 41,612 Da, with an isoelectric point of 5.51, the 375 amino acid sequence of which (SEQ ID NO:8) has 98.4% and 98.1% identity with the amino acid sequences of the γ-actin proteins of  A. nidulans  and  P. chrysogenum , respectively. In the promoter region there is found a pyrimidine-rich zone between positions 607-654, a presumed TATA box in position 747 (TTATAAAA) and a CAAT box in position 338. 
     4.2. Expression of the ble R  Gene in  A. chrysogenum  Under the PactAc 
     The plasmid pALCfleo1 (FIG.  6 ), which includes the ble R  gene expressed under the control of the PactAc, the terminator of the trpC gene under the ble R  gene, the chloramphenicol resistance gene as marker in  E. coli  and the polylinker of the plasmid pBC KS (+), was constructed for the purpose of expressing the ble R  gene under the control of the PactAc. 
     The ble R  gene without its promoter region was obtained from the plasmid pUT737 (Mullaney et al. (1985), Mol. Gen. Genet. 199: 37-45) as a 1,100 bp NcoI-ApaI fragment. This fragment was then fused in a reading frame with the PactAc, making use of the fact that the act gene has an NcoI site above the ATG which codes for the initiator methionine of the protein. To this end the ble R  gene was subcloned in the plasmid pALCact1 (carrying the PactAc) previously digested with NcoI-ApaI, giving the plasmid pALCfleo1 (FIG.  6 ). The sequencing of the fusion region between PactAc and ble R  confirmed the arrangement of the latter gene in the correct reading frame. 
     Transformations of  A. chrysogenum  were performed with the plasmid pALCfleo1, the transformants being selected on the basis of their resistance to 10 μg/ml of phleomycin. The maximum level of phleomycin resistance of the transformants was then established in a solid medium, some being obtained that were capable of growing in the presence of more than 30 μg/ml of phleomycin. In the transformants selected for their phleomycin resistance, analyses were made for the presence of the plasmid by the Southern blot technique and the existence of a transcript corresponding to the ble R  gene by the Northern blot technique, positive results being obtained in both cases. These results confirmed the possibility of expressing heterologous genes in  A. chrysogenum  under the control of the PactAc. A transformant of  E. coli  DH5α with the plasmid pALC52, which carries the act gene, has been deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT4850. The plasmid pALC53 can be obtained from the deposited plasmid pALC52 simply by subcloning the pALC52 insert in the HindIII site of pBluescript I KS(+) in the opposite orientation. 
     The introduction, in actinomycetes, Penicillium, Aspergillus, Acremomium or Saccharomyces, of the inserts present in the deposited plasmids using  E. coli  as host is only a question of technical routine and of choosing the most appropriate vectors for the transformation of said genera or families. 
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG.  1 .—Restriction map of the gdh gene of  P. chrysogenum , which codes for NADP-dependent glutamate dehydrogenase enzyme activity (EC.1.4.1.4). 
     FIG.  2 .—Restriction map of the hex gene of  P. chrysogenum , which codes for β- N -acetylhexosaminidase enzyme activity (EC.3.2.1.52). 
     FIG.  3 .—Restriction map of the act gene of  P. chrysogenum , which codes for γ-actin. 
     FIG.  4 .—Restriction map of the act gene of  A. chrysogenum , which codes for γ-actin. 
     FIG.  5 .—Vectors for the expression of the lacZ gene of  E. coli , the ble R  gene of  S. hindustanus  and the antisense fragment of the pahA gene of  P. chrysogenum  in  P. chrysogenum  and/or  A. chrysogenum  under the promoter Pgdh. 
     FIG.  6 .—Vectors for the expression of the ble R  gene of  S. hindustanus  in  P. chrysogenum  and/or  A. chrysogenum  under the promoters Phex, PactPc, PactAc. 
     
       
         
           
             20 
           
           
             
               2816 base pairs 
               nucleotides 
               double 
               linear 
             
             
               genomic DNA 
             
             NO 
             NO 
             
               Penicillim chrysogenum 
             
             
               plasmids pALP784 and pALP 785 
             
             
               coding sequence 
                join (922...970, 1131...1261, 1319   2521)
 
             
             
               intron 
                971...1130
 
             
             
               intron 
                1262...1318
 
             
             
                gdh gene
 
             
              1
GATCGCCGTT TATGGGATAG TGGGCACGTG ACAGAGCCTG CAGCCGAGTC AAATTGCCGA     60
AGTTGGCAGT TGGTGGCGGA GAACTCGAGA TTTTATTTGC GTTTATTTCG TTTATTTCGA    120
TTTTAGTTTT CCTATTTTTC CTATTTTGGT TGATTCCATC CAACTTTATA GGATACTACT    180
TCATAATAGG TCGATCATAG TACAAGCACC AACTCGTCGC ATCATGCATT TTCTGGGGTT    240
CGAATTCTTT ACTTAGAGTA AGGTTTCTCT CAGCCTCCTA ATAAACTACC TAGGTAGGTT    300
AAATTTACTT TTTAACATTT TATTTATTCA GAAGATTGTC GGAGAGGACC GATCCGAAGG    360
ACACGAATTG AACACGGAAG GGATATTAGG GACAAGGAAG ATTTAGGGAT AAAAAAACGA    420
GCTGTGATTG ATGGGAAGGT TAAAGTGTAG TAATGAAGGT GATGGGACCA AAAGGAGTGG    480
GAGAGATAAG CCAAATTCTG TGCAAATTCT GTGACCTTAA ACCATAAGAT AACATTGTTC    540
GGGCCCCGAA CTTCGGACGT TCTTCCCACG GAAAGGCAAA TCATTGGGTT TCATCGATTC    600
TCTTGGATCT TTATCCTAAT TCCCCGTGCA ACCTGGTCTT GGGGATTATT GTCGACTTGT    660
AGGCGCATTA ACCCATCTCC CGTCTTCCCT CCAATCAATC CCGGATTCTC TCGTCCGACT    720
CCGGCTTCGA CTCTCTCTCT CTCCACATCT CTATATAATT GTACACTCCC CCATCCCATT    780
CTTTTCTTCT CTTCTCATCT ACTCTCTTGA ATCTCAATTG TCTTAATACT CTCTCTGCTC    840
TTGTCTTTAT TTATAATTTA TTAGATCACT GCTTAGCATT GATCTACTTA CCTAAAAGCA    900
GAGTTAACAG TACCGGCCGA A ATG ATG CAA AAC CTT CCC TTC GAG CCT GAG      951
                        Met Met Gln Asn Leu Pro Phe Glu Pro Glu
                        1               5                   10
TTC GAG CAG GCC TAC AAG G GTATGTCTCT TTTAATTTTT CCCTTTCTTA TTTCAA   1006
Phe Glu Gln Ala Tyr Lys
                15
TTCCATATCG TCCATATCAC ACACTATTTC CCGACTCAAT TCCTTTACCC ATCGGCATCT   1066
TCCCGGCCTT TGGCTCCACC GGGGGCATAA TTTCGGGGTG ACTCAGCTAA CAATCCCGAA   1126
ACAG  AG CTC GCC TCC ACT CTC GAG AAC TCC ACT CTT TTC CAG AAG AAG    1174
     Glu Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Gln Lys Lys
                 20                  25                  30
CCC GAG TAC CGC AAG GCT CTT CAG GTC GTC TCT GTC CCC GAG CGT GTT     1222
Pro Glu Tyr Arg Lys Ala Leu Gln Val Val Ser Val Pro Glu Arg Val
            35                  40                  45
ATT CAG TTC CGT GTT GTC TGG GAA GAT GAC AAA GGC CAG GTAAGACCTT      1271
Ile Gln Phe Arg Val Val Trp Glu Asp Asp Lys Gly Gln
        50                  55                  60
TCTTTTTGAA AATGTCTAAT TAATTGCCAC ATGCTAATTC CGTTCAG GTC CAA ATC     1327
                                                    Val Gln Ile
AAC CGT GGA TAC CGT GTC CAG TTC AAC TCC GCT CTT GGC CCC TAC AAG     1375
Asn Arg Gly Tyr Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys
    65                  70                  75
GGT GGC CTC CGG TTC CAC CCC ACG GTG AAC CTT TCC ATC CTC AAG TTC     1423
Gly Gly Leu Arg Phe His Pro Thr Val Asn Leu Ser Ile Leu Lys Phe
80                  85                  90                  95
CTC GGT TTC GAG CAG ATC TTC AAG AAT GCC CTC ACC GGC CTG AAC ATG     1471
Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Asn Met
                100                 105                 110
GGC GGT GGT AAG GGT GGA TCC GAC TTC GAC CCC AAG GGC AAG ACC GAT     1519
Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Thr Asp
            115                 120                 125
AAC GAG ATC CGC CGC TTC TGT GTC TCC TTC ATG ACC GAG CTG TGC AAG     1567
Asn Glu Ile Arg Arg Phe Cys Val Ser Phe Met Thr Glu Leu Cys Lys
        130                 135                 140
CAC ATC GGT GCC GAC ACC GAT GTT CCC GCC GGT GAT ATC GGT GTG ACC     1615
His Ile Gly Ala Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Thr
    145                 150                 155
GGC CGC GAG GTT GGT TTC ATG TTC GGC CAG TAC AAG AAG ATC CGC AAC     1663
Gly Arg Glu Val Gly Phe Met Phe Gly Gln Tyr Lys Lys Ile Arg Asn
160                 165                 170                 175
CAG TGG GAG GGT GTC CTC ACC GGT AAG GGT GGC AGC TGG GGT GGT TCC     1711
Gln Trp Glu Gly Val Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser
                180                 185                 190
CTC ATC CGC CCC GAG GCC ACC GGC TAC GGT GTC GTC TAC TAC GTC GAG     1759
Leu Ile Arg Pro Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Glu
            195                 200                 205
CAC ATG ATC CAG CAC GCC TCC GGC GGC AAG GAA TCC TTC GCT GGT AAG     1807
His Met Ile Gln His Ala Ser Gly Gly Lys Glu Ser Phe Ala Gly Lys
        210                 215                 220
CGC GTC GCC ATC TCC GGT TCC GGA AAC GTC GCC CAG TAC GCC GCT CTC     1855
Arg Val Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu
    225                 230                 235
AAG GTC ATC GAG CTC GGT GGC TCC GTC ATC TCC CTC TCC GAC TCC CAG     1903
Lys Val Ile Glu Leu Gly Gly Ser Val Ile Ser Leu Ser Asp Ser Gln
240                 245                 250                 255
GGT GCT CTC GTC CTG AAC GGC GAG GAG GGC TCC TTC ACC GCT GAG GAG     1951
Gly Ala Leu Val Leu Asn Gly Glu Glu Gly Ser Phe Thr Ala Glu Glu
                260                 265                 270
ATC AAC ACC ATC GCT GAG ATC AAG GTC CAG CGC AAG CAG ATC GCC GAG     1999
Ile Asn Thr Ile Ala Glu Ile Lys Val Gln Arg Lys Gln Ile Ala Glu
            275                 280                 285
CTC GCT ACC CAG GAC GCC TTC AGC TCC AAG TTC AAG TAC ATC CCC GGT     2047
Leu Ala Thr Gln Asp Ala Phe Ser Ser Lys Phe Lys Tyr Ile Pro Gly
        290                 295                 300
GCC CGC CCC TGG ACC AAC ATC GCC GGC CGC ATC GAT GTC GCT CTC CCC     2095
Ala Arg Pro Trp Thr Asn Ile Ala Gly Arg Ile Asp Val Ala Leu Pro
    305                 310                 315
TCC GCC ACC CAG AAC GAG GTC TCC GGC GAT GAG GCC AAG GCT CTC ATC     2143
Ser Ala Thr Gln Asn Glu Val Ser Gly Asp Glu Ala Lys Ala Leu Ile
320                 325                 330                 335
GCC GCT GGC TGC AAG TTC ATC GCT GAG GGC TCC AAC ATG GGT TCC ACC     2191
Ala Ala Gly Cys Lys Phe Ile Ala Glu Gly Ser Asn Met Gly Ser Thr
                340                 345                 350
CAG GAG GCT ATC GAT GTC TTC GAG GCC CAC CGT GAT GCC AAC CCT GGT     2239
Gln Glu Ala Ile Asp Val Phe Glu Ala His Arg Asp Ala Asn Pro Gly
            355                 360                 365
GCC GCT GCC ATC TGG TAC GCC CCT GGT AAG GCC GCC AAC GCT GGT GGT     2287
Ala Ala Ala Ile Trp Tyr Ala Pro Gly Lys Ala Ala Asn Ala Gly Gly
        370                 375                 380
GTT GCC GTC TCT GGT CTC GAG ATG GCC CAG AAC TCT GCC CGT GTC AAC     2335
Val Ala Val Ser Gly Leu Glu Met Ala Gln Asn Ser Ala Arg Val Asn
    385                 390                 395
TGG TCC CGT GAG GAG GTT GAC TCC CGT CTT AAG AAG ATT ATG GAG GAC     2383
Trp Ser Arg Glu Glu Val Asp Ser Arg Leu Lys Lys Ile Met Glu Asp
400                 405                 410                 415
TGC TTC AAC AAC GGT CTC TCT ACT GCT AAG GAG TAT GTC ACC CCT GCT     2431
Cys Phe Asn Asn Gly Leu Ser Thr Ala Lys Glu Tyr Val Thr Pro Ala
                420                 425                 430
GAG GGT GTT CTT CCT TCC CTC GTG GCT GGC TCC AAC ATT GCT GGT TTC     2479
Glu Gly Val Leu Pro Ser Leu Val Ala Gly Ser Asn Ile Ala Gly Phe
            435                 440                 445
ACC AAG GTC GCT GAG GCC ATG AAG GAG CAC GGT GAC TGG TGG TAAATTA     2531
Thr Lys Val Ala Glu Ala Met Lys Glu His Gly Asp Trp Trp
        450                 455                 460
GCATCCCCAT TTATTCTGGG AGGTGTTCTG TGACGATTTC TGTCCTCTCT TAAGGAGAGG   2591
CAGCTTTGAT GCATTTTCTT TTCATTTAAA TAGCTTTTTA CCCTTTTTGT CAAGCGGGTT   2651
ACGGATAGAG GCGCTTGGTT TTCTCCACTG TTGCATTCGA TTGATATCCC CACTTGAGCA   2711
CCGCTGTTTG TTTTGGTTCT GCACTTGGGA CTGTCATGAT GATAATGAGA TACAATGAAT   2771
AACTTAAAAA TAATTGTGTG GTCTCGTAAA GTTGTAAACT CTAGA                   2816
 
           
           
             
               5240 base pairs 
               nucleotides 
               double 
               linear 
             
             
               genomic DNA 
             
             NO 
             NO 
             
               Penicillum chrysogenum 
             
             
               plasmids pALP295 and pALP 388 
             
             
               coding sequence 
                1324    3111
 
             
             
                hex gene
 
             
              2
GTCGACCTCG CAACAGTCGA GAAGCACGCC GCCTATCTCG CCCGCAGCGG GGTAACCGGC     60
CTAGTAACCC AGGGTAGCAA TGGCGAAGCC GTCCACCTAG ACCGGGAAGA ACGCAAGGCC    120
ATCACAGCCG CCACACGCCG CGCCGTGGAC GCAGCCGGCT ACAGCAACAT GCCGGTGATT    180
GCCGGCTGTG GCGCCGCCTC AACCCGTGAG ACCATCCAAT TCTGCCAGGA CTCCGGTGCA    240
GCAGGCGCCG ACGCTGTCCT CGTGCTCCCA CCCAGCTACT ACAAGTCCCT CGTGAGCACC    300
GAGTCCATGC ACGCCCACTT CCGGGCTGTG GCCGATGCCT CGCCCGTCCC TGTCCTCATC    360
TACAACTTCC CCGGCGTGCA GTCCGGCCTC GATCTCAGCT CAGATGATAT CTTAACTCTC    420
GCAGAACACC CCAATATCAT CGGCTGTAAG CTCACGTGCG GCAACACGGG TAAGTTGGCT    480
CGTGTTGCGG CGGCCAAGCC GGATTTCTTG ACTTTTGGTG GCTCGGCCGA TTTCACGCTG    540
CAGACGCTGG TTGTTGGTGG GGCGGGGATT ATCGGTGGCG TGGCTAACAT GATTCCTCGC    600
TCGTGTGTGC GTCTGATGGA GTTGTATCGT GCTGGGAAGG TTCAGGAGGC GCAGAAGGTG    660
CAGGCTATTG TTGCGCGCGC TGACTGGGCT GCTATCCATG GTGGCTTTAT CGCTGTTAAG    720
ACGGGCCTCC AAGCCTACCA CGGTTACGGT GGTCTTCCTC GGCGGCCTTG TGTCGTGCCT    780
TCTGCTAAGG ATGCGGCAGC CATTCAGGAG GAGTTCCGGG AGGGAATGGA GCTGGAGAAG    840
TCGTTGGAGT CCTAATGGAT ATAGTAGATT AAATCATGAT TACCAGAGAT CCCATGTGGA    900
GATTTCTATT CCTTTCCAGG GGTTTTCCAG GGGTTTTCCA GATGTTTTCC AGGTGTTTTC    960
CAAATGTTTC AGGTTGCTTC ATAGATCGAC AGACCGGTGT GACTGTGTCA TTTGCCAGTA   1020
GATCCGGAGA TCCCGTAGCT TTCCCCCTCT TTATCTTTTA ATATTTGTTG TTATATGGGA   1080
GTTCAAGTTG CATGTAGAGG TTGCACTCTC TCTCTCTCTC TTTCCCTTGA ATTATTTGAG   1140
TCCAAGGTGT GTTAGTTGTA TGCAATGTAA CTAGGGAGCT GTTTGTTTTT CCCCTTCCCC   1200
AGGGTTGCAT CCTGGGCCAT TCCCCATTCC GATGAAAGAT CGACAATGCA GCTAAACATA   1260
AATAGTTCTG GTTATCTCCT GGCCACAGTT TCTCTACTTT TCATCGTCAC TCACCTTATC   1320
AAC ATG AAG TTC GCC TCG GTG TTG AAT GTG CTC GGG GCC CTG ACG GCT     1368
    Met Lys Phe Ala Ser Val Leu Asn Val Leu Gly Ala Leu Thr Ala
    1               5                   10                  15
GCG TCC GCC GTC CAA GTG AAT CCA CTT CCC GCC CCC CGT AAC ATC ACC     1416
Ala Ser Ala Val Gln Val Asn Pro Leu Pro Ala Pro Arg Asn Ile Thr
                20                  25                  30
TGG GGA TCC TCC GGT CCA ATC CAA GTC AAC AAC TTG AAT CTC AAC GGT     1464
Trp Gly Ser Ser Gly Pro Ile Gln Val Asn Asn Leu Asn Leu Asn Gly
            35                  40                  45
CCT CAC TCC CCT TTG CTC ACT CAA GCT TGG GAG CGA GCA TGG GAA ACC     1512
Pro His Ser Pro Leu Leu Thr Gln Ala Trp Glu Arg Ala Trp Glu Thr
        50                  55                  60
ATC ACC ACC CTG CAA TGG GTT CCT GCT GCT GTT GAA TCC CCA ATC GCC     1560
Ile Thr Thr Leu Gln Trp Val Pro Ala Ala Val Glu Ser Pro Ile Ala
    65                  70                  75
TCC TAT CCG GCC TTC CCC ACC TCG ACC CCT GTC TCC TCT GCC CCC AAG     1608
Ser Tyr Pro Ala Phe Pro Thr Ser Thr Pro Val Ser Ser Ala Pro Lys
80                  85                  90                  95
GCC AAA CGC GCG CCC TCC GGA ATC CAT AAC GTC GAT GTT CAT GTG GTG     1656
Ala Lys Arg Ala Pro Ser Gly Ile His Asn Val Asp Val His Val Val
                100                 105                 110
GAC AAC GAT GCC GAT CTC CAA TAC GGT GTG GAT GAA TCC TAT ACA CTG     1704
Asp Asn Asp Ala Asp Leu Gln Tyr Gly Val Asp Glu Ser Tyr Thr Leu
            115                 120                 125
GTA GTG AGC GAT GGT GGC ATC AGG ATC AAT TCT CAG ACG GTC TGG GGT     1752
Val Val Ser Asp Gly Gly Ile Arg Ile Asn Ser Gln Thr Val Trp Gly
        130                 135                 140
GTG TTG CAG GCA TTC ACC ACC CTG CAG CAG ATT ATC ATC TCG GAT GGG     1800
Val Leu Gln Ala Phe Thr Thr Leu Gln Gln Ile Ile Ile Ser Asp Gly
    145                 150                 155
AAG GGC GGT TTG ATC ATT GAA CAG CCC GTC AAG ATC AAG GAT GCC CCG     1848
Lys Gly Gly Leu Ile Ile Glu Gln Pro Val Lys Ile Lys Asp Ala Pro
160                 165                 170                 175
CTG TAC CCC CAT CGT GGT ATC ATG ATA GAC ACC GGG CGC AAC TTC ATT     1896
Leu Tyr Pro His Arg Gly Ile Met Ile Asp Thr Gly Arg Asn Phe Ile
                180                 185                 190
ACC GTT CGC AAG CTC CTT GAG CAG ATC GAC GGT ATG GCC CTG TCC AAG     1944
Thr Val Arg Lys Leu Leu Glu Gln Ile Asp Gly Met Ala Leu Ser Lys
            195                 200                 205
CTC AAT GTT CTC CAC TGG CAC TTG GAC GAT TCT CAG TCG TGG CCC ATG     1992
Leu Asn Val Leu His Trp His Leu Asp Asp Ser Gln Ser Trp Pro Met
        210                 215                 220
CAG ATG AGC TCC TAC CCG GAG ATG ACC AAA GAT GCT TAC TCG CCT CGC     2040
Gln Met Ser Ser Tyr Pro Glu Met Thr Lys Asp Ala Tyr Ser Pro Arg
    225                 230                 235
GAA ATC TAC ACC GAG CAC GAC ATG CGC CGC GTG ATT GCC TAC GCA CGC     2088
Glu Ile Tyr Thr Glu His Asp Met Arg Arg Val Ile Ala Tyr Ala Arg
240                 245                 250                 255
GCG CGA GGT GTC CGC GTC ATC CCC GAG GTC GAC ATG CCC GCC CAC TCA     2136
Ala Arg Gly Val Arg Val Ile Pro Glu Val Asp Met Pro Ala His Ser
                260                 265                 270
GCC TCC GGC TGG CAG CAG GTC GAC CCG GAG ATC GTG GCA TGT GCC GAA     2184
Ala Ser Gly Trp Gln Gln Val Asp Pro Glu Ile Val Ala Cys Ala Glu
            275                 280                 285
TCC TGG TGG TCG AAC GAC GTT TGG GCG GAG CAC ACC GCC GTC CAG CCG     2232
Ser Trp Trp Ser Asn Asp Val Trp Ala Glu His Thr Ala Val Gln Pro
        290                 295                 300
AAC CCT GGC CAG CTC GAC ATT ATC TAC CCC AAG ACC TAC GAA GTT GTC     2280
Asn Pro Gly Gln Leu Asp Ile Ile Tyr Pro Lys Thr Tyr Glu Val Val
    305                 310                 315
AAC AAT GTC TAC CAG GAA TTG TCT CGC ATC TTC AGC GAC AAC TTG TTC     2328
Asn Asn Val Tyr Gln Glu Leu Ser Arg Ile Phe Ser Asp Asn Leu Phe
320                 325                 330                 335
CAC GTT GGT GCA GAC GAG ATC CAG CCC AAC TGC TAC AAC TAC AGC ACC     2376
His Val Gly Ala Asp Glu Ile Gln Pro Asn Cys Tyr Asn Tyr Ser Thr
                340                 345                 350
CAT ATC ACT AAG TGG TTT GCC GAG GAT CCC TCG CGC ACC TAC AAC GAC     2424
His Ile Thr Lys Trp Phe Ala Glu Asp Pro Ser Arg Thr Tyr Asn Asp
            355                 360                 365
CTT GCG CAG TAC TGG GTT GAC CAT TCC ATG CCC ATC TTC CGT AGT GTC     2472
Leu Ala Gln Tyr Trp Val Asp His Ser Met Pro Ile Phe Arg Ser Val
        370                 375                 380
GGC GAC CAC CGC CGT CTT ATG ATG TGG GAG GAC ATA GCT ATC GCG ACT     2520
Gly Asp His Arg Arg Leu Met Met Trp Glu Asp Ile Ala Ile Ala Thr
    385                 390                 395
GAA AGC GCC CAC GAC GTG CCC AAA GAC GTC ATC ATG CAG ACC TGG AAC     2568
Glu Ser Ala His Asp Val Pro Lys Asp Val Ile Met Gln Thr Trp Asn
400                 405                 410                 415
AGC GGC GAG GGT GAG GGT AAC ATC AAG AAA CTC ACC TCC GCC GGC TAC     2616
Ser Gly Glu Gly Glu Gly Asn Ile Lys Lys Leu Thr Ser Ala Gly Tyr
                420                 425                 430
GAC GTT GTC GTT TCG ACC TCC GAT TTC CTC TAC CTC GAC TGC GGG CGC     2664
Asp Val Val Val Ser Thr Ser Asp Phe Leu Tyr Leu Asp Cys Gly Arg
            435                 440                 445
GGC GGC TAT GTC ACC AAC GAC GCC CGC TAC AAC GTG CAG AGC AAC ACC     2712
Gly Gly Tyr Val Thr Asn Asp Ala Arg Tyr Asn Val Gln Ser Asn Thr
        450                 455                 460
GAC GGC GGA GTG AAC TTC AAC TAC GGC GGC GAC GGT GGC TCC TGG TGC     2760
Asp Gly Gly Val Asn Phe Asn Tyr Gly Gly Asp Gly Gly Ser Trp Cys
    465                 470                 475
GCC CCC TAC AAG ACC TGG CAG CGC ATC TAC GAC TAC GAC TTC CTC ACG     2808
Ala Pro Tyr Lys Thr Trp Gln Arg Ile Tyr Asp Tyr Asp Phe Leu Thr
480                 485                 490                 495
AAT CTC ACT TCC TCC GAA GCG AAG CAC ATT ATC GGC GCC GAG GCT CCT     2856
Asn Leu Thr Ser Ser Glu Ala Lys His Ile Ile Gly Ala Glu Ala Pro
                500                 505                 510
TTG TGG TCG GAG CAG GTC GAC GAT GTG ACC GTC TCC AGC GTG TTC TGG     2904
Leu Trp Ser Glu Gln Val Asp Asp Val Thr Val Ser Ser Val Phe Trp
            515                 520                 525
CCT CGC GCT GCT GCT CTG GGT GAG CTT GTC TGG TCT GGT AAC CGT GAC     2952
Pro Arg Ala Ala Ala Leu Gly Glu Leu Val Trp Ser Gly Asn Arg Asp
        530                 535                 540
GCT GCG GGT AGA AAG CGT ACC ACC AGC TTT ACT CAG CGT ATT CTG AAC     3000
Ala Ala Gly Arg Lys Arg Thr Thr Ser Phe Thr Gln Arg Ile Leu Asn
    545                 550                 555
TTC CGT GAA TAC CTC GTT GCC AAT GGT GTG ATG GCT ACT GCT CTT GTG     3048
Phe Arg Glu Tyr Leu Val Ala Asn Gly Val Met Ala Thr Ala Leu Val
560                 565                 570                 575
CCG AAG TAT TGT CTG CAG CAC CCT CAT GCT TGC GAC CTC TAT AAA AAC     3096
Pro Lys Tyr Cys Leu Gln His Pro His Ala Cys Asp Leu Tyr Lys Asn
                580                 585                 590
CAG ACT GTA ATG TCT TGATTGTGGT TAAGCTGGAC TGCTAGTGAG CCTTACAACT     3151
Gln Thr Val Met Ser
            595
GCCTGTTCGT CTGTATATAC TTATTCTATC TTCGATACCC AATTCCATTG GAATTTCTTC   3211
CAGGATACAT GTCCCTGATC AGTATACCAT TTCACGTCCA CATTCAATCT TCAGCAACAC   3271
GAATTTATCC AAACCAATCA CCACCCTAGA TCTACCACAA CACTACCTTT ATACATATCT   3331
ACTTGATACC CAATCCCATT CCAACCAGGC GCAAAAGGCG TGCCCAGTCC AAATCAAAAT   3391
CAGCCCCCCG AGCCCAACCC TCTCCACATA TCCATACCCT AATCAAAATC ACCTTAATCT   3451
AAACAAATCC ATCACGCCCA AGGACCCCAC AGACCTCCCC TTCCCAACCC ACCCAGTCCA   3511
CCTCCACAAA CCAAACCCCA AATCAGAACT GCCGTGCAAC TCTCCGTCTT AGAACTCGCC   3571
CTTCGGTCCC GTCCCGAACT TAGATGGGCT TCGGGACGGC TTGCTGTATG CACTATGCAT   3631
GTAGTACGGA GTACGCCGTA CACATGTAGT AGGGGATATA TGTATGTACT ATGTACGCAT   3691
GTTCGAGTAC GCAGTACGTA GTGTGGCATG CAGGTCAGCT AGCATTGGCA GTAGCATATA   3751
CGGCATAACC TACGCTATGC ATCTAATATT CTTCGGTATA TACCACATGG TACGGAATTA   3811
GATGCAATAC ATGTACATGT ACATGTGCAT ACCTAGGTAC AAAGTGAATC TCGTTATTGT   3871
ATGTCTAGTC GTGTATAAGT GTAGTCCCAT GTCATATATA CAAGCCCATA CCGCATCGGA   3931
GCAAACCAGC CCATTCAGAC ATCCCTGCTC GAAACCCAGT CTACGGATTG AGACCGGGCT   3991
GAGCTGGGGT TTGGGTGTTG CTGCATGCGT ACGCCTACAT ACGTAGGGAG ATATGTTGCA   4051
CAGGATGCAG GGAATGACAA ATTGACGAAT TGAGAAATAC GCGAGTGGTT AGATGTTAAT   4111
TCTCGTTCGG GATGTTTATG TTTACCTAGG TATACTGGCT GGGGGGTCGT CATACACGTG   4171
GGAATTTGTG GCAATCTGTC AGTGGCCAGG TCCTTGTTTG ATTTATATGT TTGGGATGGG   4231
GATGGTCAAT GGGTATTCCA AGGAGGATGT ATCATCTGCT TTACACCGTC CCTTGCCTGG   4291
GATTTGGATT GAATTCTTCT TTCCACGTCG ATGTAGATTC TTCCCCGGAG CTATTCGGGT   4351
ACAACCCTGG CTTCCATATA TCATGTGTCC ATACTAAGTA CAGACGCTTC GATTTCCGGT   4411
GCTGCGAGTA GATCGGGAAC TGATCTCGCA TGTCTGTACA CGAAGGGTTG TACAAGCACG   4471
CGGTCGTTCT GCGTAACCGG TTGTTTATGT TATTGGATTT GGTATTCGTC TAATATGGAT   4531
GATTTGGGAT AAGCTTCTAT CCTGGGAATG GGTGCTTGGT ATAGTTCAGC CTAGTACTTC   4591
GTCTTCTATG TGATATTTCC AAAATAGTAG TTTTCGGTAA GTATATCTCC TACCTTTGAC   4651
TTTGGTTTGT GGTTTACGTC TTACCTGGCG TTTAGAGGGA GGGATAGGTT TCTGTATCAC   4711
CGTCGTGTTT CAACGTGGAT CGGGGTCCTT TCCCTGATAT ATATCTTGGC TTATGTTTCG   4771
TGCGGTAGTG CGGGTTCGTA TAATGCATGT CTGGTATATC ATACGGCATT AGTGACTGGG   4831
ACGTTGAGGT CGAGCTTGGT TTGAGGTTAC ATATATTGAG CCAAAATGGT CGAAAATATA   4891
TATCAACATT GCCAAAACAG AACTTCATTC GTTGGATGCC ATGCCAAATT GCTAATAGGT   4951
CTTGATCTTA CTCTGACTCC TATCTCATCT CACCTTGGTT ATTCGTTACA CAGCATTAAC   5011
CCCAAGAACC AGGTATAGTC TGATCGTGGA TGTGGGCCAC GACAAAATAG AAGGTCTCGT   5071
GTTTAGGGCG ACGAATCTGG GACTGCATTC CAGACGGGCC TGCGGAGAAT TTGCAGCATT   5131
TTATATCTAC ATGGTTGTTC CCTGGTGTGT GTGGGTGTTT CATGATATAT CCTGGTCGAT   5191
TCTGACGTGC GTATGTATCG CTGGAAAGGC TCGTAGGGGC TGCGTCGAC               5240
 
           
           
             
               2994 base pairs 
               nucleotides 
               double 
               linear 
             
             
               genomic DNA 
             
             NO 
             NO 
             
               Penicillum chrysogenum 
             
             
               plasmids pALP315 and pALP316 
             
             
               coding sequence 
                join (494...500, 617...647, 846...901,
               1047...1077, 1181...1952, 2022...2249)
 
             
             
               intron 
                501...616
 
             
             
               intron 
                648...845
 
             
             
               intron 
                902...1046
 
             
             
               intron 
                1078...1180
 
             
             
               intron 
                1953...20216 
                act gene
 
             
              3
GAATTCAGCA GCCTACGGAG TCCATAAGAC ACCAAGACAC AGCCATTGTA TGGATTATAT     60
ATGCCATGTA TGCCTGACAA TGCTGTATAA GTACTGTAAT ACAAGGTAAA CCCCCAACCC    120
GGTCAAGGTA CGTGTTCCCG CCGTACCCAA AAGGGTCCCC AAGAATGTCC ACGCAATACT    180
TTTAGGTAGA CATTGAAGGA ATCCAAGTGA GAAATTCAAT GAACATGAAC AATAGTTCTG    240
CCTTATAATC TTTATAAGTA TAAAAATCAG AAAGAGAATT ATATACAAAA GGGTAGATCT    300
GGAGGGGGTT CAGAGTTAAG GCCTCAGGCA GGCGCACAAT CCCAGCCATC ACAAACCCCT    360
CTCCACTCTT CCCTCTCTCT CTCTTCCTTC TTCCTTTCTC CCCTAATCCC AACTATATCC    420
CCTCTAACCT CTTTCCATCT TTCTTTTCTT TTTTCCCCTC TTCTCCCCTA AGTCCCTTGT    480
TTAATCAGTC ACA ATG GAG G GTATGTTATT CCAGTTGTGG CCACATCAGC AGCTTCCC   538
           Met Glu
               1
CGGAAGCTCC CCCCCCTGTT GGCCACAGCT TCGATTCCAT ATTTGCGAAT GACAACTAAC    598
CCGTATATCT CATTATAG  AA GAA GTT GCT GCT CTC GTC ATC GAC AAT GG       647
                    Glu Glu Val Ala Ala Leu Val Ile Asp Asn Gly
                            5                   10
GTATGTGCTA TACTTTTCCC CGGAGCTTCT GGCTTGTGTT GGGGTCGCCA ACTCAGCCCC    707
GGTCGCAGTC GTTGCCACCC CTAATCCGCC CGCGACGGCA GATGGAATCC ATCCCAATGG    767
CTTTCCATCT CGCTCCACAA CTACCAGAGG GTGATCCAAA GACTACAAGA ACTATGATAC    827
TGATTATTTG CGATATAG T TCG GGT ATG TGT AAG GCC GGT TTC GCC GGT GAC    879
                      Ser Gly Met Cys Lys Ala Gly Phe Ala Gly Asp
                          15                  20
GAC GCA CCA CGA GCT GTT TTC C GTAAGTCCA ACCCCACAGA ATATGACACC        930
Asp Ala Pro Arg Ala Val Phe
25                  30
CCTCCTGTGC GAAGGCCGCC ATCCCACCAA CCCTTGCGTC GGATGGCTTC CCCTCTTTTG    990
CTTGGCTAGG AGGAACCTGG AACCTAGGAA ATCAAATAAC TGACAAAATT CAACAG       1046
CT TCC ATT GTC GGT CGT CCC CGC CAC CAT GG  GTAAATGATC CCCCCTTTTT    1097
Pro Ser Ile Val Gly Arg Pro Arg His His Gly
             35                  40
TTTCCGGCTC GTTTCGGCTG TATACGCTAT ACGCAGCCAA TTTGATCCCT AATGAACCAA   1157
AAAGAATACT AACATGGGCG CAG T ATT ATG ATT GGT ATG GGT CAG AAG GAC     1208
                            Ile Met Ile Gly Met Gly Gln Lys Asp
                                    45                  50
TCG TAC GTT GGT GAT GAG GCA CAG TCG AAG CGT GGT ATC CTC ACG CTC     1256
Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile Leu Thr Leu
            55                  60                  65
CGT TAC CCT ATT GAG CAC GGT GTT GTC ACC AAC TGG GAC GAC ATG GAG     1304
Arg Tyr Pro Ile Glu His Gly Val Val Thr Asn Trp Asp Asp Met Glu
        70                  75                  80
AAG ATC TGG CAC CAC ACC TTC TAC AAC GAG CTC CGT GTT GCC CCC GAA     1352
Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val Ala Pro Glu
    85                  90                  95
GAG CAC CCC ATT CTC TTG ACC GAA GCT CCC ATC AAC CCC AAG TTC AAC     1400
Glu His Pro Ile Leu Leu Thr Glu Ala Pro Ile Asn Pro Lys Phe Asn
100                 105                 110                 115
CGT GAG AAG ATG ACC CAG ATC GTG TTC GAG ACC TTC AAC GCC CCC GCC     1448
Arg Glu Lys Met Thr Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala
                120                 125                 130
TTC TAC GTC TCC ATC CAG GCC GTT CTG TCC CTG TAC GCC TCC GGT CGT     1496
Phe Tyr Val Ser Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg
            135                 140                 145
ACC ACT GGT ATC GTT CTC GAC TCC GGT GAC GGT GTC ACC CAC GTC GTC     1544
Thr Thr Gly Ile Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val
        150                 155                 160
CCC ATC TAC GAG GGT TTC TCT CTG CCC CAC GCT ATC TCG CGT GTC GAC     1592
Pro Ile Tyr Glu Gly Phe Ser Leu Pro His Ala Ile Ser Arg Val Asp
    165                 170                 175
ATG GCT GGC CGT GAT CTG ACC GAC TAC CTG ATG AAG ATC CTC GCT GAG     1640
Met Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu
180                 185                 190                 195
CGT GGT TAC ACT TTC TCC ACC ACC GCC GAG CGT GAA ATC GTC CGT GAC     1688
Arg Gly Tyr Thr Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg Asp
                200                 205                 210
ATC AAG GAG AAG CTT TGC TAC GTC GCC CTC GAC TTC GAG CAG GAG ATC     1736
Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Ile
            215                 220                 225
CAG ACC GCT TCC CAG AGC TCC AGC CTC GAG AAG TCC TAC GAG CTT CCC     1784
Gln Thr Ala Ser Gln Ser Ser Ser Leu Glu Lys Ser Tyr Glu Leu Pro
        230                 235                 240
GAT GGA CAG GTC ATC ACT ATT GGC AAC GAG CGC TTC CGT GCT CCT GAG     1832
Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg Ala Pro Glu
    245                 250                 255
GCT CTG TTC CAG CCT AAC GTT CTT GGC CTC GAG TCT GGC GGT ATC CAC     1880
Ala Leu Phe Gln Pro Asn Val Leu Gly Leu Glu Ser Gly Gly Ile His
260                 265                 270                 275
GTC ACC ACC TTC AAC TCC ATC ATG AAG TGT GAT GTT GAT GTC CGT AAG     1928
Val Thr Thr Phe Asn Ser Ile Met Lys Cys Asp Val Asp Val Arg Lys
                280                 285                 290
GAT CTC TAC GGC AAC ATT GTC ATG GTAAGAAAAA AGCCTCCAGA GCTGATGTTG    1982
Asp Leu Tyr Gly Asn Ile Val Met
            295
CGCAAAGATC CCCACTAACA TACAACTCCT TTTTTTTAG TCT GGT GGT ACC ACC      2036
                                           Ser Gly Gly Thr Thr
                                           300
ATG TAC CCC GGT ATC TCC GAC CGT ATG CAG AAG GAG ATC ACT GCT CTT     2084
Met Tyr Pro Gly Ile Ser Asp Arg Met Gln Lys Glu Ile Thr Ala Leu
305                 310                 315                 320
GCT CCT TCT TCC ATG AAG GTC AAG ATC ATC GCT CCC CCC GAG CGC AAG     2132
Ala Pro Ser Ser Met Lys Val Lys Ile Ile Ala Pro Pro Glu Arg Lys
                325                 330                 335
TAC TCC GTC TGG ATC GGT GGA TCC ATT CTG GCC TCC CTG TCG ACC TTC     2180
Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe
            340                 345                 350
CAG CAG ATG TGG ATC TCC AAG CAG GAG TAC GAC GAG AGC GGT CCT TCC     2228
Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser
        355                 360                 365
ATC GTT CAC CGC AAG TGC TTC TAAGCTTCTT GCAGCACTTT ACTACTCGTA        2279
Ile Val His Arg Lys Cys Phe
    370                 375
TTCGCTCGTA CTTTCCTGGT GTATCAAAAA GCAGGATGGA GGCACTGGTG GATTGCAAGC   2339
GTTGTTGGAC TCGCATTATC AAGCGGATAG CCTGAAAATG GAATCTCGAT TTTAGTGGAA   2399
TAGAGTCGGT CGTTTTCTTT TTGTTACTCT TTACCTTACT CTTTACTCGA TCTCTATCCA   2459
TCCATTTCTG CTTTGAACCA TTTCACCTTT ACTCCATCTT TTTCCCTTTC CTCATTCGAA   2519
TCCGCTGTCC CGTCCACCTC TCTGATTGTT TTGCCTGGAC GGGTCTCTGG CGATGCGGCA   2579
TCAACAGTGT ACCTGTAGGG CAAGGATGTA TATGGAGTTG GTTGGCTATA GGGATTAGGT   2639
TGCGTTGTCC TTTTCGACGT CTTCTACGTC TTTGTTCTAG CCCCTTGCGT TGTCTTCAAC   2699
TAAACTGCCC TTGTCCGTAG CTTTTAACGT GACTTTGACT TCAAATATTC CACTGGTTCC   2759
TTGTATTCTG CTAGAAACGC TGGTTCAACG CTTGTTGAAT GTCTTCTATG TCCAACATCT   2819
ACAAGACGTA TCCGAGAAGA CAACAAAAAG GCTCTGAGGA AAGTCTACTA AAAACTTGGC   2879
CAGGCCGGAT TAGGCCTTTG TCATGGTTAT TGTACTGTCA TTCGATCAGT CCATATTGAT   2939
ATTCTGGGAA TATGTAGGCT GACGAGATAA ATGGCACGCA TTGGGTGTGT ATCTT        2994
 
           
           
             
               3240 base pairs 
               nucleotides 
               double 
               linear 
             
             
               genomic DNA 
             
             NO 
             NO 
             
               Penicillum chrysogenum 
             
             
               &lt;Unknown&gt; 
             
             
               intron 
                794...920
 
             
             
               intron 
                952...1123
 
             
             
               intron 
                1180...1289
 
             
             
               intron 
                1321...1410
 
             
             
               intron 
                2183...2249 
                act gene
 
             
              4
GCCAGGCTGG CACCGGCCTG CCTTGATGCG AGATGCCTAC TCGTACTATG CCTACAGGTA     60
TGGGCTTTCC GCGTGTCGTC AGCTTGCGAC CGCGCGGCTG CTGACGACCC AAGGCAAGCT    120
GGTAACATGG CGGCACGAAA TTTCTCTCTG CCTGCTCGTC CTCTTGGTGT GGAGGGGTAC    180
GAGTGCAGGT ATGATGGGAC GGCAGAGGAG TGACGGAGGC TGTGCGGTTG GCACGAGTAC    240
TGTACGAGTA CTCGTACTGT AGGTGCAGCG ACTGTGGTGG TACTGCTAGG TGGAATTGGG    300
TCCAGCAGGC ATGCAGCTCC CAGCCACCGT CGTTAACCAA TCAGTTAAAG CAGCAACGCA    360
ACCCGCCCCC GTTTTTCTGC CAGAAATTTG GGCGGTGTCG TGCCCCCAGT CGCTGTTGCC    420
CGCCCTTGTC TGGTCGCCTA CAGGCTGCAC CACAGGTAAC AACAGCCCGC CCCAGGTCCT    480
TGTAGGTGCC CAGTGAGTGC CCGGTGCCCA CAAGTTTCTC GTGGCATCCA CTGGCGGACT    540
TGGAAGCCCA TCAGTGATGC TTCCCTCCTT TCCCCCTCCA CATCTCACTC AGCTCACGCA    600
AGCCAACCCT CTCTCCCCCC GTCTCCATTC CATCTTCTTC TCTCCACGAC CCTTAAGAGT    660
CCCTCCTGCT CACGTCGACC ATCCTTCGCT CCCAGCCCCA CGACATCTGC ATCGTCTGGG    720
CTTCTTGACA CTCTGTCATT TCTTCCTTAT AAAACCTCTT TACCGCTCTT CCCGTAATCC    780
GACGCC ATG GAG G GTACGTGTCG CCGCAACGCA CTCCCGCTTC CCCTACTACC CCTA    837
       Met Glu
       1
TCGCGC ATCCACACGG CGCCGCGATG CCTAGCCATC GCGAGGGTGC ATCGCAACGA CTT    896
GGCTAAC TGTTCTTCGC TTCACAG  AG GAG GTC GCC GCC CTC GTT ATC GAC       946
                           Glu Glu Val Ala Ala Leu Val Ile Asp
                                   5                   10
AAT GG  GTAAGCTCGC CCGCTGTCTC ACCGACATCC ATCGTCCCCC TGGCCTCTGT      1001
Asn Gly
CGAGATGGGA GCCTCCAGGG GTCCCTTCGA CGAGCGCGTC GATTGCCAAA ATCCAACGAG   1061
ATCGGGCCAT ACTGAGCCGA CACTCGTGTG TTTTCTGGAC ATTAGGACTG ACTTGATTCT   1121
AG T TCG GGT ATG TGC AAG GCC GGT TTC GCC GGT GAT GAT GCT CCC CGA    1169
     Ser Gly Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg
         15                  20                  25
GCT GTT TTC C GTAAGTACCC CACTTCCACC CGTCGAGCTC CCCAATTGTC CACCGCCAGG1229
Ala Val Phe
    30
GCGAGAAGGG GGCAGAACGG GGCAAACTGC ATCGCAAACA TGGCTAATTC GATGCGACAG   1289
CG TCC ATT GTC GGT CGT CCC CGC CAC CAT GG  GTAAGTTTCC GGCCGCAGCC    1340
Pro Ser Ile Val Gly Arg Pro Arg His His Gly
            35                  40
GACACCTCTC ACCCCCCCCC GGGGGGCTCC TAAGCGAGTC AGCGCTGGTT CTGACCGCTG   1400
GATACTATAG C ATC ATG ATC GGC ATG GGC CAG AAG GAC TCG TAC GTC GGT    1450
             Ile Met Ile Gly Met Gly Gln Lys Asp Ser Tyr Val Gly
                     45                  50                  55
GAC GAG GCT CAG TCC AAG CGT GGT ATC CTC ACC CTG CGC TAC CCC ATT     1498
Asp Glu Ala Gln Ser Lys Arg Gly Ile Leu Thr Leu Arg Tyr Pro Ile
                60                  65                  70
GAG CAC GGT GTT GTC ACC AAC TGG GAC GAC ATG GAG AAG ATC TGG CAC     1546
Glu His Gly Val Val Thr Asn Trp Asp Asp Met Glu Lys Ile Trp His
            75                  80                  85
CAC ACC TTC TAC AAC GAG CTG CGT GTT GCC CCC GAG GAG CAC CCG GTC     1594
His Thr Phe Tyr Asn Glu Leu Arg Val Ala Pro Glu Glu His Pro Val
        90                  95                  100
CTG CTC ACC GAG GCG CCC ATC AAC CCC AAG TCC AAC CGT GAG AAG ATG     1642
Leu Leu Thr Glu Ala Pro Ile Asn Pro Lys Ser Asn Arg Glu Lys Met
    105                 110                 115
ACC CAG ATC GTC TTC GAG ACC TTC AAC GCC CCT GCC TTC TAC GTC TCC     1690
Thr Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala Phe Tyr Val Ser
120                 125                 130                 135
ATC CAG GCC GTC CTG TCA CTG TAC GCC TCC GGC CGT ACG ACC GGT ATC     1738
Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg Thr Thr Gly Ile
                140                 145                 150
GTC CTG GAC TCT GGT GAT GGT GTC ACC CAC GTT GTC CCC ATC TAC GAG     1786
Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val Pro Ile Tyr Glu
            155                 160                 165
GGT TTC GCC CTG CCC CAC GCC ATT GCC CGT GTC GAC ATG GCT GGT CGT     1834
Gly Phe Ala Leu Pro His Ala Ile Ala Arg Val Asp Met Ala Gly Arg
        170                 175                 180
GAT CTC ACC GAC TAC CTC ATG AAG ATC CTG GCC GAG CGC GGC TAC ACC     1882
Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu Arg Gly Tyr Thr
    185                 190                 195
TTC TCC ACC ACG GCC GAG CGT GAG ATT GTC CGT GAC ATC AAG GAG AAG     1930
Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg Asp Ile Lys Glu Lys
200                 205                 210                 215
CTC TGC TAC GTC GCC CTC GAC TTC GAG CAG GAG ATC CAG ACT GCC GCC     1978
Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Ile Gln Thr Ala Ala
                220                 225                 230
CAG AGC TCC AGC CTG GAG AAG TCC TAC GAG CTT CCC GAC GGC CAG GTC     2026
Gln Ser Ser Ser Leu Glu Lys Ser Tyr Glu Leu Pro Asp Gly Gln Val
            235                 240                 245
ATC ACC ATT GGC AAT GAG CGC TTC CGT GCT CCC GAG GCT CTC TTC CAG     2074
Ile Thr Ile Gly Asn Glu Arg Phe Arg Ala Pro Glu Ala Leu Phe Gln
        250                 255                 260
CCC TCC GTC CTG GGT CTC GAG AGC GGC GGC ATC CAC GTC ACC ACC TTC     2122
Pro Ser Val Leu Gly Leu Glu Ser Gly Gly Ile His Val Thr Thr Phe
    265                 270                 275
AAC TCC ATC ATG AAG TGC GAC GTC GAT GTC CGT AAG GAT CTG TAC GGC     2170
Asn Ser Ile Met Lys Cys Asp Val Asp Val Arg Lys Asp Leu Tyr Gly
280                 285                 290                 295
AAC ATT GTC ATG GTAAGTCAGA TGCCGGGCCT GGAAGACACC TCATTTAGGA TCT     2225
Asn Ile Val Met
TGCTAAC ACCAATTTTT TTTTTAG TCT GGT GGT ACC ACC ATG TAC CCT GGC      2276
                           Ser Gly Gly Thr Thr Met Tyr Pro Gly
                           300                 305
CTC TCT GAC CGT ATG CAG AAG GAG ATC ACT GCT CTT GCT CCT TCT TCC     2324
Leu Ser Asp Arg Met Gln Lys Glu Ile Thr Ala Leu Ala Pro Ser Ser
    310                 315                 320
ATG AAG GTC AAG ATC ATT GCT CCC CCG GAG CGC AAG TAC TCC GTC TGG     2372
Met Lys Val Lys Ile Ile Ala Pro Pro Glu Arg Lys Tyr Ser Val Trp
325                 330                 335                 340
ATC GGT GGT TCC ATT CTG GCG TCT CTG TCC ACC TTC CAG CAG ATG TGG     2420
Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe Gln Gln Met Trp
                345                 350                 355
ATC TCG AAG CAG GAG TAC GAC GAG AGC GGC CCC TCC ATC GTC CAC CGC     2468
Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg
            360                 365                 370
AAG TGC TTC TAAGGTATGT TGTCGTCGGG AAGCCGGATA CCCGAATGTA AGGTTGACAG  2527
Lys Cys Phe
        375
GTTCGAAAAG ACAAGGCAAC CGGCCAGAAC CAAATCCTTC CACCCTCCGC AAAAGAACGC   2587
CAAGATGTCG GAGTCGGTGG CGACCGATGC AACGTCTACT CACGTGCGCG CGTATCCCAC   2647
TCAAGTCTCA TATTTACGAA AAGTTATTTC ACATGGTCAG GCGGTGGTGG GCGTTGCCTT   2707
TTCTCGGAAC AGACATGACG GCGGCCACTT TTGTAGTCGG ATGCGTTTAG GGATGCGAGC   2767
CTAGGGGTGT AGGAAGCTGA GGTTGATATA CAATAACTTT TTTTGCTTTC CGTTCTAGAC   2827
TCGTTCAATG GGAAGACGTG ACGGAATCGC TTGGCTGTCT AATAGCCAGC TTGATCAGGC   2887
GAGTCGGGTT GTTGTGTTTC GATGTTGAGA GGTGCACCAG CGTATTTGTA TGGCCGAGGT   2947
AGGTATTATG GTCTCGTATT TGCAACACTA GAGCTCGCTT GCTCGTTTTT ACCAGCAGTG   3007
TCCTCTGCCA TGCCGCGGCT CCGACTCTCG TCTGGCTTCT CAGACCGTGC CTCGTCAATA   3067
GTATTATCCC CCGTAGTAAC CTCCGCACTA GCCGGTTCTT TGTCGTCTTC CTGCTCGCCG   3127
ATGAGCTTCC TGTACTTGCG CCTCTTCTTC TTGTCGGCGC TGGCAGCCCT CTTCTGCTTG   3187
ATGCGCCCGA CCATGGCGGA CCGGCTCTGC TCCCCGTTGA GCAGCTCGTC GAC          3240
 
           
           
             
               461 amino acids 
               amino acids 
               single 
               linear 
             
             
               peptide 
             
             
               Penicillum chrysogenum 
             
             
                amino acid sequence of the
               glutamate dehydrogenase enzyme
          (EC.1.4.1.4) with a molecular weight
               of 49837 Da.
 
             
              5
Met Met Gln Asn Leu Pro Phe Glu Pro Glu Phe Glu Gln Ala Tyr
1               5                   10                  15
Lys Glu Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Gln Lys
                20                  25                  30
Lys Pro Glu Tyr Arg Lys Ala Leu Gln Val Val Ser Val Pro Glu
                35                  40                  45
Arg Val Ile Gln Phe Arg Val Val Trp Glu Asp Asp Lys Gly Gln
                50                  55                  60
Val Gln Ile Asn Arg Gly Tyr Arg Val Gln Phe Asn Ser Ala Leu
                65                  70                  75
Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Thr Val Asn Leu
                80                  85                  90
Ser Ile Leu Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala
                95                  100                 105
Leu Thr Gly Leu Asn Met Gly Gly Gly Lys Gly Gly Ser Asp Phe
                110                 115                 120
Asp Pro Lys Gly Lys Thr Asp Asn Glu Ile Arg Arg Phe Cys Val
                125                 130                 135
Ser Phe Met Thr Glu Leu Cys Lys His Ile Gly Ala Asp Thr Asp
                140                 145                 150
Val Pro Ala Gly Asp Ile Gly Val Thr Gly Arg Glu Val Gly Phe
                155                 160                 165
Met Phe Gly Gln Tyr Lys Lys Ile Arg Asn Gln Trp Glu Gly Val
                170                 175                 180
Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser Leu Ile Arg Pro
                185                 190                 195
Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Glu His Met Ile
                200                 205                 210
Gln His Ala Ser Gly Gly Lys Glu Ser Phe Ala Gly Lys Arg Val
                215                 220                 225
Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys
                230                 235                 240
Val Ile Glu Leu Gly Gly Ser Val Ile Ser Leu Ser Asp Ser Gln
                245                 250                 255
Gly Ala Leu Val Leu Asn Gly Glu Glu Gly Ser Phe Thr Ala Glu
                260                 265                 270
Glu Ile Asn Thr Ile Ala Glu Ile Lys Val Gln Arg Lys Gln Ile
                275                 280                 285
Ala Glu Leu Ala Thr Gln Asp Ala Phe Ser Ser Lys Phe Lys Tyr
                290                 295                 300
Ile Pro Gly Ala Arg Pro Trp Thr Asn Ile Ala Gly Arg Ile Asp
                305                 310                 315
Val Ala Leu Pro Ser Ala Thr Gln Asn Glu Val Ser Gly Asp Glu
                320                 325                 330
Ala Lys Ala Leu Ile Ala Ala Gly Cys Lys Phe Ile Ala Glu Gly
                335                 340                 345
Ser Asn Met Gly Ser Thr Gln Glu Ala Ile Asp Val Phe Glu Ala
                350                 355                 360
His Arg Asp Ala Asn Pro Gly Ala Ala Ala Ile Trp Tyr Ala Pro
                365                 370                 375
Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu
                380                 385                 390
Met Ala Gln Asn Ser Ala Arg Val Asn Trp Ser Arg Glu Glu Val
                395                 400                 405
Asp Ser Arg Leu Lys Lys Ile Met Glu Asp Cys Phe Asn Asn Gly
                410                 415                 420
Leu Ser Thr Ala Lys Glu Tyr Val Thr Pro Ala Glu Gly Val Leu
                425                 430                 435
Pro Ser Leu Val Ala Gly Ser Asn Ile Ala Gly Phe Thr Lys Val
                440                 445                 450
Ala Glu Ala Met Lys Glu His Gly Asp Trp Trp
                455                 460
 
           
           
             
               596 amino acids 
               amino acids 
               single 
               linear 
             
             
               peptide 
             
             
               Penicillum chrysogenum 
             
             
                  amino acid sequence of the  -N-
               acetylhexosaminidase enzyme
          (EC.3.2.1.52) with a
               molecular weight of 66545 Da.
 
             
              6
Met Lys Phe Ala Ser Val Leu Asn Val Leu Gly Ala Leu Thr Ala
1               5                   10                  15
Ala Ser Ala Val Gln Val Asn Pro Leu Pro Ala Pro Arg Asn Ile
                20                  25                  30
Thr Trp Gly Ser Ser Gly Pro Ile Gln Val Asn Asn Leu Asn Leu
                35                  40                  45
Asn Gly Pro His Ser Pro Leu Leu Thr Gln Ala Trp Glu Arg Ala
                50                  55                  60
Trp Glu Thr Ile Thr Thr Leu Gln Trp Val Pro Ala Ala Val Glu
                65                  70                  75
Ser Pro Ile Ala Ser Tyr Pro Ala Phe Pro Thr Ser Thr Pro Val
                80                  85                  90
Ser Ser Ala Pro Lys Ala Lys Arg Ala Pro Ser Gly Ile His Asn
                95                  100                 105
Val Asp Val His Val Val Asp Asn Asp Ala Asp Leu Gln Tyr Gly
                110                 115                 120
Val Asp Glu Ser Tyr Thr Leu Val Val Ser Asp Gly Gly Ile Arg
                125                 130                 135
Ile Asn Ser Gln Thr Val Trp Gly Val Leu Gln Ala Phe Thr Thr
                140                 145                 150
Leu Gln Gln Ile Ile Ile Ser Asp Gly Lys Gly Gly Leu Ile Ile
                155                 160                 165
Glu Gln Pro Val Lys Ile Lys Asp Ala Pro Leu Tyr Pro His Arg
                170                 175                 180
Gly Ile Met Ile Asp Thr Gly Arg Asn Phe Ile Thr Val Arg Lys
                185                 190                 195
Leu Leu Glu Gln Ile Asp Gly Met Ala Leu Ser Lys Leu Asn Val
                200                 205                 210
Leu His Trp His Leu Asp Asp Ser Gln Ser Trp Pro Met Gln Met
                215                 220                 225
Ser Ser Tyr Pro Glu Met Thr Lys Asp Ala Tyr Ser Pro Arg Glu
                230                 235                 240
Ile Tyr Thr Glu His Asp Met Arg Arg Val Ile Ala Tyr Ala Arg
                245                 250                 255
Ala Arg Gly Val Arg Val Ile Pro Glu Val Asp Met Pro Ala His
                260                 265                 270
Ser Ala Ser Gly Trp Gln Gln Val Asp Pro Glu Ile Val Ala Cys
                275                 280                 285
Ala Glu Ser Trp Trp Ser Asn Asp Val Trp Ala Glu His Thr Ala
                290                 295                 300
Val Gln Pro Asn Pro Gly Gln Leu Asp Ile Ile Tyr Pro Lys Thr
                305                 310                 315
Tyr Glu Val Val Asn Asn Val Tyr Gln Glu Leu Ser Arg Ile Phe
                320                 325                 330
Ser Asp Asn Leu Phe His Val Gly Ala Asp Glu Ile Gln Pro Asn
                335                 340                 345
Cys Tyr Asn Tyr Ser Thr His Ile Thr Lys Trp Phe Ala Glu Asp
                350                 355                 360
Pro Ser Arg Thr Tyr Asn Asp Leu Ala Gln Tyr Trp Val Asp His
                365                 370                 375
Ser Met Pro Ile Phe Arg Ser Val Gly Asp His Arg Arg Leu Met
                380                 385                 390
Met Trp Glu Asp Ile Ala Ile Ala Thr Glu Ser Ala His Asp Val
                395                 400                 405
Pro Lys Asp Val Ile Met Gln Thr Trp Asn Ser Gly Glu Gly Glu
                410                 415                 420
Gly Asn Ile Lys Lys Leu Thr Ser Ala Gly Tyr Asp Val Val Val
                425                 430                 435
Ser Thr Ser Asp Phe Leu Tyr Leu Asp Cys Gly Arg Gly Gly Tyr
                440                 445                 450
Val Thr Asn Asp Ala Arg Tyr Asn Val Gln Ser Asn Thr Asp Gly
                455                 460                 465
Gly Val Asn Phe Asn Tyr Gly Gly Asp Gly Gly Ser Trp Cys Ala
                470                 475                 480
Pro Tyr Lys Thr Trp Gln Arg Ile Tyr Asp Tyr Asp Phe Leu Thr
                485                 490                 495
Asn Leu Thr Ser Ser Glu Ala Lys His Ile Ile Gly Ala Glu Ala
                500                 505                 510
Pro Leu Trp Ser Glu Gln Val Asp Asp Val Thr Val Ser Ser Val
                515                 520                 525
Phe Trp Pro Arg Ala Ala Ala Leu Gly Glu Leu Val Trp Ser Gly
                530                 535                 540
Asn Arg Asp Ala Ala Gly Arg Lys Arg Thr Thr Ser Phe Thr Gln
                545                 550                 555
Arg Ile Leu Asn Phe Arg Glu Tyr Leu Val Ala Asn Gly Val Met
                560                 565                 570
Ala Thr Ala Leu Val Pro Lys Tyr Cys Leu Gln His Pro His Ala
                575                 580                 585
Cys Asp Leu Tyr Lys Asn Gln Thr Val Met Ser
                590                 595
 
           
           
             
               375 amino acids 
               amino acids 
               single 
               linear 
             
             
               peptide 
             
             
               Penicillium chrysogenum 
             
             
                amino acid sequence of the  -actin
               protein with a molecular weight of
               41760 Da.
 
             
              7
Met Glu Glu Glu Val Ala Ala Leu Val Ile Asp Asn Gly Ser Gly
1               5                   10                  15
Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val
                20                  25                  30
Phe Pro Ser Ile Val Gly Arg Pro Arg His His Gly Ile Met Ile
                35                  40                  45
Gly Met Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser
                50                  55                  60
Lys Arg Gly Ile Leu Thr Leu Arg Tyr Pro Ile Glu His Gly Val
                65                  70                  75
Val Thr Asn Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe
                80                  85                  90
Tyr Asn Glu Leu Arg Val Ala Pro Glu Glu His Pro Ile Leu Leu
                95                  100                 105
Thr Glu Ala Pro Ile Asn Pro Lys Phe Asn Arg Glu Lys Met Thr
                110                 115                 120
Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala Phe Tyr Val Ser
                125                 130                 135
Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg Thr Thr Gly
                140                 145                 150
Ile Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val Pro Ile
                155                 160                 165
Tyr Glu Gly Phe Ser Leu Pro His Ala Ile Ser Arg Val Asp Met
                170                 175                 180
Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu
                185                 190                 195
Arg Gly Tyr Thr Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg
                200                 205                 210
Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln
                215                 220                 225
Glu Ile Gln Thr Ala Ser Gln Ser Ser Ser Leu Glu Lys Ser Tyr
                230                 235                 240
Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe
                245                 250                 255
Arg Ala Pro Glu Ala Leu Phe Gln Pro Asn Val Leu Gly Leu Glu
                260                 265                 270
Ser Gly Gly Ile His Val Thr Thr Phe Asn Ser Ile Met Lys Cys
                275                 280                 285
Asp Val Asp Val Arg Lys Asp Leu Tyr Gly Asn Ile Val Met Ser
                290                 295                 300
Gly Gly Thr Thr Met Tyr Pro Gly Ile Ser Asp Arg Met Gln Lys
                305                 310                 315
Glu Ile Thr Ala Leu Ala Pro Ser Ser Met Lys Val Lys Ile Ile
                320                 325                 330
Ala Pro Pro Glu Arg Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile
                335                 340                 345
Leu Ala Ser Leu Ser Thr Phe Gln Gln Met Trp Ile Ser Lys Gln
                350                 355                 360
Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg Lys Cys Phe
                365                 370                 375
 
           
           
             
               375 amino acids 
               amino acids 
               single 
               linear 
             
             
               Peptide 
             
             
               Acremonium chrysogenum 
             
             
                  amino acid sequence of the  -actin
               protein with a molecular weight of
               41612 Da.
 
             
              8
Met Glu Glu Glu Val Ala Ala Leu Val Ile Asp Asn Gly Ser Gly
1               5                   10                  15
Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val
                20                  25                  30
Phe Pro Ser Ile Val Gly Arg Pro Arg His His Gly Ile Met Ile
                35                  40                  45
Gly Met Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser
                50                  55                  60
Lys Arg Gly Ile Leu Thr Leu Arg Tyr Pro Ile Glu His Gly Val
                65                  70                  75
Val Thr Asn Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe
                80                  85                  90
Tyr Asn Glu Leu Arg Val Ala Pro Glu Glu His Pro Val Leu Leu
                95                  100                 105
Thr Glu Ala Pro Ile Asn Pro Lys Ser Asn Arg Glu Lys Met Thr
                110                 115                 120
Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala Phe Tyr Val Ser
                125                 130                 135
Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg Thr Thr Gly
                140                 145                 150
Ile Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val Pro Ile
                155                 160                 165
Tyr Glu Gly Phe Ala Leu Pro His Ala Ile Ala Arg Val Asp Met
                170                 175                 180
Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu
                185                 190                 195
Arg Gly Tyr Thr Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg
                200                 205                 210
Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln
                215                 220                 225
Glu Ile Gln Thr Ala Ala Gln Ser Ser Ser Leu Glu Lys Ser Tyr
                230                 235                 240
Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe
                245                 250                 255
Arg Ala Pro Glu Ala Leu Phe Gln Pro Ser Val Leu Gly Leu Glu
                260                 265                 270
Ser Gly Gly Ile His Val Thr Thr Phe Asn Ser Ile Met Lys Cys
                275                 280                 285
Asp Val Asp Val Arg Lys Asp Leu Tyr Gly Asn Ile Val Met Ser
                290                 295                 300
Gly Gly Thr Thr Met Tyr Pro Gly Leu Ser Asp Arg Met Gln Lys
                305                 310                 315
Glu Ile Thr Ala Leu Ala Pro Ser Ser Met Lys Val Lys Ile Ile
                320                 325                 330
Ala Pro Pro Asp Gly Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile
                335                 340                 345
Leu Ala Ser Leu Ser Thr Phe Gln Gln Met Trp Ile Ser Lys Thr
                350                 355                 360
Glu Tyr Asp Glu Glu Arg Pro Ser Ile Val His Arg Lys Cys Phe
                365                 370                 375
 
           
           
             
               22 amino acids 
               amino acids 
               single 
               linear 
             
             
               Peptide 
             
             
               Penicillum chrysogenum 
             
              9
Ala Pro Ser Gly Ile His Asn Val Asp Val His Val Val Asp Asn Asp Ala
                  5                  10                  15
Gln Tyr Gly
 20
 
           
           
             
               22 amino acids 
               amino acids 
               single 
               linear 
             
             
               Peptide 
             
             
               Penicillum chrysogenum 
             
              10
Val Gln Val Asn Pro Leu Pro Ala Pro Arg Arg Ile Thr Xaa Gly
                  5                  10                  15
Ser Ser Gly Pro Xaa Xaa Val
                 20
 
           
           
             
               32 base pairs 
               nucleotides 
               single 
               linear 
             
              11
TCGACGACGT GSACGTCSAC GTTGTGGATG CC                                   32
 
           
           
             
               32 base pairs 
               nucleotides 
               single 
               linear 
             
              12
CCGTAYTGSA GGTCRGCGTC GTTGTCGACG AC                                   32
 
           
           
             
               23 base pairs 
               nucleotides 
               single 
               linear 
             
              13
GGGGCVGGSA GVGGGTTGAC YTG                                             23
 
           
           
             
               28 base pairs 
               nucleotides 
               single 
               linear 
             
              14
CTCCATGGTG ATAAGGTGAG TGACGATG                                        28
 
           
           
             
               18 base pairs 
               nucleotides 
               single 
               linear 
             
              15
GTAAAACGAC GGCCAGTG                                                   18
 
           
           
             
               18 amino acids 
               amino acids 
               single 
               linear 
             
             
               peptide 
             
             
               Penicillum chrysogenum 
             
              16
Met Lys Phe Ala Ser Val Leu Asn Val Leu Gly Ala Leu Thr Ala Ala Ser
                  5                  10                  15
 
           
           
             
               8 amino acids 
               amino acids 
               single 
               linear 
             
             
               Penicillum chrysogenum 
             
              17
Phe Ala Ser Val Leu Asn Val Leu
                  5
 
           
           
             
               8 amino acids 
               amino acids 
               single 
               linear 
             
             
               Penicillum chrysogenum 
             
              18
Gly Ala Leu Thr Ala Ala Ser Ala
                  5
 
           
           
             
               28 base pairs 
               nucleotides 
               single 
               linear 
             
              19
CTCCATGGTG ACTGATTAAA CAAGGGAC                                        28
 
           
           
             
               18 base pairs 
               nucleotides 
               single 
               linear 
             
              20
GTAAAACGAC GGCCAGTG                                                   18