EP0656943A1 - Fungal promoters active in the presence of glucose - Google Patents

Abstract

A method is described for the identification and cloning of promoters that express under a defined environmental condition, such as growth in glucose medium. Using this method, five (Trichodermal) promoters capable of the high expression of operably linked coding sequences are identified, one of which is the promoter for (T. reesei tef1). Also provided are altered (cbh1) promoters, altered so that glucose no longer represses expression from such promoter. The invention further provides vectors and hosts that utilize such promoters, and unique fungal enzyme compositions from such hosts.

Description

Title of the Invention

Fungal Promoters Active in the Presence of Glucose

Cross-Reference to Related Applications

This application is a continuation-in-part of U.S. Application No. 07/496,155 filed March 19, 1990.

Background of the Invention

I. Methods for the Identification of Promoters

Many systems have been used to isolate genes and their promoters located immediately upstream of the translation start site of a gene. The techniques can roughly be divided in two categories, namely (1) where the aim is to isolate genomic DNA fragments containing promoter activity randomly by so-called promoter probe vector systems and (2) where the aim is to isolate a gQiie perse from a genomic bank (library) and isolation of the corresponding promoter follows therefrom. In promoter probe vector systems, genomic DNA fragments are randomly cloned in front of the coding sequence of a reporter gene that is expressed only when the cloned fragment contains promoter activity (Neve, R.L. et al, Nature 277:324-325 (1979)). Promoter probe vectors have been designed for cloning of promoters in E. coli (An, G. et al, J. Bad. 740:400- 407 (1979)) and other bacterial hosts (Band, L. et al , Gene 26:313-315 (1983); Achen, M.G., Gene 45:45-49 (1986)), yeast (Goodey, A.R. et al , Mol. Gen. Genet. 204:505-511 (1986)) and mammalian cells (Pater, M.M. et al, J. Mol. App. Gen. 2:363-371 (1984)). Because it is well known in the art that Trichoderma promoters fail to work in E. coli and yeast (e.g. Penttila, M.Ε. et al, Mol. Gen. Genet. 794:494-499 (1984)), these organisms cannot be used as hosts to isolate Trichoderma promoters. Due to the fact that, during the transformation of Trichoderma, the transforming DNA integrates into the fungal genome in varying copies in random locations, application of this method by using Trichoderma itself as a cloning host is also unlikely to succeed and would not be practical for efficient isolation of Trichoderma promoters with the desired properties.

Known genes can be isolated from either a cDNA or chromosomal gene bank (library) using hybridization as a detection method. Such hybridization may be with a corresponding, homologous gene from another organism (e.g., Vanhanen et al , Curr. Genet. 75:181-186 (1989)) or with a probe designed on the basis of expected similarities in amino acid sequence. If amino acid sequence is available for the corresponding protein, an oligonucleotide can also be designed which can be used in hybridization for isolation of the gene. If the gene is cloned into an expression bank, the expression product of gene can be also detected from such expression bank by using specific antibodies or an activity test.

Specific genes can be isolated by using complementation of mutations in E. coli or yeast (e.g., Keesey, J.K. et al , J. Bad. 752:954-958 (1982); Kaslow, D.C., J. Biol. Chem. 265:12337-12341 (1990); Kronstad, J.W., Gene 79:97-106 (1989)), or complementation of corresponding mutants of filamentous fungi for instance by using SIB selection (Akins et al. , Mol Cell. Biol. 5:2272-2278 (1985)).

However, a major concern is how to isolate specific genes that have the desired promoter properties, for example genes which would be most highly expressed when glucose is present in the medium. There is no information available in literature to indicate which genes are the most highly expressed in an organism, and especially not from filamentous fungi. The phosphoglyceratekinase (PGK) promoter from the yeast Saccharomyces cerevisiae is considered to be a strong promoter for protein production. However, results obtained by the inventors have shown that the corresponding Trichoderma promoter is not suitable for such protein production. Thus, the identification of specific Trichoderma genes for their isolation in order to obtain the best possible promoter for protein production in certain desired conditions is unknown and cannot be predicted. Consequently one cannot rely on any previous nucleotide or amino acid sequence information, nor complement any previously known mutations, in gene isolation for such purpose in Trichoderma.

Differential hybridization has been used for cloning of genes expressed under certain conditions. The method relies on the screening of a bank separately with an induced and noninduced cDNA probe. By this method e.g., Trichoderma reesei genes strongly expressed during production of cellulolytic enzymes have been isolated (Teeri, T. et al, Bio/Technology 7:696-699 (1983)). The differential hybridization methods used are based on the idea that the genes searched for are expressed in certain conditions (like cellulases on cellulose) but not in some other conditions (like cellulases on glucose) which enables picking up clones hybridizing with only one of the cDNA probes used. However, for isolation of the genes expressed strongly on glucose, this approach (expression on glucose and not on some other media) is not a suitable one, and might in fact result in not finding the most highly expressed genes. This is because when differentially screening a chromosomal bank, only induced genes are selected. Such induced genes are not necessarily the most strongly expressed genes. Thus, no method is known in the art which would permit the identification of promoters which function strongly in Trichoderma on glucose medium.

Another option for obtaining a promoter with desired properties is to modify the already existing ones. This is based on the fact that the function of a promoter is dependent on the interplay of regulatory proteins which bind to specific, discrete nucleotide sequences in the promoter, termed motifs. Such interplay subsequently affects the general transcription machinery and regulates transcription efficiency. These proteins are positive regulators or negative regulators (repressors), and one protein can have a dual role depending on the context (Johnson, P.F. and McKnight, S.L. Annu. Rev. Biochem. 58:799-839 (1989)). However, even a basic understanding of the regions responsible for regulation of a promoter requires a considerable amount of experimental data, and data obtained from the corresponding promoter of another organism is usually not useful (see Vanhanen, S. et al , Gene 706: 129-133 (1991)), or at least not sufficient, to explain the function of a promoter originating from another organism.

//. Translation Elongation Factors

Translation Elongation Factors (TEFs) are universally conserved proteins that promote the GTP-dependent binding of an aminoacyl-tRNA to ribosomal A-site in protein synthesis. Especially conserved is the N-terminus of the protein containing the GTP binding domain. TEFs are known as very abundant proteins in cells comprising about 4-6% of total soluble proteins (Miyajima, I. et al , J. Biochem. 53:453-462 (1978); Thiele, D. et al, J. Biol. Chem. 260:3084-3089 (1985)). tef genes have been isolated from several organisms. In some of them they constitute a multigene family. Also a number of pseudogenes have been isolated from some organisms. The promoter of the human tef gene can direct transcription in vitro at least 2-fold more effectively than the adenovirus major late promoter, which indicates that the tef promoter is a strong promoter in mammalian expression systems (Uetsuki et al. , J. Biol. Chem. 264:5191-519 (1989)). Both the human and the A. thaliana tefl promoter (for translation elongation factor EF-lα) has been used in an expression system with high efficiency of gene expression (Kim et al , Gene 97:217-223 (1990); Curie et al , Nucl Acid Res. 79: 1305-1310 (1991)). In both cases the full expression of the promoter was dependent on the presence of the intron in the 5' noncoding region. tef is quite constitutively expressed, the major exception being its expression in aging and quiescent cells. It is not known to be regulated by the growth substrates of the host. ///. Expression of Recombinant Proteins in Trichoderma

The filamentous fungus Trichoderma reesei is an efficient producer of hydrolases, especially of different cellulose degrading enzymes. Due to its excellent capacity for protein secretion and developed methods for industrial cultivations, Trichoderma is a powerful host for production of heterologous, recombinant proteins in large scale. The efficient production of both homologous and heterologous proteins in fungi relies on fungal promoters. The promoter of the main cellulase gene of Trichoderma, cellobiohydrolase 1 (cbhl), has been used for production of heterologous proteins in Trichoderma grown on media containing cellulose or its derivatives (Harkki et al , Bio/Technology 7:596-603 (1989); Saloheimo etal , Bio/Technology 9:987-990 (1991)). The cbhl promoter cannot be used when the Trichoderma are grown on glucose containing media due to glucose repression of cbhl promoter activity. This regulation occurs at the transcriptional level and thus glucose repression could be mediated through the promoter sequences. It is also known that cellulase genes cbhl, cbh2, egll and egl2 are coexpressed in various growth conditions, thus it is presumable that same regulatory factors operate on fairly similar promoter sequences mediating similar functions. However, nothing is yet known of the mechanism of glucose repression at the promoter level in filamentous fungi.

Glucose repression in the yeast Saccharomyces cerevisiae has been studied for many years. These studies have however failed, until recently, to identify binding sequences in promoters or regulatory proteins binding to promoters which would mediate glucose repression. The first ever published glucose repressor protein and the binding sequence in eukaryotic cells was published by Nehlin and Ronne (Nehlin, J.O. and Ronne, H. EMBO J. 9:2891-2899 (1990)). This MIG1 protein seems to be responsible of one fifth of the glucose repression of GAL genes in Saccharomyces cerevisiae, other factors still being required to obtain full glucose repression effect (Nehlin, J.O. et al, EMBO J. 70:3373-3377 (1991)). Thus, it is desirable to be able to produce proteins in Trichoderma grown on glucose. Not only is the substrate glucose cheap and readily available, but also Trichoderma produces less protease activity when grown on glucose. Further, cellulase production is repressed when Trichoderma is grown on glucose, thus allowing for the easier purification of the desired product from the Trichoderma medium. Nevertheless, to date there has been no identification or characterization of any promoter that is highly functional in Trichoderma grown on glucose. In addition, no modifications of the normally glucose repressed promoter, the cbhl promoter, have been identified which would allow the use of this strong promoter for expression of heterologous genes in Trichoderma grown on glucose.

Summary of the Invention

This invention is first directed to the identification of the motif, the DNA element, that imparts glucose repression onto the Trichoderma cbhl promoter.

The invention is further directed to a modified Trichoderma cbhl promoter, such modified promoter lacking such glucose repression element and such modified promoter being useful for the production of proteins, including cellulases, when the host is grown on glucose medium. The invention is further directed to a method for the isolation of genes that are highly expressed on glucose, especially from filamentous fungal hosts such as Trichoderma.

The invention is further directed to five such previously undescribed genes and their promoters from Trichoderma reesei. The invention is further directed to specific cloning vectors for

Trichoderma containing the above mentioned sequences.

The invention is further directed to filamentous fungal strains transformed with said vectors, which strains thus are able to produce proteins such as cellulases on glucose. The invention is further directed to a process for producing cellulases or other useful enzymes on glucose.

Brief Description of the Drawings

Figure 1 shows the plasmid pTHNl which carries the tefl promoter and 5' part of the coding region and shows the relevant features of the te 7 gene and the sequenced areas. Figure IA is the nucleotide sequence of the tefl promoter and coding sequence [TEF001; SEQ ID 1]. The promoter sequence stops at base number 1234. The methionine codon of the start site of translation is located at base numbers 1235-1237 and is underlined. The total number of bases shown is 3461. The DNA sequence composition is 850A, 1044C, 860G, 697T, and 10 other.

Figure 2 shows the plasmid pEA33 which carries the tefl promoter and the coding region with relevant features.

Figure 3 shows the plasmid pTHN3 which carries the promoter and coding region of the clone cDNAl and shows the relevant features. Figure 3 A is the nucleotide sequence of the cDNAl promoter and coding sequence [SEQ ID 2]. The promoter sequence stops at base number 1157. The methionine codon of the start site of translation is located at base numbers 1158-1160 as numbered in Figure 3A and is underlined. Figure 4 shows the plasmid pEAlO which carries the promoter and coding region of the clone cDNAlO and the relevant regions and sequenced areas. Diagonally hatched = insert; solid line = sequenced region (genomic DNA); squared criss-crossed = sequenced region (cDNA). Not all EcoRV and Ndel sites are shown. Figure 4A is the nucleotide sequence of the cDNAlO promoter and coding sequence [CDNAIOSEQ; SEQ ID 3]. The promoter sequence stops at base number 1522. The methionine codon of the start site of translation is located at base numbers 1523-1525 and is underlined. The total number of bases shown is 2868. The DNA sequence composition is 760A, 765C, 675G and 668T. Figure 5 shows the plasmid pEA12 which carries the clone cDNA12 and relevant features and sequenced areas. Diagonally hatched = insert; solid line = sequenced region (genomic DNA); squared criss-crossed = sequenced region (cDNA). ? = unsequenced intron region. Note: Aval is not a unique site. Figure 5 A is the nucleotide sequence of the cDNA12 promoter and coding sequence [A12DNA; SEQ ID 4]. The promoter sequence stops at base number 1101. The methionine codon of the start site of translation is located at base numbers 1102-1104 and is underlined. The total number of bases is 2175. The DNA sequence composition is 569A, 602C, 480G, 519T and 5 other.

Figure 6 shows the plasmid pEA155 which carries the promoter and coding region of the clone cDNA15 and the relevant features and sequenced areas. Diagonally hatched = insert; solid line = sequenced region (genomic DNA); squared criss-crossed = sequenced region (cDNA). Not all Pstl and EcoRI sites are shown. Figure 6A is the nucleotide sequence of the cDNA15 promoter and coding sequence [SΕQ ID 5]. The total number of bases is 2737. The DNA composition is 647A, 695C, 742G, 649T and 4 other.

Figure 7 shows plasmid pPLΕ3 which carries the eg/7 cDNA. Just above the plasmid map is the sequence of the adaptor molecule [SEQ ID 25] that was constructed to remove the small Sacll and Asp718 fragment from the plasmid so as to construct an exact joint [SEQ ID 26, SEQ ID 27] between the cbhl promoter and the egll signal sequences [SEQ IDs 18 and 16]. Figure 7A shows the 1588 bp sequence of the egll cDNA (369A, 527C, 418G and 274T) [SEQ ID 16]. Figure 7B shows the sequence of the 745 bp cbhl terminator of pPLE131 (198A, 191C, 177G, and 179T) [SEQ ID 23].

Figure 8 shows construction of plasmid pEM-3A and SEQ ID 28. The "A" on the plasmid maps denotes the EGI tail sequence and the "B" denotes the EGI hinge sequence.

Figure 9 shows the plasmid pTHNlOOB for expression of the EGIcore under the tefl promoter and SEQ ID 28. Figure 10 shows production of EGIcore from the plasmid pTHNlOOB into the culture medium of the host strain QM9414 analyzed by EGI specific antibodies from a slot blot. Lane 1: pTHN100B-16b, 200 μl glucose supernatant; lane 2: QM9414, 200 μl glucose supernatant; lane 3: TBS; lane 4: QM9414, 200 μl solka floe 1 :500 diluted supernatant; lane 5: QM9414, 200 μl solka floe 1:5,000 diluted supernatant; lane 6: QM9414, 200 μl solka floe 1:10,000 diluted supernatant; lane 7: pTHN100B-16b, 200 μl glucose 1:5 diluted supernatant; lane 8: QM9414, 200 μl glucose 1:5 diluted supernatant; lane 9: 200 ng EGI protein; lane 10: 100 ng EGI protein; lane 11: 50 ng EGI protein; and lane 12: 25 ng EGI protein.

Figure 11 shows Western blotting with EGI specific antibodies of culture medium of the strain pTHN100B-16c grown in whey-spent grain or glucose medium, and of EGIcore purified from the glucose medium. Lane 1: pTNH100B-16c, 10 μl whey spent grain supernatant; lane 2: pTNH100B-16c, 5 μl whey spent grain supernatant; lanes 3-5: EGIcore purified from pTHN100B-16c glucose fermentation; lane 6: pTHN100B-16c, 15 μl glucose fermenter supernatant, concentrated lOOx; lane 7: pTHN100B-16c, 7.5 μl glucose fermenter supernatant, concentrated lOOx; and lane 8: low molecular weight markers at 94kDa, 67 kDa, 43 kDa, 30 kDa and 20.1 kDa (bands 1-5 starting from lane 8, top of gel).

Figure 12 shows Western blotting of culture medium of the strain pTHN100B-16c grown on glucose medium. Lane 1: EGI protein, about 540 ng; lane 2, EGI protein, about 220 ng; lane 3, EGI protein, about 110 ng; lane 4: pTHN100B-16c, 30 μl glucose fermenter supernatant; lane 5: pTHN100B-16c, 30 μl glucose fermenter supernatant, concentrated 4.2x; lane 6: low molecular weight markers at 94kDa, 67 kDa, 43 kDa, 30 kDa and 20.1 kDa (bands 1-5 starting from lane 6, top of gel).

Figure 13 diagrams the elements of the plasmid pMLO16. Figure 13A is the sequence of the cbhl promoter of plasmid pML016 [SEQ ID18]. Figure 13B is the sequence of the T. reesei cbhl terminator on plasmid pML016 and plasmids derived from it [SEQ ID24]. JO-

Figure 14 shows the expression of -galactosidase on glucose medium in pMLO16del5(l l)-transformants of Trichoderma reesei QM 9414 (A2-F5).

Al: QM 9414 host strain; Cl and El: QM 9414 transformant in which one copy of 0-galactosidase expression cassette with intact cbhl promoter has replaced the cbhl locus; Bl, Dl and FI: empty wells.

Figure 15 shows the restriction map of the plasmid pMLO16del5(l l), which carries the shortened form of the cbhl promoter fused to the lacZ gene and the cbhl terminator. Figure 15A is the sequence of the truncated cbhl promoter [(pMLO16del5(l l)); SEQ ID19]. The polylinker is underlined. The arrow denotes the deletion site.

Figure 16 shows the restriction map of the plasmid pMLO17, which carries the shortened form of the cbhl promoter fused to the cbhl chromosomal gene. The restriction sites marked with a superscripted cross "⁺" are not single sites. There are two additional EcøRI sites in the cbhl gene that are not shown. Figure 16A shows the sequence of the Kspl-Xmal fragment (the underlined portion) that contains the chromosomal cbhl gene [SEQ ID 17].

Figure 17 shows the expression of CBHI on glucose medium in pMLO17 transformants of Trichoderma reesei QM 9414. A collection of single spore cultures (number and a letter-code) and different control samples are shown.

Figure 18 shows specific mutations of mig-like sequences (M) in cbhl promoters of pMI-24, pMI-25, pMI-26, pMI-27 and pMI-28. The promoters shown here were fused to lacZ gene and cbhl terminator as described for pMLO16 (see Figure 13) or pMLO16del0(2) (see Figure 19). *: sequence alteration made in cbhl promoter in different combinations. At position -1505-1500 the genomic sequence is 5'-CTGGGG and the altered sequence is 5'-TCTAAA. At position -1001-996 the genomic sequence is 5'-CTGGGG and the altered sequence is 5'-TCTAAA. At position -720-715 the genomic sequence is 5^'-GTGGGG and the altered sequence is 5^'-TCTAGA. pMLO16delO(2) was used as a starting vector for pMI-25, pMI-26, pMI-27 and pMI-28, pMLO16 for pMI-24. v = the polylinker. Figure 18A is the sequence of the altered cbhl promoter of pMI-24 (PMI27PROM) ([SEQ ID20]). The total number of bases is 1776. The sequence composition is 487A, 399C, 434G, and 456T. The polylinker is underlined and the sequence alteration is boxed. Figure 18B is the sequence of the altered cbhl promoter of pMI-27 ([SEQ ID21]). The polylinker is underlined, the arrow denotes the deletion point and the sequence alterations are boxed. Figure 18C is the sequence of the altered cbhl promoter of pMI-28 (PMI28PROM) ([SEQ ID22]). The polylinker is underlined, the arrow denotes the deletion point and the sequence alterations are boxed. The total number of bases is 1776. The sequence composition if 490A, 399C, 430G and 457T.

Figure 19 shows the restriction map of the plasmid pMLO16delO(2), which carries the shortened form of the cbhl promoter fused to lacZ gene and the cbhl terminator. Figure 20 shows the expression of β-galactosidase on indicated medium in Trichoderma reesei QM9414 transformed with pMLO16del0(2), pMI-25, pMI-27, pMI-28, pMLOlό and pMI-24.

Detailed Description of the Preferred Embodiments

I. Identification of Fungal Genes that Express on Glucose Medium

In the following description, reference will be made to various methodologies known to those of skill in the art of molecular genetics and biology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. General principles of the biochemistry and molecular biology of the filamentous fungi are set forth, for example, in Finkelstein, D.B. et al, eds., Biotechnology of Filamentous Fungi: Technology and Products, Butterworth- Heinemann, publishers, Stoneham, MA (1992) and Bennett, J.W. et al , More Gene Manipulations in Fungi, Academic Press - Harcourt Brace Jovanovich, publishers, San Diego CA (1991).

To be able to develop versatile systems for protein production from

Trichoderma, especially when Trichoderma are grown on glucose, a method has been developed for the isolation of previously unknown Trichoderma genes which are highly expressed on glucose, and their promoters. The method of the invention requires the use of only one cDNA population of probes.

It is to be understood that the method of the invention would be useful for the identification of promoter sequences that are active under any desired environmental condition to which a cell could be exposed, and not just to the exemplified isolation of promoters that are capable of expression in glucose medium. By "environmental condition" is meant the presence of a physical or chemical agent, such agent being present in the cellular environment, either extracellularly or intracellularly. Physical agent would include, for example, certain growth temperatures, especially a high or low temperature. Chemical agents would include any compound or mixtures including carbon growth substrates, drugs, atmospheric gases, etc.

According to the method of the invention, the organism is first grown under the desired growth condition, such as the use of glucose as a carbon source. Total mRNA is then extracted from the organism and preferably purified through at least a polyA+ enrichment of the mRNA from the total RNA population. A cDNA bank is made from this total mRNA population using reverse transcriptase and the cDNA population cloned into any appropriate vector, such as the commercially available lambda-ZAP vector system (Stratagene). When using the lambda-ZAP vector system, or any lambda vector system, the cDNA is packaged such that it is suitable for infection of any E. coli strain susceptable to lambda bacteriophage infection.

The cDNA bank is transferred by standard colony hybridization techniques onto nitrocellulose filters for screening. The bank is plated and plaque lifts are taken onto nitrocellulose. The bank is screened with a population of labelled cDNAs that had been synthesized against the same RNA population from which the cloned cDNA bank was constructed, using stringent hybridization conditions. It should be noted that the genes are not expressed in any way during this selection process. This results in clones hybridizing with varying intensity and the ones showing the strongest signals are picked. Genes that are most strongly expressed in the original population comprise the majority of the total mRNA pool and thus give a strong signal in this selection.

The inserts in clones with the strongest signals are sequenced from the 3 'end of the insert using any standard DNA sequencing technique as known in the art. This provides a first identification of each clone and allows the exclusion of identical clones. The frequency with which each desired clone is represented in the cDNA lambda-bank is determined by hybridizing the bank against a clone-specific PCR probe. The desired clones are those which, in addition to having the strongest signals as above, are also represented at the highest frequencies in the cDNA bank, since this implies that the abundancy of the mRNA in the population was relatively high and thus that the promoter for that gene was highly active under the growth conditions. Thus, the relevance of this approach and any clone identified therefrom can be double- checked: the intensity of the hybridization signal of a specific clone should correlate positively with the frequency with which that clone is found in the cDNA bank. The inserts of the clones selected in this manner, such inserts corresponding to the cDNA sequences, may be used as probes to isolate the corresponding genes and their promoters from a chromosomal bank, such as one cloned into lambda as above. The method of the invention is not limited to Trichoderma, but would be using for cloning genes from any host, or from a specific tissue with such host, from which a cDNA bank may be constructed, including, prokaryote (bacterial) hosts, and any eukaryotic host plants, mammals, insects, yeast, and any cultured cell populations. For example, using the method of the invention, five genes that express relatively high levels of mRNA in Trichoderma reesei when such Trichoderma are grown on glucose were identified. These genes were sequenced and identified as clone cDNA33, cDNAl, cDNAlO, cDNA12, and cDNA15. When used to screen a Trichoderma chromosomal lambda-bank, the corresponding genes and their promoters were identified. Such genes and promoters (or portions thereof) may then be subcloned into any desired vector, such as the pSP73 vector (Promega, Madison, WI, USA).

According to the invention, the clones containing the genes and their promoters (or parts of them) highly expressed in Trichoderma grown on glucose are represented as follows:

One of the genes isolated according to the invention as being highly expressed when Trichoderma was grown on glucose has been identified as the one encoding Trichoderma translation elongation factor lα (tefl). In addition, four other, new genes have been identified for the first time that are highly expressed on glucose in Trichoderma.

These data show that the method used in this invention resulted in isolating five genes, one of which (tef7) is known to be efficiently expressed in other organisms. However, the tefl gene was not the most highly expressed of the five genes isolated from the Trichoderma cDNA bank by the method of the invention.

Of the five genes isolated, only tef7 shows a relevant degree of homology to any known protein sequences. All of the genes isolated are also expressed on other carbon sources and would not have been found with the classical method of differential cloning. This shows the importance of the method used in this invention in isolation of the most suitable genes for a specific purpose, such as for isolation of strong promoters for expression on glucose containing medium. The promoter of any of these genes may be operably linked to a sequence heterologous to such promoter, and especially heterologous to the host Trichoderma, for expression of such gene from a Trichoderma host that is grown on glucose. Preferably, the coding sequence provides a secretion signal for secretion of the recombinant protein into the medium. Use of the promoters of the invention allow for the expression of genes from Trichoderma under conditions in which there are no cellulases and relatively few proteases. Thus, for the first time, recombinant genes can be highly expressed on Trichoderma using a glucose-based growth medium.

The promoters of the invention, while being strongly expressed on glucose (that is, when the filamentous fungal host is grown on medium providing glucose as a carbon and energy source), are not repressed in the absence of glucose. In addition, they are active when the Trichoderma host is grown on carbon sources other than glucose.

The glucose promoters of the invention, and those identified by the methods of the invention, can be used to produce enzymes native to Trichoderma itself, especially of those capable of hydrolysing different kinds of plant material. On glucose, the fungus does not naturally produce these enzymes and consequently one or more specific hydrolytic enzymes could be produced on glucose medium free from other plant material hydrolyzing enzymes. This would result in an enzyme preparate or enzyme mixtures for specific applications. II. Modification of the Cellobiohydrolase I Promoter

This invention also describes a method for the modification of the cellobiohydrolase 1 promoter (cbhl) such that the activity of the promoter is retained but the promoter no longer is repressed when cells are grown on glucose-containing medium. Essentially, the DNA motif that imparted glucose repression has been identified and removed from this promoter, allowing production of desired proteins whose coding sequences are operably linked to the promoter in suitable hosts, such as Trichoderma. Such a modified cbhl promoter is termed a derepressed cbhl promoter. As above, when the recombinant organisms obtained from transformation with such constructs are cultivated on glucose containing medium, any protein, including a cellulase may be produced without production of other plant material hydrolysing enzymes, especially of native cellulases.

Isolated glucose promoters or derepressed cbhl promoter can be used for instance to produce separate individual cellulases in hosts grown on glucose without any simultaneous production of other hydrolases such as other cellulases, hemicellulases, xylanases etc. or to produce heterologous proteins in varying growth media.

III. Preparation of Coding Sequences Operably Linked to the Promoter Sequences of the Invention

The process for genetically engineering a coding sequence, for expression under a promoter of the invention, is facilitated through the isolation and partial sequencing of pure protein encoding an enzyme of interest or by the cloning of genetic sequences which are capable of encoding such protein with polymerase chain reaction technologies; and through the expression of such genetic sequences. As used herein, the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences that are capable of encoding a protein are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof. The preferred source of genomic DNA is a fungal genomic bank. The preferred source of the cDNA is a cDNA bank prepared from fungal mRNA grown in conditions known to induce expression of the desired gene to produce mRNA or protein. However, since the genetic code is universal, a coding sequence from any host, including prokaryotic (bacterial) hosts, and any eukaryotic host plants, mammals, insects, yeasts, and any cultured cell populations would be expected to function (encode the desired protein). Genomic DNA may or may not include naturally occurring introns.

Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the gene sequences and/or with the 3' transcriptional termination region. According to the invention however, the native promoter region would be replaced with a promoter of the invention. Such genomic DNA may also be obtained in association with the genetic sequences which encode the 5' non-translated region of the mRNA and/or with the genetic sequences which encode the 3' non-translated region. To the extent that a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5' and/or 3' non-transcribed regions of the native gene, and/or, the 5' and/or 3' non-translated regions of the mRNA may be retained and employed for transcriptional and translational regulation.

Genomic DNA can be extracted and purified from any host cell, especially a fungal host cell, which naturally expresses the desired protein by means well known in the art. A genomic DNA sequence may be shortened by means known in the art to isolate a desired gene from a chromosomal region that otherwise would contain more information than necessary for the utilization of this gene in the hosts of the invention. For example, restriction digestion may be utilized to cleave the full-length sequence at a desired location. Alternatively, or in addition, nucleases that cleave from the 3'-end of a DNA molecule may be used to digest a certain sequence to a shortened form, the desired length then being identified and purified by gel electrophoresis and DNA sequencing. Such nucleases include, for example, Exonuclease III and fiα/31. Other nucleases are well known in the art.

For cloning into a vector, such suitable DNA preparations (either genomic DNA or cDNA) are randomly sheared or enzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) bank.

A DNA sequence encoding a desired protein or its functional derivatives may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., (Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, second edition, 1988) and are well known in the art.

Libraries containing sequences coding for the desired gene may be screened and the desired gene sequence identified by any means which specifically selects for a sequence coding for such gene or protein such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone.

Oligonucleotide probes specific for a certain protein which can be used to identify clones to this protein can be designed from the knowledge of the amino acid sequence of the protein or from the knowledge of the nucleic acid sequence of the DNA encoding such protein or a related protein.

Alternatively, antibodies may be raised against purified forms of the protein and used to identify the presence of unique protein determinants in transformants that express the desired cloned protein. When an amino acid sequence is listed horizontally, unless otherwise stated, the amino terminus is intended to be on the left end and the carboxy terminus is intended to be at the right end. Similarly, unless otherwise stated or apparent from the context, a nucleic acid sequence is presented with the 5' end on the left.

Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Peptide fragments may be analyzed to identify sequences of amino acids that may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.

Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide sequence, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide. Using the genetic code, one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the desired protein. The probability that a particular oligonucleotide will, in fact, constitute the actual protein encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Using "codon usage rules," a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the protein sequences is identified.

The suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of a certain gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., 1992, IRL Press, New York) and employed as a probe to identify and isolate a clone to such gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, T., et al , in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), and by Hames, B.D., et al, in: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)). Those members of the above-described gene bank which are found to be capable of such hybridization are then analyzed to determine the extent and nature of coding sequences which they contain.

To facilitate the detection of a desired DNA coding sequence, the above-described DNA probe is labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as ³²P, ³H, ^MC, ^3SS, ^,2SI, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. If single stranded, the oligonucleotide may be radioactively labelled using kinase reactions. Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group. Thus, in summary, the elucidation of a partial protein sequence, permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe(s) for the identification and isolation of clones containing a gene.

In an alternative way of cloning a gene, a bank is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing the protein into an expression vector. The bank is then screened for members which express the desired protein, for example, by screening the bank with antibodies to the protein.

The above discussed methods are, therefore, capable of identifying genetic sequences that are capable of encoding a protein or biologically active or antigenic fragments of this protein. The desired coding sequence may be further characterized by demonstrating its ability to encode a protein having the ability to bind antibody in a specific manner, the ability to elicit the production of antibody which are capable of binding to the native, non- recombinant protein, the ability to provide a enzymatic activity to a cell that is a property of the protein, and the ability to provide a non-enzymatic (but specific) function to a recipient cell, among others.

In order to produce the recombinant protein in the vectors of the invention, it is desirable to operably link such coding sequences to the glucose regulatable promoters of the invention. When the coding sequence and the operably linked promoter of the invention are introduced into a recipient eukaryotic cell (preferably a fungal host cell) as a non-replicating DNA (or

RNA), non-integrating molecule, the expression of the encoded protein may occur through the transient (nonstable) expression of the introduced sequence.

Preferably the coding sequence is introduced on a DNA molecule, such as a closed circular or linear molecule that is incapable of autonomous replica- tion, Preferably, a linear molecule that integrates into the host chromosome. Genetically stable transformants may be constructed with vector systems, or transformation systems, whereby a desired DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or, be assisted by transformation with a vector which functionally inserts itself into the host chromosome. The gene encoding the desired protein operably linked to the promoter of the invention may be placed with a transformation marker gene in one plasmid construction and introduced into the host cells by transformation, or, the marker gene may be on a separate construct for co-transformation with the coding sequence construct into the host cell. The nature of the vector will depend on the host organism. In the practical realization of the invention the filamentous fungus Trichoderma has been employed as a model. Thus, for Trichoderma and especially for T. reesei, vectors incorporating DNA that provides for integration of the expression cassette (the coding sequence operably linked to its transcriptional and translational regulatory elements) into the host's chromosome are preferred. It is not necessary to target the chromosomal insertion to a specific site. However, targeting the integration to a specific locus may be achieved by providing specific coding or flanking sequences on the recombinant construct, in an amount sufficient to direct integration to this locus at a relevant frequency. Cells that have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co- transformation. A genetic marker especially for the transformation of the hosts of the invention is amdS, encoding acetamidase and thus enabling Trichoderma to grow on acetamide as the only nitrogen source. Selectable markers for use in transforming filamentous fungi include, for example, acetamidase (the amdS gene), benomyl resistance, oligomycin resistance, hygromycin resistance, aminoglycoside resistance, bleomycin resistance; and, with auxotrophic mutants, ornithine carbamoyltransferase (OCTase or the argB gene). The use of such markers is also reviewed in Finkelstein, D.B. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 6, Finkelstein, D.B. et al, eds., Butterworth-Heinemann, publishers, Stoneham, MA, (1992), pp. 113-156).

To express a desired protein and/or its active derivatives, transcriptional and translational signals recognizable by an appropriate host are necessary. The cloned coding sequences, obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell, either prokaryote or eukaryote, to produce recombinant protein or a functional derivative thereof. Depending upon which strand of the coding sequence is operably linked to the sequences controlling transcriptional expression, it is also possible to express antisense RNA or a functional derivative thereof.

Expression of the protein in different hosts may result in different post- translational modifications which may alter the properties of the protein. Preferably, the present invention encompasses the expression of the protein or a functional derivative thereof, in eukaryotic cells, and especially in fungus.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are

"operably linked" to the nucleotide sequence which encodes the polypeptide. An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence. Two DNA sequences (such as a coding sequence and a promoter region sequence linked to the 5' end of the coding sequence) are said to be operably linked if induction of promoter function results in the transcription of mRNA encoding the desired protein and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the protein, antisense RNA, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably linked to a DNA sequence if the promoter was capable of effecting transcription of that DNA sequence.

The precise nature of the regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like, with those elements necessary for the promoter sequence being provided by the promoters of the invention. Such transcriptional control sequences may also include enhancer sequences or upstream activator sequences, as desired. Expression of a protein in eukaryotic hosts such as fungus requires the use of regulatory regions functional in such hosts, and preferably fungal regulatory systems. A wide variety of transcriptional and translational regu¬ latory sequences can be employed, depending upon the nature of the host. Preferably, these regulatory signals are associated in their native state with a particular gene which is capable of a high level of expression in the host cell.

In eukaryotes, where transcription is not linked to translation, such control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. Promoters from filamentous fungal genes which encode a mRNA product capable of translation are preferred, and especially, strong promoters can be employed provided they also function as promoters in the host cell.

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the desired protein, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine. The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the protein-coding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the protein-coding sequence).

It may be desired to construct a fusion product that contains a partial coding sequence (usually at the amino terminal end) of a protein and a second coding sequence (partial or complete) of a second protein. The first coding sequence may or may not function as a signal sequence for secretion of the protein from the host cell. For example, the sequence coding for desired protein may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host. Such fusion protein sequences may be designed with or without specific protease sites such that a desired peptide sequence is amenable to subsequent removal. In a preferred embodiment, the native signal sequence of a fungal protein is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the peptide that is operably linked to it. Aspergillus leader/secretion signal elements also function in Trichoderma. If desired, the non-transcribed and/or non-translated regions 3' to the sequence coding for a desired protein can be obtained by the above-described cloning methods. The 3 '-non-transcribed region may be retained for its transcriptional termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells. Where the native expression control sequences signals do not function satisfactorily in a host cell, then sequences functional in the host cell may be substituted.

The vectors of the invention may further comprise other operably linked regulatory elements such as DNA elements which confer antibiotic resistance, or origins of replication for maintenance of the vector in one or more host cells. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

Once the vector or DNA sequence containing the construct(s) is prepared for expression, the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. If this medium includes glucose, expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein as desired. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner, for example, by induction of expression.

Fungal transformation is carried out also accordingly to techniques known in the art, for example, using, for example, homologous recombination to stably insert a gene into the fungal host and/or to destroy the ability of the host cell to express a certain protein.

Fungi- useful as recombinant hosts for the purpose of the invention include, e.g., Trichoderma, Aspergillus, Claviceps purpurea, Penicillium chrysogenum, Magnaporthe grisea, Neurospora, Mycosphaerella spp. , Collectotrichum trifolii, the dimorphic fungus Histoplasmia capsulatum, Nectia haematococca (anamorph:F«αri#rn solani f. sp. phaseoli and f. sp. pisi), Ustilago violacea, Ustilago maydis, Cephalosporium acremonium, Schizophyllum commune, Podospora anserina, Sordaria macrospora, Mucor circinelloides, and Collectotrichum capsici. Transformation and selection techniques for each of these fungi have been described (reviewed in Finkelstein, D.B. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 6, Finkelstein, D.B. et al, eds., Butterworth-Heinemann, publishers, Stoneham, MA, (1992), pp. 113-156). Especially preferred are Trichoderma reesei, T. harzianum, T. longibrachiatum, T. viride, T. koningii, Aspergillus nidulans, A. niger, A. terreus, A. ficum, A. oryzae, A. awamori and Neurospora crassa. The hosts of the invention are meant to include all Trichoderma.

Trichoderma are classified on the basis of morphological evidence of similarity. T. reesei was formerly known as T. viride Pers. or T. koningii Oudem; sometimes it was classified as a distinct species of the T. longibrachiatum group. The entire genus Trichoderma, in general, is characterized by rapidly growing colonies bearing tufted or pustulate, repeatedly branched conidiophores with lageniform phialides and hyaline or green conidia borne in slimy heads (Bissett, J., Can. J. Bot. 62:924-931 (1984)).

The fungus called T. reesei is clearly defined as a genetic family originating from the strain QM6a, that is, a family of strains possessing a common genetic background originating from a single nucleus of the particular isolate QM6a. Only those strains are called T. reesei.

Classification by morphological means is problematic and the first recently published molecular data from DNA-fingerprint analysis and the hybridization pattern of the cellobiohydrolase 2 (cbh2) gene in T. reesei and T. longibrachiatum clearly indicates a differentiation of these strains (Meyer, W. et al, Curr. Genet. 27:27-30 (1992); Morawetz, R. et al , Curr. Genet. 27:31-36 (1992).

However, there is evidence of similarity between different Trichoderma species at the molecular level that is found in the conservation of nucleic acid and amino acid sequences of macromolecular entities shared by the various Trichoderma species. For example, Cheng, C, et al, Nucl. Acids. Res. 18:5559 (1990), discloses the nucleotide sequence of T. viride cbhl. The gene was isolated using a probe based on the T. reesei sequence. The authors note that there is a 95% homology between the amino acid sequences of the T. viride and T. reesei gene. Goldman, G.H. et al, Nucl. Acids Res. 18:6111 (1990), discloses the nucleotide sequence of phosphoglycerate kinases from T. viride and notes that the deduced amino acid sequence is 81 % homologous with the phosphoglycerate kinase gene from T. reesei. Thus, the species classified to T. viride and T. reesei must genetically be very close to each other.

In addition, there is a high similarity of transformation conditions among the Trichoderma. Although practically all the industrially important species of Trichoderma can be found in the formerly discussed Trichoderma section Longbrachiatum, there are some other species of Trichoderma that are not assigned to this section. Such a species is, for example, Trichoderma harzianum, which acts as a biocontrol agent against plant pathogens. A transformation system has also been developed for this Trichoderma species (Herrera-Estrella, A. et al, Molec. Microbiol. 4:839-843 (1990) that is essentially the same as that taught in the application. Thus, even though Trichoderma harzianum is not assigned to the section Longibrachiatum, the method used by Herrera-Estrella in the preparation of spheroplasts before transformation is the same. The teachings of Herrera-Estrella show that there is not a significant diversity of Trichoderma spp. such that the transformation system of the invention would not be expected to function in all Trichoderma. Further, there is a common functionality of fungal transcriptional control signals among fungal species. At least three A. nidulans promoter sequences, amdS, argB, and gpd, have been shown to give rise to gene expression in T. reesei. For amdS and argB, only one or two copies of the gene are sufficient to being about a selectable phenotypes (Penttila et al, Gene 67: 155-164 (1987). Gruber, F. et al, Curr. Genetic 18:11-16 (1990) also notes that fungal genes can often be successfully expressed across different species. Therefore, it is to be expected that the glucose regulated promoters identified herein would be also regulatable by glucose in other fungi. Except for cbhl, it is understood that the glucose regulated promoters of the invention may not be directly regulated by glucose, but rather that they function regardless of its presence. Many species of fungi, and especially Trichoderma, are available from a wide variety of resource centers that contain fungal culture collections. In addition, Trichoderma species are catalogued in various databases. These resources and databases are summarized by O'Donnell, K. et al, in Biochemistry of Filamentous Fungi: Technology and Products, D.B. Fingelstein et al, eds., Butterworth-Heinemann, Stoneham, MA, USA, 1992, pp. 3-39.

After the introduction of the vector and selection of the transformant, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the synthesis and secretion of the desired heterologous or homologous protein, or in the production of a fragment of this protein, into the medium of the host cell.

In a preferred embodiment, the coding sequence is the sequence of an enzyme that is capable of hydrolysing lignocellulose. Examples of such sequences include a DNA sequence encoding cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII), endoglucanase I (EGI), endoglucanase II (EGII), endoglucanase III (EGIII), /3-glucosidases, xylanases (including endoxylanases and 3-xylosidase), side-group cleaving activities, (for example, a- arabinosidase, α-D-glucuronidase, and acetyl esterase) , mannanases, pectinases (for example, endo-polygalacturonase, exo-polygalacturonase, pectinesterase, or, pectin and pectin acid lyase), and enzymes of lignin polymer degradation, (for example, lignin peroxidase LIII from Phlebia radiata (Saloheimo et al, Gene 55:343-351 (1989)), or the gene for another ligninase, laccase or Mn peroxidase (Kirk, In: Biochemistry and Genetics of Cellulose Degradation, Aubert et al. (eds.), FEMS Symposium No. 43, Academic Press, Harcourt, Brace Jovanovitch Publishers, London, pp. 315-332 (1988))). The cloning of the cellulolytic enzyme genes has been described and recently reviewed (Teeri, T.T. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 14, Finkelstein, D.B. etal. , eds. , Butterworth-Heinemann, publishers, Stoneham, MA, (1992), pp. 417-445). The gene for the native cellobiohydrolase CBHI sequence has been cloned by Shoemaker et al. (Shoemaker, S., et al, Bio/Technology 7:691-696 (1983)) and Teeri et al. (Teeri, T., et al , Bio/Technology 7:696-699 (1983)) and the entire nucleotide sequence of the gene is known (Shoemaker, S., et al , Bio/Technology 7:691- 696 (1983)). From T. reesei, the gene for the major endoglucanase (EGI) has also been cloned and characterized (Penttila, M., et al , Gene 45:253-263 (1986); Patent Application EP 137,280; Van Arstel, J.N.V., et al , Bio/Technology 5:60-64). Other isolated cellulase genes include cbhl (Patent Application WO 85/04672; Chen, CM., et al, Bio /Technology 5:274-278 (1987)) and egl3 (Saloheimo, M., et al, Gene 65: 11-21 (1988)). The genes for the two endo-/3-xylanases of T. reesei (xlnl and xln2 have been cloned and described in applicants' copending application, U.S. 07/889,893, filed May 29, 1992. The xylanase proteins have been purified and characterized (Tenkanen, M. et al, Proceeding of the Xylans and Xylanases Symposium, Wageningen, Holland (1991)).

The expressed protein may be isolated and purified from the medium of the host in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. For example, the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid- agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.

The manner and method of carrying out the present invention may be more fully understood by those of skill by reference to the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto. Example 1 Isolation of Trichoderma reesei Genes Strongly Expressed on Glucose

For the isolation of glucose induced mRNA Trichoderma reesei strain QM9414 (Mandels, M. et al. , Appl. Microbiol. 27: 152-154 (1971)) was grown in a 10 liter fermenter in glucose medium (glucose 60 g/1, Bacto-Peptone 5 g/1, Yeast extract 1 g/1, KH₂PO₄4 g/1, (NH₄)₂SO₄4 g/1, MgSO₄0.5 g/1, CaCl₂ 0.5 g/1 and trace elements FeSO₄*7H₂O 5 mg/1, MnSO₄ ^«H₂O 1.6 mg/1, ZnSO₄»7H₂O 1.4 mg/1, and CoCl₂ ^«6H₂O 3.7 mg/1, pH 5.0-4.0). Glucose feeding (465g/20h) was started after 30 hours of growth. Mycelium was harvested at 45 hours of growth and RNA was isolated according to Chirgwin, J.M. et al , Biochem. J. 78:5294-5299 (1979)). Poly A+ RNA was isolated from the total RNA by oligo(dT)-cellulose chromatography (Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)) and cDNA synthesis and cloning of the cDNAs was carried out according to manufacturer's instructions into lambda-ZAP vector (ZAP-cDNA synthesis kit, Stratagene). The cDNA bank was transferred onto nitrocellulose filters and screened with ³²P-labelled single- stranded cDNA synthesized (Teeri, T.T. et al, Anal. Biochem. 764:60-67 (1987)) from the same poly A+ RNA from which the bank was constructed. The labelled cDNA was relabelled with ³²P-dCTP (Random Primed DNA Labeling kit, Boehringer-Mannheim). The hybridization conditions were as described in Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Fifty clones giving the strongest positive reaction were isolated and the cDNAs were subcloned in vivo into Bluescript SK(-) plasmid according to manufacturer's instructions (ZAP-cDNA synthesis kit, Stratagene).

To identify the clones and exclude the same ones they were all sequenced from the 3' end by using standard methods. The frequency of each specific clone in the cDNA lambda-bank was determined by hybridizing the bank with a clone specific PCR probe. The clones cDNA33, cDNAl , cDNAlO, cDNA12, cDNA15, showing the five highest frequencies corresponded to 1-3% of the total mRNA pool.

Example 2

Characterization of Isolated Glucose Expressed Trichoderma Genes and Their Promoters

The cDNAs of the clones cDNA33, cDNAl, cDNAlO, cDNA12, and cDNA15 were used as probes to isolate the corresponding genes and promoters from a Trichoderma chromosomal lambda-bank prepared earlier (Vanhanen, S. et al, Curr. Genet. 75: 181-186 (1989)). On the basis of Southern analysis of restriction enzyme digestions carried out for the chromosomal lambda clones, the promoters and either the 5' parts of the chromosomal genes or the whole genes were subcloned into pSP73 vector (Promega, Madison, USA) using appropriate restriction enzymes yielding the plasmids pTHNl (Figure 1), pEA33 (Figure 2), pTHN3 (Figure 3), pEAlO (Figure 4), pEA12 (Figure 5) and pEA155 (Figure 6), corresponding to the clones cDNA33, cDNAl, cDNAlO, cDNA12 and cDNA15, respectively. Sequences were obtained from the 5' ends of the genes and from the promoters using primers designed from previously obtained sequences. The sequences of the isolated promoters and genes or parts of them (either obtained from cDNA or chromosomal DNA) are shown in SEQ ID1 for cDNA33, SEQ ID2 for cDNAl, SEQ ID3 for cDNAlO, SEQ ID4 for cDNA12, and SEQ ID5 for cDNA15. Based on sequence similarity to known sequences in a protein data bank the clone cDNA33 could be identified as a translation elongation factor, TEFlα. Example 3

Construction of Vectors for Expression ofEGI-core under the tefl-Promoter in Trichoderma

A Xhol + Dralll fragment that is internal to the egll cDNA [SEQ ID 16 and Figure 7 A] sequence of plasmid pPLE3 (Figure 7) carrying the EcøRI - BamHl fragment of egll cDNA from pTTcll (Penttila et al, Gene 45:253- 263 (1986); Penttila et al, Yeast 3: 175-185 (1987) inbetween the cbhl promoter and c. 700 nt long Avail terminator fragment was replaced by a Xhol-Drάlll fragment of cDNA from plasmid pΕG131 (Nitisinprasert, S. , Reports from Department of Microbiology, University of Helsinki (1990)). The pPEG131 insert sequence is egll cDNA in which a STOP codon is constructed just before the hinge region of the egll gene. The cbhl terminator sequence is Figure 7B [SEQ ID 23]. SEQ ID 23 is a shortened cbhl terminator sequence, similar to SEQ ID 24 (the "long" cbhl terminator but lacking 30 nucleotides at the 5' end). pPLE3 contains a pUC18 backbone, and carries the cbhl promoter inserted at the EcoRl site. The cbhl promoter is operably linked to the full length egll cDNA coding sequence and to the cbhl transcriptional terminator. The ori and amp genes are from the bacterial plasmid. The resulting plasmid pEM-3 (Figure 8) now carries a copy of egll cDNA with a translational stop codon after the egll core region (EGI amino acids 1-22 are the EGI signal sequence; EGI amino acids 23-393, terminating at a Thr, are considered the 'core' sequence). pEM-3 was then digested with EcøRI and Sphl and the released Bluescribe M13+ moiety (Vector Cloning Systems, San Diego, USA) of the plasmid was replaced by EcøRI and Sphl digested pAMD (Figure 8) containing a 3.4 kb amdS fragment from plasmid p3SR2 (Hynes, M.J. et al, Mol. Cell. Biol. 3: 1430-1439 (1983); Tilburn, J. et al, Gene 26:205-221 (1983). This resulting plasmid pΕM-3A (Figure 8) was digested with EcøRI and partially with Kspl to release the 2.3 kb fragment carrying the cM7-promotor and the 8.6 kb fragment carrying the rest of the plasmid was purified from agarose gel. Based on the sequence data of the tefl promoter (SEQ ID1 bases 1-1234), two primers were designed (SEQ ID6 and SEQ 1D7) and used in a PCR reaction to isolate a 1.2 kb promoter fragment adjacent to the translational start site of the tefl gene. The 5' primer was ACCGGAATTCATATCTAGAGGAGCCCGCGAGTTTGGATACGCC (SEQ ID6) and the 3' primer was

ACCGCCGCGGTTTGACGGTΓTGTGTGATGTAGCG (SEQ ID7). The bold and underlined GAATTC in the 5' primer is an EcøRI site. The bold and underlined TCTAGA in the 5' primer is an Xbal site. The bold and underlined CCGCGG in the _.' primer is a SACII site. This fragment was digested with EcøRI and partially with Kspl and purified from agarose gel and ligated to the 8.6 kb pΕM-3A fragment resulting in plasmid pTHNlOOB (Figure 9). This expression vector carries DNA encoding the EGI-core construction operably linked to the tefl promoter; this plasmid also carries an amdS marker gene for selection of Trichoderma transformants.

Example 4

Transformation of Trichoderma, Purification of the EGI-Core Producing Clones and Their Analysis

Trichoderma reesei strain QM9414 was transformed essentially as described (Penttila, M. et al, Gene 67: 155-164 (1987) using 6-10 μg of the plasmid pTHNlOOB. The Amd⁺ transformants obtained were streaked twice onto slants containing acetamide (Penttila, M. et al. Gene 67: 155-164 (1987)). Thereafter spore suspensions were made from transformants grown on Potato Dextrose agar (Difco). EGI-core production was tested by slot blotting with EGI specific antibody from 50 ml shake flask cultures carried out in minimal medium (Penttila, M. et al. Gene 67: 155-164 (1987)) supplemented with 5 % glucose and using additional glucose feeding (total amount of fed glucose was 6 ml of 20% glucose). The spore suspensions of the EGI-core producing clones were purified to single spore cultures on Potato Dextrose agar plates. EGI-core production was analyzed again from these purified clones as described above (Figure 10).

Example 5 Characterization of EGI-core produced by Trichoderma Grown on Glucose

EGI-core producing strain pTHN100B-16c was grown in a 10 liter fermenter in glucose medium as described earlier in Example 1 except that yeast extract was left out and glucose feeding was 555g/22h. The culture supernatant was separated from the mycelium by centrifugation. The secretion of EGI-core by Trichoderma was verified by Western blotting by conventional methods running concentrated culture supematants on SDS-PAGE and treating the blotted filter with monoclonal EGI-core specific antibodies (Figure 11 and Figure 12). The enzyme activity was shown semiquantitatively in a microtiter plate assay by using the concentrated culture supematants and 3 mM chloronitrophenyl lactocide as a substrate and measuring the absorbance at 405 nm (Clayessens, M. et al., Biochem. J. 267:819-825 (1989).

Example 6

Construction of β-Galactosidase Expression Vectors with Truncated Fragments of the cbhl-Promoter

The vector pMLO16 (Figure 13) contains a 2.3 kb cbhl promoter fragment ([SEQ ID18, Figure 13A) starting at 5' end from the EcøRI site, isolated from chromosomal gene bank of Trichoderma reesei (Teeri, T. et al., J. Bio/Technology 7:696-699 (1983)), a 3J kb BamWl fragment of the lacZ gene from plasmid pAN924-21 (van Gorcom et al, Gene 40:99-106 (1985)) and a 1.6 kb cbhl terminator (Figure 13B, [SΕQ ID 24]) starting from 84 bp upstream from the translation stop codon and extending to a BamHl site at the 3' end (Shoemaker, S. et al, Bio/Technology 7:691-696 (1983); Teeri, T. et al, Bio/Technology 7:696-699 (1983)). These pieces were linked to a 2.3 kb long EcoRI-TVttll region of pBR322 (Sutcliffe, J.G., Cold Spring Harbor Symp. Quant. Biol. 43:11-90 (1979)) generating junctions as shown in Figure 13. The exact in frame joint between the 2.3 kb cbhl promoter and the 3.1 kb lacZ gene was constructed by using an oligo depicted in Figure 13. A polylinker shown in Figure 13 was cloned into the single internal Xbal site in the cbhl promoter for the purpose of promoter deletions. A short 5c7I linker shown in Figure 13 was cloned into the joint between the pBR322 and cbhl promoter fragments so that the expression cassette can be released from the vector by restriction digestion with Sail and Sphl. Progressive unidirectional deletions were introduced to the cM7 promoter by cutting the vector with Kpnl and Xhol and using the Εrase-A-Base System (Promega, Madison, USA) according to manufacturer's instructions. Plasmids obtained from different deletion time points were transformed into the E. coli strain DH5α (BRL) by the method described in (Hanahan D., J. Mol. Biol. 766:557-580 (1983)) and the deletion end points were sequenced by using standard methods.

Example 7

Transformation of Trichoderma, Isolation of the β-Galactosidase Producing Clones and Their Analysis

Trichoderma reesei strain QM9414 was transformed with expression vectors for β-galactosidase containing either the intact 2.3 kb cbhl promoter or truncated versions of it, generated as explained in Example 6. Twenty μg of the plasmids were digested with Sail and Sphl to release the expression cassettes from the vectors and these mixtures were cotransformed to Trichoderma together with 3 μg of plasmid p3SR2 (Hynes, M.J. et al , Mol. Cell. Biol 3: 1430-1439 (1983)) containing the acetamidase gene. The transformation method was that described in (Penttila, M. et al. Gene 67: 155- 164 (1987)) and the Amd⁺ transformants were screened as described earlier in Example 4. The β-galactosidase production of the Amd⁺ transformants was tested by inoculating spore suspensions on microtiter plate wells containing solid minimal medium (Penttila, M. et al. Gene 67: 155-164 (1987)) supplemented with 2% glucose, 2% fructose and 0.2% peptone and pH adjusted to 7. After 24 h incubation in 28 °C, 10 μl of the chromogenic substrate X-gal (20 mg/ml) was added to each well and the formation of blue color was followed as an indication of 0-galactosidase activity. An intense blue color could be detected in transformants transformed with a plasmid pMLO16del5(l l) (Figure 14) containing a 1110 bp deletion in the cbhl promoter beginning from the promoter internal polylinker and ending 385 bp before the translation initiation site (Figure 15). The sequence of this truncated promoter is provided as SEQ ID 19 (Figure 15 A).

Example 8

Production of CBHI on Glucose with the Glucose-Derepressed cbhl- Promoter

For the production of CBHI on glucose an expression plasmid pMLO 17 (Figure 16) was constmcted. The plasmid pMLO16del5(ll) was digested with the enzymes Kspl (the first nucleotide of the recognition sequence is at the position -16 from the ATG) and Xmal (the first nucleotide of the recognition sequence is 76 nucleotides downstream from the translation stop codon of the cbhl gene). The vector part containing the shortened cbhl promoter, the cbhl terminator and the pBR322 sequence was ligated to the chromosomal cbhl gene isolated as a £spI-XmαI-fragment from the chromosomal gene bank of Trichoderma reesei (Teeri, T. et al , Bio/Technology 7:696-699 (1983)). The sequence of this fragment is provided as the underlined portion of Figure 16A ([SEQ ID17]). The plasmid pMLO17 was transformed to the Trichoderma reesei strain QM 9414 and the Amd⁺ transformants were screened as described earlier in example 7. CBHI production was tested from 40 transformants in microtiter plate cultures (200 μl; 3 days) carried out in minimal medium (Penttila, M. et al. Gene 67: 155- 164 (1987) supplemented with 3 % glucose and using additional glucose feeding (total amount of fed glucose was 6 mg/200 μl culture). The culture supematants were slot blotted on nitrocellulose filters and CBHI was detected with specific antibody. The spore suspensions of the 10 best CBHI producing transformants were purified to single spore cultures on plates containing acetamide and Triton X-100 (Penttila, M. et al, Gene 67: 155-164 (1987)). Thirty single spore cultures were tested for CBHI production in shake flask cultivations (50 ml; 6 days) carried out in the same medium as described above. The total amount of fed glucose was 1.8g/50ml culture. Dilutions of the culture supematants were slot blotted and CBHI was detected with specific antibody (Figure 17).

Example 9 β-Galactosidase Expression Vectors with Specific Mutations in cbhl Promoter to Release Glucose Repression

Three 6 bp sequences found in cbhl promoter similar to binding sites of Saccharomyces cerevisiae glucose repressor protein MIGl (Nehlin & Ronne, EMBO J. 9:2891-2899 (1990); Nehlin et al, EMBO J. 10:3313-3311 (1991)) were changed into other nucleotides to study the functionality of these mig-like sequences in mediating the glucose repression of the native cbhl promoter of Trichoderma reesei. To construct 3-galactosidase expression vectors with cbhl promoters carrying specific mutations, sequence alterations were made into primers (specifically: TCT TCA AGA ATT GCT CGA CCΆ ATT CTC ACG GTG AAT GTA GG (SEQ ID 8); ACA CAT CTA GAG GTG ACC TAG GCA

TTC TGG CCA CTA GAT ATA TAT TTA GAA GGT TCT TGT AGC TCA AAA GAG c (SEQ ID 9); GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID 10); GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT

GAC c (SEQID 11); GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG

CTT GAC CGA TCT AAA CTG TTC GAA GCC CGA ATG AG G (SEQ ID 12); GGG

AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA CCC (SEQ ID 13); TAG CGA ATT CTA GGT CAC CTC TAA AGG TAC CCT GCA GCT CGA GCT AG (SEQID 14); andGGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID 15); these primers were specific for the cbhl promoter and the cbhl promoter internal polylinker and were used in PCR amplification of cbhl promoter sequences for cloning. pMLOlό (Figure 13) was used as a PCR template with the appropriate primers to yield a 770 bp fragment A (primers TAG CGA ATT CTA GGT CAC CTC TAA AGG TAC ccT GCA GCT CGA GCT AG (SEQ ID 14) and GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID 10), beginning at the polylinker at -1500 and ending at -720 upstream of ATG, and a 720 bp fragment B (primers GGG AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA ccc (SEQ ID 13) and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID 15)), beginning at -720 and ending at Kspl at -16. Fragments A and B were purified from agarose gel and digested with BstEll-Xbal and Xbal-Kspl respectively, ligated to the 7.8 kb fragment of pMLOlό to produce pMI-24. The resulting cbhl promoter carries a sequence alteration (genomic sequence 5' GTGGGG, altered sequence: 5' TCTAGA) at position -720 to -715 upstream of the translation initiation codon of intact cbhl promoter (Figure 18). The sequence of the altered cbhl promoter in pMI-24 is provided in Figure 18A and SEQ ID20. pMLO16delO(2) (Figure 19) containing a 460 bp deletion in the cM7 promoter beginning from the promoter internal polylinker and ending 1025 bp before the translation initiation site was constmcted as described in Example 6 and used as a PCR template with primers (TCT TCA AGA ATT GCT CGA CCA

ATT CTC ACG GTG AAT GTA GG (SEQ ID 8) and ACA CAT CTA GAG GTG ACC TAG GCA TTC TGG CCA CTA GAT ATA TAT TTA GAA GGT TCT TGT AGC TCA AAA GAG c (SEQ ID 9)) to yield a 800 bp fragment C, beginning from the 5' end of cbhl promoter and ending at the promoter internal polylinker. Fragment C was purified from agarose gel, digested with SaR-Xbal and ligated to the 7.6 kb SalJ-Xbal fragment of pMLO16delO(2) to produce pMI-25. The cbhl promoter of pMI-25 has a sequence alteration (genomic sequence: 5'GTGGGG, altered sequence: 5TCTAAA) at position -1505-1500 upstream of the translation initiation codon of intact cbhl promoter (Figure 18). pMLO16delO(2) was used as a PCR template to yield a 750 bp fragment D (primers GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG

CTT GAC CGA TCT AAA CTG TTC GAA GCC CGA ATG TAG G (SEQ ID 12) and

GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID 15)), beginning from the promoter intemal polylinker and ending at Kspl at -16. Fragment D was purified from agarose gel, digested with BstEll-Kspl and ligated to the 7.8 kb BstEll-Kspl fragment of pMI-25 to produce pMI-26. The cbhl promoter of pMI-26 has sequence alterations at positions -1505-1500 (genomic sequence: 5'GTGGGG, altered sequence: 5TCTAAA) and -1001-996 (genomic sequence: 5'CTGGGG, altered sequence: 5TCTAAA) upstream of the translation initiation codon of intact cbhl promoter (Figure 18). pMLO16delO(2) was used as a PCR template to yield a 280 bp fragment E (primers GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID 10) and GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC C (SEQ ID 11)), beginning from the promoter intemal polylinker and ending at -720 and a 720 bp fragment F (primers GGG AAT TCT TCT AGA

TTG CAG AAG CAC GGC AAA GCC CAC TTA CCC (SEQ ID 13) and GGG AAT

TCA TGA TGC GCA GTC CGC GG (SEQ ID 15)), beginning at -720 and ending at Kspl at -16. Fragments D and E were purified from agarose gel, digested with BstEll-Xbal and Xbal-Kspl respectively and ligated to the 7.8 kb BstEII- Kspl fragment of pMI-25 to produce pMI-27. The cbhl promoter of pMI-27 has sequence alterations at positions -1505-1500 (genomic sequence: 5'GTGGGG, altered sequence: 5TCTAAA) and -720-715 (genomic sequence: 5'GTGGGG, altered sequence: 5TCTAGA) upstream of the translation initiation codon of intact cbhl promoter (Figure 18). The sequence of the altered cbhl promoter of pMI-27 is shown in Figure 18C and SEQ ID21. pMLO16delO(2) was used as a PCR template to yield a 280 bp fragment G (primers GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID 10) and GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC CGA TCT AAA CTG TTC GAA GCC CGA ATG TAG G (SEQ ID 12)), beginning from the promoter intemal polylinker and ending at -720 and a 720 bp fragment H (primers GGG AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA ccc (SEQ ID 13) and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID 15)), beginning at -720 and ending at Kspl at -16. Fragments G and H were purified from agarose gel, digested with BstEII-Xbal and Xbal -Kspl respectively and ligated to the 7.8 kb BstEII-KspI fragment of pMI-25 to produce pMI-28. The cbhl promoter of pMI-28 has sequence alterations at positions -1505-1500 (genomic sequence: 5'GTGGGG, altered sequence: 5TCTAAA), -1001-996 (genomic sequence: 5'CTGGGG, altered sequence: 5TCTAAA), and -720-715 (genomic sequence: 5'GTGGGG, altered sequence: 5TCTAGA) upstream of the translation initiation codon of intact cbhl promoter (Figure 18). The sequence of the altered cbhl promoter of pMI-28 is shown in Figure 18C and SEQ ID22.

All PCR amplified DNA fragments and ligation joints were sequenced using standard methods to ensure that the mutations were present and no other nucleotides were changed. Transformation of Trichoderma reesei QM9414 with the vectors mentioned above, isolation of /3-galactosidase producing clones and their analysis was done as described in Example 7. After addition of X-gal, an intense blue color was detected on glucose grown transformant colonies as an indication of (8-galactosidase activity in transformants transformed with the plasmids pMI-24, pMI-27 and pMI-28 (Figure 20), indicating that altering the cbhl promoter according to any of those mutations was sufficient to allow for expression of proteins in Trichoderma under the cM7 promoter in the presence of glucose.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: ALKO Ltd

(B) STREET: Salmisaarenranta 7 H

(C) CITY: Helsinki

(D) COUNTRY: Finland

(E) POSTAL CODE: FIN-00180

(ii) TITLE OF INVENTION: Fungal Promoters Active In The Presence Of Glucose

(iii) NUMBER OF SEQUENCES: 28

(iv)

(v) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/932,485

(B) FILING DATE: 19-AUG-1992

(vi) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 358-0-13311

(B) TELEFAX: 358-0-1333346

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3461 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

CGCCGTGACG ACAGAAACGG AGCCCGCGAG TTTGGATACG CCGCTGAAAT GGGGCTTGAC 60

GGTGAAGGAG AAGCCGAGCG CGGTGCCAGA GGACAAGATG GATGTAGAGC CAGGCGACGA 120

CGACCAAACG CAACCATCAA ATCAATCAGA TGGCAATGAC GCACCACCGC CCCAGCAGCG 180

CGAACCGCCG ACGAAGAAGC CATGGACGCG CTCCTCGGCA AGACGCCCAA GGAACAGAAA 240

AAAGTAATCT CCGCACCCGT ATCAGAAGAC GACGCCTACC GCCGCGACGT CGAAGCCTCC 300

GGCGCGGTGT CCACGCTCCA GGATTACGAA GACATGCCCG TCGAGGAGTT TGGCGCCGCC 360

CTCCTCCNNN GCATGGGCTG GAACGGGGAA GCCCGCGGCC CGCCGGTCAA GCAGGTCAAG 420 AGGCGGCAGA ACAGGCTCGG CCTCGGCGCC AAGGAGCTCA AGGAGGAAGA GGACCTCGGC 480

GGGTGGAACC AGAACGGCAA GAAAAAGTCG AGGCCSCGCG GCTGAGCGAG TATCGGAGGG 540

AGGAGAGCAA GCGCAAGGAA GGCCGGGGGC ATGAGGACAG CTATAAACGA GAGAGGGAGC 600

GCGAACGGAT CGCGAGAGGG ATCACTACAG GGAGCGAGAC CGGGACAGGG ATCGCGATTA 660

TAGGGATCGG GATAGGGATA GACATCGGGA CCACGATAGG CACAGGGACC GACATCGCGA 720

CTCTGACCGG CACCATCGAC GATGAAGGAG CTTTTGCATT CTTCTCTTCG TCAACCACTT 780

TTGAGACTAA CATTAACCAT GCCGTTTTCT TGAAAAGCTT GTACTCATCA TGATGTTTTT 840

AAGCAAATAG GCGACAGGCG TACAGACACC TTAATATCAC ATAGAGGCAC GGCACACATA 900

CGTCTTGGAG AAGACACGTA CTTACGAATG ATGGGAGAAT TACCTACTCT GACTTGTGTA 960

AATTAGAATA TCAATGACAC TATGTATATT CAGTCGAGCT GCGAATGGTC ACACATTGTC 1020

TGATCTGCGA ATTTGTATGT GCTGCCTCTC CCTCTGACCT TCTGGTCTGG TGATACCATC 1080

CTCCCTCAGT TTGGATCATC GCCTTATTCT TCTTCCCTCT TCTGCATCTG CTTCCTGCTC 1140

GTTTGAGGAA CATCGCCAGC TGACTCTGCT TGCCTCGCAG CGATCTAGTC AAGAACAACA 1200

CNAGCTCTCA CGCTACATCA CACAAACCGT CAAAATGGGT AAGGAGGACA AGACTCACAT 1260

CAACGTGGTC GTCATCGTAC GTATTTTCCG ATCCCTCATC GGCNGTCATC TGNCCAGTCT 1320

GATTCCAAGA ATCACCGTGC TAACCATATA CCATCTANGG GTGCGTATTC CATCAATCAT 1380

CTTGAGCCAG ATCGACCGAA CATACGATAC TGACTTTGCT ACGACAGCCA CGTCGACTCC 1440

GGCAAGTCTA CCACCGTGAG TAAACACCCA TTCCACTCCA CGACCGCAAG CTCCATCTTG 1500

CGCGTGGCGT CTCTGCGATG AACATCCGAA ACTGACGTTC TGTTACAGAC TGGTCACTTG 1560

ATCTACCAGT GCGGTGGTAT CGACAAGCGT ACCATTGAGA AGTTCGAGAA GGTAAGCTTC 1620

GTTCCTTAAA TCTCCAGACG CGAGCCCAAT CTTTGCCCAT CTGCCCAGCA TCTGGCGAAC 1680

GAATGCTGTG CCGACACGAT TTTTTTTTTC ATCACCCCGC TTTCTCCTAC CCCTCCTTCG 1740

AGCGACGCAA ATTTTTTTTG CTGCCTTACG AGTTTTAGTG GGGTCGCACC TCACAACCCC 1800

ACTACTGCTC TCTGGCCGCT CCCCAGTCAC CCAACGTCAT CAACGCAGCA GTTTTCAATC 1860

AGCGATGCTA ACCATATTCC CTCGAACAGG AAGCCGCCGA ACTCGGCAAG GGTTCCTTCA 1920

AGTACGCGTG GGTTCTTGAC AAGCTCAAGG CCGAGCGTGA GCGTGGTATC ACCATCGACA 1980

TTGCCCTCTG GAAGTTCGAG ACTCCCAAGT ACTATGTCAC CGTCATTGGT ATGTTGGCAG 2040

CCATCACCTC ACTGCGTCGT TGACACATCA AACTAACAAT GCCCTCACAG ACGCTCCCGG 2100

CCACCGTGAC TTCATCAAGA ACATGATCAC TGGTACTTCC CAGGCCGACT GCGCTATCCT 2160

CATCATCGCT GCCGGTACTG GTGAGTTCGA GGCTGGTATC TCCAAGGATG GCCAGACCCG 2220

TGAGCACGCT CTGCTCGCCT ACACCCTGGG TGTCAAGCAG CTCATCGTCG CCATCAACAA 2280

GATGGACACT GCCAACTGGG CCGAGGCTCG TTACCAGGAA ATCATCAAGG AGACTTCCAA 2340

CTTCATCAAG AAGGTCGGCT TCAACCCCAA GGCCGTTGCT TTCGTCCCCA TCTCCGGCTT 2400

CAACGGTGAC AACATGCTCA CCCCCTCCAC CAACTGCCCC TGGTACAAGG GCTGGGAGAA 2460

GGAGACCAAG GCTGGCAAGT TCACCGGCAA GACCCTCCTT GAGGCCATCG ACTCCATCGA 2520

GCCCCCCAAG CGTCCCACGG ACAAGCCCCT GCGTCTTCCC CTCCAGGACG TCTACAAGAT 2580 CGGTGGTATC GGAACAGTTC CCGTCGGCCG TATCGAGACT GGTGTCCTCA AGCCCGGTAT 2640

GGTCGTTACC TTCGCTCCCT CCAACGTCAC CACTGAAGTC AAGTCCGTCG AGATGCACCA 2700

CGAGCAGCTC GCTGAGGGCC AGCCTGGTGA CAACGTTGGT TTCAACGTGA AGAACGTTTC 2760

CGTCAAGGAA ATCCGCCGTG GCAACGTTGC CGGTGACTCC AAGAACGACC CCCCCATGGG 2820

CGCCGCTTCT TTCACCGCCC AGGTCATCGT CATGAACCAC CCCGGCCAGG TCGGTGCCGG 2880

CTACGCCCCC GTCCTCGACT GCCACACTGC CCACATTGCC TGCAAGTTCG CCGAGCTCCT 294 0

CGAGAAGATC GACCGCCGTA CCGGTAAGGC TACCGAGTCT GCCCCCAAGT TCATCAAGTC 3 000

TGGTGACTCC GCCATCGTCA AGATGATCCC CTCCAAGCCC ATGTGCGTTG AGGCTTTCAC 3 060

CGACTACCCT CCCCTGGGTC GTTTCGCCGT CCGTGACATG CGCCAGACCG TCGCTGTCGG 3120

TGTCATCAAG GCCGTCGAGA AGTCCTCTGC CGCCGCCGCN AAGGTCACCA AGTCCGCTGC 3180

CAAGGCCGCC AAGAAATAAG CGATACCCAT CATCAACACC TGATGTTCTG GGGTCCCTCG 324 0

TGAGGTTTCT CCAGGTGGGC ACCACCATGC GCTCACTTCT ACGACGAAAC GATCAATGTT 33 00

GCTATGCATG AGSACTCGAC TATGAATCGA GGCACGGTTA ATTGAGAGGC TGGGAATAAG 33 60

GGTTCCATCA GAACTTCTCT GGGAATGCAA AACAAAAGGG AACAAAAAAA CTAGATAGAA 3420

GTGAATTCAT GACTTCGACA ACCAAAAAAA AAAAAAAAAA A 3461 (2 ) INFORMATION FOR SEQ ID N0 : 2 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 1636 base pairs

(B) TYPE : nucleic acid

( C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(xi ) SEQUENCE DESCRI PTION : SEQ ID Nθ : 2 :

GGTCTGAAGG ACGTGGAATG ATGGACTTAA TGACAAGAGT TGCCTGGCTA TTGAGCTCTG 60

GTACATGGAT CTCGAACTGA GAGCGTACAA GTTACATGTA GTAAATCTAG TAGATCTCGC 120

TGAAAGCCCT CTTTCCCGGT AGAAACACCA CCAGCGTCCC GTAGGACAAG ATCCTGTCGA 180

TCTGAGCACA TGAATTGCTT CCCTGGATCT GGCGCTGCAT CTGTTTCCCC AGACAATGAT 240

GGTAGCAGCG CATGGAAGAA CCCGGTTGTT CGGAATGTCC TTGTGCTAAC AGTGGCATGA 3 00

TTTTACGTTG CGGCTCATCT CGCCTTGGCA CCGGACCTCA GCAAATCTTG TCACAACAGC 36 0

AATCTCAAAC AGCCTCATGG TTCCCAGATT CCCTGATTCA GAACTCTAGA GCGGCAGATG 420

TCAAACGATT CTGACCTAGT ACCTTGAGCA TCCCTTTCGG ATCCGGCCCA TGTTCTGCCT 48 0

GCCCTTCTGA GCACAGCAAA CAGCCCAAAA GGCGCCGGCC GATTCCTTTC CCGGGATGCT 540

CCGGAGTGGC ACCACCTCCC AAAACAAGCA ACCTTGAACC CCCCCCCCAA ATCAACTGAA 6 00

GCGCTCTTCG CCTAACCAGC ATAAGCCCCC CCCAGGATCG TTAGGCCAAG TGGTAGGGCC 66 0

AGCCAATTAG CGAGNGGCCA TTTGGAGGTC ATGGGCGCAG AATGTCCTGA CAGTGGTATG 720

ATATTGACTG CCCGGTGTGT GTGGCATCTG GCCATAATCG CAGGCTGAGG CGAGGAAGTC 780

TCGTGAGGAT GTCCCGACTT TGACATCATG AGGGAGTGAG AAACTGAAGA GAAGGAAAGC 840

TTCGAAGGTT CGATAAGGGA TGATTTGCAT GGCGGGCGAC AGGATGCGAT GGCTCGTTGG 9 00

GATACATAAT GCTTGGGTTG GAAGCGATTC CAGGTCGTCT TTTTTTGGTT CATCATCACA 960 GCATCAACAA GCAACGATAC AAGCAATCCA CTGAGGATTA CCTCTCAACT CAACCACTTT 1020

CCAAACCATC TCAACTCCCT AAGATTCTTT CAGTGTATTA TCACTAGGAT TTTTCCCAAG 1080

CCGGCTTCAA AACACACAGA TAAACCACCA ACTCTACAAC CAAAGACTTT TTGATCAATC 1140

CAACAACTTC TCTCAACATG TCTGCTGCAA CCGTCACCCG CACTGCAACC GCCGCTGTTC 1200

GCAGACCCGG CTTCTTCATG CAAGTCCGAC GGATGGGACG CTCATTCGAG CACCAGCCCT 1260

TTGAGCGACT CTCCGCCACC ATGAAGCCTG CACGACCCGA CTATGCTAAG CAAGTCGTCT 1320

GGACGGCTGG CAAGTTTGTC ACTTATGTTC CTCTTTTCGG CGCCATGCTT ACCTGGCCTG 1380

CGCTCGCCAA STGGGCTCTG GACGGACACA TCGGACGGTG GTAAAAGATC AGACTCTTGT 1440

CGAGGCAACG GGGAATAGAC AGGACAGCAA AAAAGATATC TCCGGATAGA AGTGTCCATC 1500

TTTCGACTTG TATATATATA TATGCTATAC TCTGGGGGCG TTTGGATGGA CTTTGGGCAC 1560

GAAGCATACT TTGGCGCAAC GCAGATACTT TAATCTGATT CCTTTTGTTA ATTCAAAAAA 1620

AAAAAAAAAA AAAAAA 1636 (2 ) INFORMATION FOR SEQ ID Nθ : 3 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 2868 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(xi ) SEQUENCE DESCRIPTION : SEQ ID Nθ : 3 :

TTTGTATGGC TGGATCTCGA AAGGCCCTTG TCATCGCCAA GCGTGGCTAA TATCGAATGA 60

GGGACACCCA CTTGCATATC TCCTGATCAT TCAAACGACA AGTGTGAGGT AGGCAATCCT 120

CGTATCCCAT TGCTGGGCTG AAAGCTTCAC ACGTATCGCA TAAGCGTCTC CAACCAGTGC 180

TTAGGTGACC CTTAAGGATA CTTACAGTAA GACTGTATTA AGTCAGTCAC TCTTTCACTC 240

GGGCTTTGAA TACGATCCTC AATACTCCCG ATAACAGTAA GAGGATGATA CAGCCTGCAG 300

TTGGCAAATG TAAGCGTAAT TAAACTCAGC TGAACGGCCC TTGTTGAAAG TCTCTCTCGA 360

TCAAAGCAAA GCTATCCACA GACAAGGGTT AAGCAGGCTC ACTCTTCCTA CGCCTTGGAT 420

ATGCAGCTTG GCCAGCATCG CGCATGGCCA ATGATGCACC CTTCACGGCC CAACGGATCT 480

CCCGTTAAAC TCCCCTGTAA CTTGGCATCA CTCATCTGTG ATCCCAACAG ACTGAGTTGG 540

GGGCTGCGGC TGGCGGATGT CGGAGCAAAG GATCACTTCA AGAGCCCAGA TCCGGTTGGT 600

CCATT-^CCAA TGGATCTAGA TTCGGCACCT TGATCTCGAT CACTGAGACA TGGTGAGTTG 660

CCCGGACGCA CCACAACTCC CCCTGTGTCA TTGAGTCCCC ATATGCGTCT TCTCAGCGTG 720

CAACTCTGAG ACGGATTAGT CCTCACGATG AAATTAACTT CCAGCTTAAG TTCGTAGCCT 780

TGAATGAGTG AAGAAATTTC AAAAACAAAC TGAGTAGAGG TCTTGAGCAG CTGGGGTGGT 840

ACGCCCCTCC TCGACTCTTG GGACATCGTA CGGCAGAGAA TCAACGGATT CACACCTTTG 900

GGTCGAGATG AGCTGATCTC GACAGATACG TGCTTCACCA CAGCTGCAGC TACCTTTGCC 960

CAACCATTGC GTTCCAGGAT CTTGATCTAC ATCACCGCAG CACCCGAGCC AGGACGGAGA 1020

GAACAATCCG GCCACAGAGC AGCACCGCCT TCCAACTCTG CTCCTGGCAA CGTCACACAA 1080

CCTGATATTA GATATCCACC TGGGTGATTG CCATTGCAGA GAGGTGGCAG TTGGTGATAC 1140 CGACTGGCCA TGCAAGACGC GGCCGGGCTA GCTGAAATGT CCCCGAGAGG ACAATTGGGA 1200

GCGTCTATGA CGGCGTGGAG ACGACGGGAA AGGACTCAGC CGTCATGTTG TGTTGCCAAT 1260

TTGAGATTGT TGACCGGGAA AGGGGGGACG AAGAGGATGG CTGGGTGAGG TGGTATTGGG 1320

AGGATGCATC ATTCGACTCA GTGAGCGATG TAGAGCTCCA AGAATATAAA TATCCCTTCT 1380

CTGTCTTCTC AAAATCTCCT TCCATCTTGT CCTTCATCAG CACCAGAGCC AGCCTGAACA 1440

CCTCCAGTCA ACTTCCCTTA CCAGTACATC TGAATCAACA TCCATTCTTT GAAATCTCAC 1500

CACAACCACC ATCTTCTTCA AAATGAAGTT CTTCGCCATC GCCGCTCTCT TTGCCGCCGC 1560

TGCCGTTGCC CAGCCTCTCG AGGACCGCAG CAACGGCAAC GGCAATGTTT GCCCTCCCGG 1620

CCTCTTCAGC AACCCCCAGT GCTGTGCCAC CCAAGTCCTT GGCCTCATCG GCCTTGACTG 1680

CAAAGTCCGT AAGTTGAGCC ATAACATAAG AATCCTCTTG ACGGAAATAT GCCTTCTCAC 1740

TCCTTTACCC CTGAACAGCC TCCCAGAACG TTTACGACGG CACCGACTTC CGCAACGTCT 1800

GCGCCAAAAC CGGCGCCCAG CCTCTCTGCT GCGTGGCCCC CGTTGTAAGT TGATGCCCCA 1860

GCTCAAGCTC CAGTCTTTGG CAAACCCATT CTGACACCCA GACTGCAGGC CGGCCAGGCT 1920

CTTCTGTGCC AGACCGCCGT CGGTGCTTGA GATGCCCGCC CGGGGTCAAG GTGTGCCCGT 1980

GAGAAAGCCC ACAAAGTGTT GATGAGGACC ATTTCCGGTA CTGGGAAAGT TGGCTCCACG 2040

TGTTTGGGCA GGTTTGGGCA AGTTGTGTAG ATATTCCATT CGTACGCCAT TCTTATTCTC 2100

CAATATTTCA GTACACTTTT CTTCATAAAT CAAAAAGACT GCTATTCTCT TTGTGACATG 2160

CCGGAAGGGA ACAATTGCTC TTGGTCTCTG TTATTTGCAA GTAGGAGTGG GAGATTCGCC 2220

TTAGAGAAAG TAGAGAAGCT GTGCTTGACC GTGGTGTGAC TCGACGAGGA TGGACTGAGA 2280

GTGTTAGGAT TAGGTCGAAC GTTGAAGTGT ATACAGGATC GTCTGGCAAC CCACGGATCC 2340

TATGACTTGA TGCAATGGTG AAGATGAATG ACAGTGTAAG AGGAAAAGGA AATGTCCGCC 2400

TTCAGCTGAT ATCCACGCCA ATGATACAGC GATATACCTC CAATATCTGT GGGAACGAGA 2460

CATGACATAT TTGTGGGAAC AACTTCAAAC AGCGAGCCAA GACCTCAATA TGCACATCCA 2520

AAGCCAAACA TTGGCAAGAC GAGAGACAGT CACATTGTCG TCGAAAGATG GCATCGTACC 2580

CAAATCATCA GCTCTCATTA TCGCCTAAAC CACAGATTGT TTGCCGTCCC CCAACTCCAA 2640

AACGTTACTA CAAAAGACAT GGGCGAATGC AAAGACCTGA AAGCAAACCC TTTTTGCGAC 2700

TCAATTCCCT CCTTTGTCCT CGGAATGATG ATCCTTCACC AAGTAAAAGA AAAAGAAGAT 2760

TGAGATAATA CATGAAAAGC ACAACGGAAA CGAAAGAACC AGGAAAAGAA TAAATCTATC 2820

ACGCACCTTG TCCCCACACT AAAAGCAACA GGGGGGGTAA AATGAAAT 2868 (2) INFORMATION FOR SEQ ID Nθ:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2175 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:

AAAAAGCTAG AACGAGACGA TTCCGGCCCG GCAAACCAGG CCGAGTGACG GGAGCATTTC 60 CATGATTTCA CTCGGCAAAC TCTGGCTACA ATTTTCAGGC GGCGAGTTCC GATACAAGGG 120

AAATCTATTA CCCACAGACG AACGGGAATC GGTGATGAGT GGTTTCTTGT AAGTCAACAT 180

TGAGCTAGAT AATTCCGGGC GAGATCAAGA TGCCATACTT TGATTGATGA AAAATCAATG 240

TCAGGCGTAA GTCTCTTCAA GCTCGCCCAG TCCTCTGTAT GTAACAGCAA TCGCAATTCC 300

GAAATGTGCC GAGCCAATGG AACATGCGTG TCTTTCTCTT TTCACACACA TCCAGTTCGA 360

GAGTCTTCTC TTCATCGTTT CATCGAATCC CTTCCCCTCC AGCTATTCAC CCAGCCGAGC 420

CCTTCAGCGC ACCAGCGTAT GTATGTACCC TCGGCTAAGA CGCAACAGAA GCATCATCAA 480

TATACCTGAT GTACTACTAT CTACTATGAA GCCCAAAAAC CCCTTCGCAG CCCAAATGTA 540

ACCCAAGCAA CGAATCCCCA ATAAGAGACA ATCCTCAGTG ACCCCCAGAA GAGCACAGAA 600

TCGAGCTGGT CCTGGTGGGT CGCATTGAGA CCGGTGGAGA TGCGTTCGAT TCGACTGCCG 660

GAGCTCCCGG GAAGCCGGCA GATGGTCCCA TGCGATGCCC TGCACCGTTT TTGTGAATCG 720

TCGGCATCGC GAGAAGTGGC CTGCTATGAC GTCGCTTGCA GCTTGGCCGC TCTGTTCGAA 780

GTTTTTCGAT GTTTTTCTTC ATGCGGGAGA AAGAAAACAT CAGATGACAT GATTATCCGA 840

ATGGATGGCG GGAGTTATCG TGGTGACGGC TGCTTCATGA GATGAGTATA AATGAGCTTG 900

TTCGCTCAGC GTGTCATGGA TCTTGTCCAG CTCCAAAGCA TCGGCTTCAG CATCCATCCG 960

CTTGAACAGA CAGGCACCAG CTTGAATCAG AAGCATACCC TTGATTTGAT ACTCTCTTGG 1020

GAAAAAACAC CACCATCTGT GTAATACTTT GATACCCCCA AAGCTCAAAC GACCGCTTGT 1080

ACATACAATA ACACCGCCAC AATGTTCGCC AACTTGACGC ACGCTACCCT GCGATTCATC 1140

GCCTTCTTCA ACCACCTGAT GATCCTGGCC TCATCAGCCA TCGTCACCGG CCTCGTATCC 1200

TGGTTCCTCG ACAAGTACGA CTACCGCGGC GTGAACATTG TCTACCAGGA AGTCATCGTA 1260

TGTCCTCCCA AGCACCACAT CAAACACACC CCATACCTTG GCTCTCCTCA GCTCCGTCGA 1320

AGCACATAAT ACTAACGCAT GCAACAACTA GGCCACCATA ACTCTGGGCT TCTGGCTCGT 1380

TGGTGCCGTC TTGCCCCTCG TTGGCAGATA CCGCGGCCAC CTGGCCCCTC TCAACCTCAT 1440

CTTCTCCTAC CTCTGGCTCA CCTCTTTCAT CTTCTCCGCG CAGGACTGGA GCAGCGACAA 1500

GTGCAGCTTC GGCCAGCCTG GCGAGGGCCA CTGCAGCCGC AAGAAGGCCA TTGAATCCTT 1560

CAACTTTATC GCATTGTAAG TGCCTACAAG TAATTTGCTA TGTATATGGG AGAGAGAGAG 1620

AAGAAGAAGA ATATGGCTCT AACATGGCAT CTCTACAGCT TCTTCCTCCT CTGCAACACC 1680

CTGGTTGAGA TGCTCCTGCT CCGCGCCGAG TATGCTACCC CCGTTGCTGC TGCTCACAAC 1740

AAGGAGATTT CTGCCGGCCG CCCCTCTGAC AACTCTGTCT AAATAACAAT AGACATGCAT 1800

AGATGAACGG AGACCACTTC TACTTTCTTT GCGAGTTCCT GATCCGTTGA CCTGCAGGTC 1860

GACBBBBBCC GCGCTCGCAT GGTTCATCTG CTACAACAAC ACAATGACAA TCCGAACCAG 1920

TCAATAAACC TCGACAACAC GACGAGTACT TTTGCGGATA GAAAGATACC CATTACACAG 1980

GAGATCAAAT GGGGAAATTG GAAGTGTATG GATGGACGCC CGTGTATAAT GAGGTTGTGA 2040

ACGGGATGGG AGGCAATGAA TAATGGATAA TGAGGTAATG GATAGATTCG GTCGTTTTGA 2100

TACCACAGCT GCACTCTGCT CTACGTCTGT CATTAATGAT ACATACAAAT GATACCTTAT 2160

ACGCTAAAAA AAAAA 2175 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2737 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

TCTAGAATCT CTTCGAGATG GCCGAGAAAG GCTTGTTTTT CTCTCCTTCT TCAAACTGGC 60

CACTGTTTGT TTTCAAACTT GGGGTTTCGT GGGGCTTTTG GGGGCATGTC TGCCAGGTCT 120

CCCGTAGGCT GGACAGCCAA AGCCTCACTA CAAACAGGCA GTTGTCAATA GATTGATGTC 180

TGAGATGGAT GGTTTTATGT TTGGGGGAGG TCATGTATGT ATTTATCTAT ATTTGCAAAG 240

ATGATCCATG AGTCAGACTT GCACAGGTTT CTCGTGCGCT GGATAAATCT TGTTGGAGTG 300

CGGGTGAGGT GGTGGATGGC ATTCAACCCA CAGCAACACT TGCCCAGGGG GATGTACTGC 360

AGCGATTTGT TTCCCTTCGA GTATTAGATG ATGATGCCGA ACAGACAAAT TTGAGCCTCG 420

CTGCTCTCGG ATGTCGGGTT TCTCTTGTGT GCCGGTGATG TGTGATGGCC TGGCCCGCAA 480

AGAGAGCGAA AAACATGCTC AAAATGTAGC ACACGGCGAC TTCTCGGACA CTTGCGTACC 540

TTGAGAGACA AGCAGACTAC AGGGATGACG AGTAATACGA CAGAGCGATA CGACACAGCT 600

ATACGACACA GCTAAGAAAA TAAAGGTATT AGTACTACTA ATTGATTACC TACTACCTAG 660

ATATATACTA TACCTTATAT TTTATATGTG TGTGTGTGTG TATGTATATG CCTTACCTTA 720

TGCTTCGCAA AGAAGAGAAA CTAAAACGCC TCCTGGCTAC CTACCTACCT CTACCTTGTA 780

AGAGATGGAA TAATGTGGCC GCGCGTAAAG TAGGTACTGG ATATACAGGT CCTGAACATG 840

GCCCTGAATC CTGCCAGGCA GCCACCTCAC CCCTTCCGCA GGTATTTATG TAGCCCACAG 900

CTCCTCCAGA GACGATGCCG AGATGCCTCA TGCAGTCTAC CTACAAAGCC AGCAGTTTCA 960

CGCTTGACTC TCACTCTTGA TTGAATTCCC TCCCTCCCAT AATACCAATT GGCGTTCAAC 1020

GATTGCCAGC AGAATGGCCG CCCAACACGA CGTCGAGGCC ATGGCAAAGT CCATGTCCGA 1080

CTTTTTCAAG GACACGGCCC AAAAGCAGGA CTCGACCAAG CATGACTTTG TCCAAGCCTC 1140

GCACGGCATC ATGAGGGCCA TTGTCGAGCC GCTCGTCACC CAGATGGGCT TCCGCGAGAC 1200

CCTCACCGAG CCCGTCGTCT TGCTCGACAG CGCGTGCGGA GCGGGCGTGC TGACGCAGGA 1260

GGTGCAGGCG GCGCTGCCAA AGGAGCTTCT GGAGAGGAGC TCGTTTACGT GTGCGGACAA 1320

TGCCGAGGGC TTGGTGGACG TGGTGAAGAG GAGGATTGAT GAGGAGAAGT GGGTGAATGC 1380

AGAGGCCAAG GTCCTTGATG CCCTGGTGAG TATATACATA TATATCTATA TCTATATAGA 1440

TATATATATG CCTTTGACTC CCCCCTTTAC ATGTCCTACG GCTGCTGATT GATTGATTGA 1500

TGTGGTGATG GTGATGTCCC AGAACACGGG GCTCCCAGAC AACTCCTTCA CCCATGTGGG 1560

CATTGCCCTG GCACTGCACA TCATCCCCGA TCCAGATGCC GTCGTCAAAG GTAAACAATC 1620

ACCAGCGTCA CTGCAAAGAG AGATTACGGG ATATCATATA CTGAAACCAA AGCCCAGACT 1680

GCATCAGAAT GCTCAAGCCA GGCGGCATCT TTGGCGCATC GACATGGCCC AAGGCCAGCG 1740

CCGACATGTT CTGGATCGCC GACATGCGCA CCGCCCTGCA GTCGCTCCCC TTTGACGCGC 1800 CGCTGCCAGA CCCGTTCCCC ATGCAGCTGC ACACCTCGGG CCACTGGGAC GACGCCGCCT 1860

GGGTCGAGAA GCATCTCGTC GAGGATCTGG GGCTGGCCAA CGTCTGTGTG AGGGAGCCGG 1920

CGGGCGAGTA CAGCTTTGCG AGCGCGGACG AGTTCATGGC GACGTTTCAG ATGATGCTGC 1980

CGTGGATTAT GAAGACGTTT TGGAGCGAGG AGGTGAGGGA GAAGCATTCG GTCGACGAGG 2040

TCAAGGAGTT GGTGAAGAGG CATCTGGAGG ACAAGTATGG GGGGAAGGGA TGGACCATTA 2100

AGTGGCGGGT GATTACCATG ACTGCGACTG CGAGCAAGTG AGGGAGGGCA TCTGCTCATG 2160

ATTATGTGAC AGCGAGCCAG TAGAGAGCCA TATTGTTGTC TTCAGAATGT GAGGACCGTG 2220

ATGGTTGGTG TTTGTTGGAG TGATAACTCG TGGGTGTTGC TATTTGCATG TGAGACGATG 2280

AACCATGCGC ACCAGCCACA ATCACTGTCC CCCACCTTAC CTACCAACTT CAAGTTACCA 2340

CCTTACCTTT ACCTGATCTA GCACTGTGGC GCAGCTTGGT TTGACTGCTA GGTACCTACC 2400

TAGTAGTAAT CAGGTACATT CTTCATCCCT GTGTCCTGGT GTCGCAGTTG CAGCTTGTCT 2460

TATCGCTGTG GCCACGCATC GAGTGGCAGC ATCTTCAACT TCAAGTCCCG TCGGTCGCAC 2520

TCTGGCCACG TCGCAGATGG ATCGCAGCGG GATCTGAACC GCTCGCTCGG CAACTGATAC 2580

CAAGTCAACA AACACACGAG ACGACGGGAC GCTGATATAA NNNNGAGGAG GGTAAGAGAA 2640

CTCTACGAGG GGCGGAAACT TGGTCCGACA ATTTCCCTCC CATCTTCACC CTCGACTCGA 2700 ACTCGAACTC GATAGCCGCA CCCTCGACCG ATTGCCC 2737

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : ACCGGAATTC ATATCTAGAG GAGCCCGCGA GTTTGGATAC GCC 43

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : ACCGCCGCGG TTTGACGGTT TGTGTGATGT AGCG 34

(2) INFORMATION FOR SEQ ID Nθ:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : TCTTCAAGAA TTGCTCGACC AATTCTCACG GTGAATGTAG G 41

(2). INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 :

ACACATCTAG AGGTGACCTA GGCATTCTGG CCACTAGATA TATATTTAGA AGGTTCTTGT 60

AGCTCAAAAG AGC 73 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GGGAATTCTC TAGAAACGCG TTGGCAAATT ACGGTACG 38

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:ll: GGGAATTCGG TCACCTCTAA ATGTGTAATT TGCCTGCTTG ACC 43

(2) INFORMATION FOR SEQ ID NO:12 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAATTCGG TCACCTCTAA ATGTGTAATT TGCCTGCTTG ACCGATCTAA ACTGTTCGAA 60 GCCCGAATGT AGG 73

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGGAATTCTT CTAGATTGCA GAAGCACGGC AAAGCCCACT TACCC 45

(2) INFORMATION FOR SEQ ID NO:14 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TAGCGAATTC TAGGTCACCT CTAAAGGTAC CCTGCAGCTC GAGCTAG 47

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GGGAATTCAT GATGCGCAGT CCGCGG 26

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1588 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CCCCCCTATC TTAGTCCTTC TTGTTGTCCC AAAATGGCGC CCTCAGTTAC ACTGCCGTTG 60

ACCACGGCCA TCCTGGCCAT TGCCCGGCTC GTCGCCGCCC AGCAACCGGG TACCAGCACC 120

CCCGAGGTCC ATCCCAAGTT GACAACCTAC AAGTGTACAA AGTCCGGGGG GTGCGTGGCC 180

CAGGACACCT CGGTGGTCCT TGACTGGAAC TACCGCTGGA TGCACGACGC AAACTACAAC 240

TCGTGCACCG TCAACGGCGG CGTCAACACC ACGCTCTGCC CTGACGAGGC GACCTGTGGC 300

AAGAACTGCT TCATCGAGGG CGTCGACTAC GCCGCCTCGG GCGTCACGAC CTCGGGCAGC 360

AGCCTCACCA TGAACCAGTA CATGCCCAGC AGCTCTGGCG GCTACAGCAG CGTCTCTCCT 420

CGGCTGTATC TCCTGGACTC TGACGGTGAG TACGTGATGC TGAAGCTCAA CGGCCAGGAG 480

CTGAGCTTCG ACGTCGACCT CTCTGCTCTG CCGTGTGGAG AGAACGGCTC GCTCTACCTG 540

TCTCAGATGG ACGAGAACGG GGGCGCCAAC CAGTATAACA CGGCCGGTGC CAACTACGGG 600

AGCGGCTACT GCGATGCTCA GTGCCCCGTC CAGACATGGA GGAACGGCAC CCTCAACACT 660

AGCCACCAGG GCTTCTGCTG CAACGAGATG GATATCCTGG AGGGCAACTC GAGGGCGAAT 720

GCCTTGACCC CTCACTCTTG CACGGCCACG GCCTGCGACT CTGCCGGTTG CGGCTTCAAC 780

CCCTATGGCA GCGGCTACAA AAGCTACTAC GGCCCCGGAG ATACCGTTGA CACCTCCAAG 840 ACCTTCACCA TCATCACCCA GTTCAACACG GACAACGGCT CGCCCTCGGG CAACCTTGTG 900

AGCATCACCC GCAAGTACCA GCAAAACGGC GTCGACATCC CCAGCGCCCA GCCCGGCGGC 960

GACACCATCT CGTCCTGCCC GTCCGCCTCA GCCTACGGCG GCCTCGCCAC CATGGGCAAG 1020

GCCCTGAGCA GCGGCATGGT GCTCGTGTTC AGCATTTGGA ACGACAACAG CCAGTACATG 1080

AACTGGCTCG ACAGCGGCAA CGCCGGCCCC TGCAGCAGCA CCGAGGGCAA CCCATCCAAC 1140

ATCCTGGCCA ACAACCCCAA CACGCACGTC GTCTTCTCCA ACATCCGCTG GGGAGACATT 1200

GGGTCTACTA CGAACTCGAC TGCGCCCCCG CCCCCGCCTG CGTCCAGCAC GACGTTTTCG 1260

ACTACACGGA GGAGCTCGAC GACTTCGAGC AGCCCGAGCT GCACGCAGAC TCACTGGGGG 1320

CAGTGCGGTG GCATTGGGTA CAGCGGGTGC AAGACGTGCA CGTCGGGCAC TACGTGCCAG 1380

TATAGCAACG ACTACTACTC GCAATGCCTT TAGAGCGTTG ACTTGCCTCT GGTCTGTCCA 1440

GACGGGGGCA CGATAGAATG CGGGCACGCA GGGAGCTCGT AGACATTGGG CTTAATATAT 1500

AAGACATGCT ATGTTGTATC TACATTAGCA AATGACAAAC AAATGAAAAA GAACTTATCA 1560

AGCAAAAAAA AAAAAAAAAA AAAAAAAA 1588 (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1820 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

CCGCGGACTG CGCATCATGT 1740

ATCGGAAGTT GGCCGTCATC TCGGCCTTCT TGGCCACAGC TCGTGCTCAG TCGGCCTGCA 1800

CTCTCCAATC GGAGACTCAC CCGCCTCTGA CATGGCAGAA ATGCTCGTCT GGTGGCACTT 1860

GCACTCAACA GACAGGCTCC GTGGTCATCG ACGCCAACTG GCGCTGGACT CACGCTACGA 1920

ACAGCAGCAC GAACTGCTAC GATGGCAACA CTTGGAGCTC GACCCTATGT CCTGACAACG 1980

AGACCTGCGC GAAGAACTGC TGTCTGGACG GTGCCGCCTA CGCGTCCACG TACGGAGTTA 2040

CCACGAGCGG TAACAGCCTC TCCATTGGCT TTGTCACCCA GTCTGCGCAG AAGAACGTTG 2100

GCGCTCGCCT TTACCTTATG GGCAGCGACA CGACCTACCA GGAATTCACC CTGCTTGGCA 2160

ACGAGTTCTC TTTCGATGTT GATGTTTCGC AGCTGCCGTA AGTGACTTAC CATGAACCCC 2220

TGACGTATCT TCTTGTGGGC TCCCAGCTGA CTGGCCAATT TAAGGTGCGG CTTGAACGGA 2280

GCTCTCTACT TCGTGTCCAT GGACGCGGAT GGTGGCGTGA GCAAGTATCC CACCAACACC 2340

GCTGGCGCCA AGTACGGCAC GGGGTACTGT GACAGCCAGT GTCCCCGCGA TCTGAAGTTC 2400

ATCAATGGCC AGGCCAACGT TGAGGGCTGG GAGCCGTCAT CCAACAACGC AAACACGGGC 2460

ATTGGAGGAC ACGGAAGCTG CTGCTCTGAG ATGGATATCT GGGAGGCCAA CTCCATCTCC 2520

GAGGCTCTTA CCCCCCACCC TTGCACGACT GTCGGCCAGG AGATCTGCGA GGGTGATGGG 2580

TGCGGCGGAA CTTACTCCGA TAACAGATAT GGCGGCACTT GCGATCCCGA TGGCTGCGAC 2640

TGGAACCCAT ACCGCCTGGG CAACACCAGC TTCTACGGCC CTGGCTCAAG CTTTACCCTC 2700 GATACCACCA AGAAATTGAC CGTTGTCACC CAGTCCGAGA CGTCGGGTGC CATCAACCGA 2760

TACTATGTCC AGAATGGCGT CACTTTCCAG CAGCCCAACG CCGAGCTTGG TAGTTACTCT 2820

GGCAACGAGC TCAACGATGA TTACTGCACA GCTGAGGAGG CAGAATTCGG CGGATCCTCT 2880

TTCTCAGACA AGGGCGGCCT GACTCAGTTC AAGAAGGCTA CCTCTGGCGG CATGGTTCTG 2940

GTCATGAGTC TGTGGGATGA TGTGAGTTTG ATGGACAAAC ATGCGCGTTG ACAAAGAGTC 3000

AAGCAGCTGA CTGAGATGTT ACAGTACTAC GCCAACATGC TGTGGCTGGA CTCCACCTAC 3060

CCGACAAACG AGACCTCCTC CACACCCGGT GCCGTGCGCG GAAGCTGCTC CACCAGCTCC 3120

GGTGTCCCTG CTCAGGTCGA ATCTCAGTCT CCCAACGCCA AGGTCACCTT CTCCAACATC 3180

AAGTTCGGAC CCATTGGCAG CACCGGCAAC CCTAGCGGCG GCAACCCTCC CGGCGGAAAC 3240

CCGCCTGGCA CCACCACCAC CCGCCGCCCA GCCACTACCA CTGGAAGCTC TCCCGGACCT 3300

ACCCAGTCTC ACTACGGCCA GTGCGGCGGT ATTGGCTACA GCGGCCCCAC GGTCTGCGCC 3360

AGCGGCACAA CTTGCCAGGT CCTGAACCCT TACTACTCTC AGTGCCTGTA AAGCTCCGTG 3420

CGAAAGCCTG ACGCACCGGT AGATTCTTGG TGAGCCCGTA TCATGACGGC GGCGGGAGCT 3480

ACATGGCCCC GGGTGATTTA TTTTTTTTGT ATCTACTTCT GACCCTTTTC AAATATACGG 3540 (2) INFORMATION FOR SEQ ID Nθ:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2211 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:18:

GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120

TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180

GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360

GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480

TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540

TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTGTGGGGT ATATATCTAG 720

AGTTGTGAAG TCGGTAATCC CGCTGTATAG TAATACGAGT CGCATCTAAA TACTCCGAAG 780

CTGCTGCGAA CCCGGAGAAT CGAGATGTGC TGGAAAGCTT CTAGCGAGCG GCTAAATTAG 840

CATGAAAGGC TATGAGAAAT TCTGGAGACG GCTTGTTGAA TCATGGCGTT CCATTCTTCG 900

ACAAGCAAAG CGTTCCGTCG CAGTAGCAGG CACTCATTCC CGAAAAAACT CGGAGATTCC 960 TAAGTAGCGA TGGAACCGGA ATAATATAAT AGGCAATACA TTGAGTTGCC TCGACGGTTG 1020

CAATGCAGGG GTACTGAGCT TGGACATAAC TGTTCCGTAC CCCACCTCTT CTCAACCTTT 1080

GGCGTTTCCC TGATTCAGCG TACCCGTACA AGTCGTAATC ACTATTAACC CAGACTGACC 1140

GGACGTGTTT TGCCCTTCAT TTGGAGAAAT AATGTCATTG CGATGTGTAA TTTGCCTGCT 1200

TGACCGACTG GGGCTGTTCG AAGCCCGAAT GTAGGATTGT TATCCGAACT CTGCTCGTAG 1260

AGGCATGTTG TGAATCTGTG TCGGGCAGGA CACGCCTCGA AGGTTCACGG CAAGGGAAAC 1320

CACCGATAGC AGTGTCTAGT AGCAACCTGT AAAGCCGCAA TGCAGCATCA CTGGAAAATA 1380

CAAACCAATG GCTAAAAGTA CATAAGTTAA TGCCTAAAGA AGTCATATAC CAGCGGCTAA 1440

TAATTGTACA ATCAAGTGGC TAAACGTACC GTAATTTGCC AACGCGTTGT GGGGTTGCAG 1500

AAGCAACGGC AAAGCCCACT TCCCACGTTT GTTTCTTCAC TCAGTCCAAT CTCAGCTGGT 1560

GATCCCCCAA TTGGGTCGCT TGTTTGTTCC GGTGAAGTGA AAGAAGACAG AGGTAAGAAT 1620

GTCTGACTCG GAGCGTTTTG CATACAACCA AGGGCAGTGA TGGAAGACAG TGAAATGTTG 1680

ACATTCAAGG AGTATTTAGC CAGGGATGCT TGAGTGTATC GTGTAAGGAG GTTTGTCTGC 1740

CGATACGACG AATACTGTAT AGTCACTTCT GATGAAGTGG TCCATATTGA AATGTAAGTC 1800

GGCACTGAAC AGGCAAAAGA TTGAGTTGAA ACTGCCTAAG ATCTCGGGCC CTCGGGCTTC 1860

GGCTTTGGGT GTACATGTTT GTGCTCCGGG CAAATGCAAA GTGTGGTAGG ATCGACACAC 1920

TGCTGCCTTT ACCAAGCAGC TGAGGGTATG TGATAGGCAA ATGTTCAGGG GCCACTGCAT 1980

GGTTTCGAAT AGAAAGAGAA GCTTAGCCAA GAACAATAGC CGATAAAGAT AGCCTCATTA 2040

AACGAAATGA GCTAGTAGGC AAAGTCAGCG AATGTGTATA TATAAAGGTT CGAGGTCCGT 2100

GCCTCCCTCA TGCTCTCCCC ATCTACTCAT CAACTCAGAT CCTCCAGGAG ACTTGTACAC 2160

CATCTTTTGA GGCACAGAAA CCCAATAGTC AACCGCGGAC TGCGCATCAT G 2211 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1137 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:19:

GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120

TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180

GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360

GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480

TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540 TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTGTGGGGT ATATATCTAG 720

TGGCCAGAAT GCCTAGGTCA CCTCTAGAGA GTTGAAACTG CCTAAGATCT CGGGCCCTCG 780

GGCTTCGGCT TTGGGTGTAC ATGTTTGTGC TCCGGGCAAA TGCAAAGTGT GGTAGGATCG 840

ACACACTGCT GCCTTTACCA AGCAGCTGAG GGTATGTGAT AGGCAAATGT TCAGGGGCCA 900

CTGCATGGTT TCGAATAGAA AGAGAAGCTT AGCCAAGAAC AATAGCCGAT AAAGATAGCC 960

TCATTAAACG AAATGAGCTA GTAGGCAAAG TCAGCGAATG TGTATATATA AAGGTTCGAG 1020

GTCCGTGCCT CCCTCATGCT CTCCCCATCT ACTCATCAAC TCAGATCCTC CAGGAGACTT 1080

GTACACCATC TTTTGAGGCA CAGAAACCCA ATAGTCAACC GCGGACTGCG CATCATG 1137 (2) INFORMATION FOR SEQ ID NO: 0:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2261 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120

TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180

GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360

GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480

TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540

TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTGTGGGGT ATATATCTAG 720

TGGCCAGAAT GCCTAGGTCA CCTCTAAAGG TACCCTGCAG CTCGAGCTAG AGTTGTGAAG 780

TCGGTAATCC CGCTGTATAG TAATACGAGT CGCATCTAAA TACTCCGAAG CTGCTGCGAA 840

CCCGGAGAAT CGAGATGTGC TGGAAAGCTT CTAGCGAGCG GCTAAATTAG CATGAAAGGC 900

TATGAGAAAT TCTGGAGACG GCTTGTTGAA TCATGGCGTT CCATTCTTCG ACAAGCAAAG 960

CGTTCCGTCG CAGTAGCAGG CACTCATTCC CGAAAAAACT CGGAGATTCC TAAGTAGCGA 1020

TGGAACCGGA ATAATATAAT AGGCAATACA TTGAGTTGCC TCGACGGTTG CAATGCAGGG 1080

GTACTGAGCT TGGACATAAC TGTTCCGTAC CCCACCTCTT CTCAACCTTT GGCGTTTCCC 1140

TGATTCAGCG TACCCGTACA AGTCGTAATC ACTATTAACC CAGACTGACC GGACGTGTTT 1200 TGCCCTTCAT TTGGAGAAAT AATGTCATTG CGATGTGTAA TTTGCCTGCT TGACCGACTG 1260

GGGCTGTTCG AAGCCCGAAT GTAGGATTGT TATCCGAACT CTGCTCGTAG AGGCATGTTG 1320

TGAATCTGTG TCGGGCAGGA CACGCCTCGA AGGTTCACGG CAAGGGAAAC CACCGATAGC 1380

AGTGTCTAGT AGCAACCTGT AAAGCCGCAA TGCAGCATCA CTGGAAAATA CAAACCAATG 1440

GCTAAAAGTA CATAAGTTAA TGCCTAAAGA AGTCATATAC CAGCGGCTAA TAATTGTACA 1500

ATCAAGTGGC TAAACGTACC GTAATTTGCC AACGCGTTTC TAGATTGCAG AAGCACGGCA 1560

AAGCCCACTT ACCCACGTTT GTTTCTTCAC TCAGTCCAAT CTCAGCTGGT GATCCCCCAA 1620

TTGGGTCGCT TGTTTGTTCC GGTGAAGTGA AAGAAGACAG AGGTAAGAAT GTCTGACTCG 1680

GAGCGTTTTG CATACAACCA AGGGCAGTGA TGGAAGACAG TGAAATGTTG ACATTCAAGG 1740

AGTATTTAGC CAGGGATGCT TGAGTGTATC GTGTAAGGAG GTTTGTCTGC CGATACGACG 1800

AATACTGTAT AGTCACTTCT GATGAAGTGG TCCATATTGA AATGTAAGTC GGCACTGAAC 1860

AGGCAAAAGA TTGAGTTGAA ACTGCCTAAG ATCTCGGGCC CTCGGGCTTC GGCTTTGGGT 1920

GTACATGTTT GTGCTCCGGG CAAATGCAAA GTGTGGTAGG ATCGACACAC TGCTGCCTTT 1980

ACCAAGCAGC TGAGGGTATG TGATAGGCAA ATGTTCAGGG GCCACTGCAT GGTTTCGAAT 2040

AGAAAGAGAA GCTTAGCCAA GAACAATAGC CGATAAAGAT AGCCTCATTA AACGAAATGA 2100

GCTAGTAGGC AAAGTCAGCG AATGTGTATA TATAAAGGTT CGAGGTCCGT GCCTCCCTCA 2160

TGCTCTCCCC ATCTACTCAT CAACTCAGAT CCTCCAGGAG ACTTGTACAC CATCTTTTGA 2220

GGCACAGAAA CCCAATAGTC AACCGCGGAC TGCGCATCAT G 2261 (2) INFORMATION FOR SEQ ID Nθ:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1776 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

CAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120

TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180

GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360

GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480

TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540

TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTTCTAAAT ATATATCTAG 720 TGGCCAGAAT GCCTAGGTCA CCTCTAAATG TGTAATTTGC CTGCTTGACC GACTGGGGCT 780

GTTCGAAGCC CGAATGTAGG ATTGTTATCC GAACTCTGCT CGTAGAGGCA TGTTGTGAAT 840

CTGTGTCGGG CAGGACACGC CTCGAAGGTT CACGGCAAGG GAAACCACCG ATAGCAGTGT 900

CTAGTAGCAA CCTGTAAAGC CGCAATGCAG CATCACTGGA AAATACAAAC CAATGGCTAA 960

AAGTACATAA GTTAATGCCT AAAGAAGTCA TATACCAGCG GCTAATAATT GTACAATCAA 1020

GTGGCTAAAC GTACCGTAAT TTGCCAACGC GTTTCTAGAT TGCAGAAGCA CGGCAAAGCC 1080

CACTTACCCA CGTTTGTTTC TTCACTCAGT CCAATCTCAG CTGGTGATCC CCCAATTGGG 1140

TCGCTTGTTT GTTCCGGTGA AGTGAAAGAA GACAGAGGTA AGAATGTCTG ACTCGGAGCG 1200

TTTTGCATAC AACCAAGGGC AGTGATGGAA GACAGTGAAA TGTTGACATT CAAGGAGTAT 1260

TTAGCCAGGG ATGCTTGAGT GTATCGTGTA AGGAGGTTTG TCTGCCGATA CGACGAATAC 1320

TGTATAGTCA CTTCTGATGA AGTGGTCCAT ATTGAAATGT AAGTCGGCAC TGAACAGGCA 1380

AAAGATTGAG TTGAAACTGC CTAAGATCTC GGGCCCTCGG GCTTCGGCTT TGGGTGTACA 1440

TGTTTGTGCT CCGGGCAAAT GCAAAGTGTG GTAGGATCGA CACACTGCTG CCTTTACCAA 1500

GCAGCTGAGG GTATGTGATA GGCAAATGTT CAGGGGCCAC TGCATGGTTT CGAATAGAAA 1560

GAGAAGCTTA GCCAAGAACA ATAGCCGATA AAGATAGCCT CATTAAACGA AATGAGCTAG 1620

TAGGCAAAGT CAGCGAATGT GTATATATAA AGGTTCGAGG TCCGTGCCTC CCTCATGCTC 1680

TCCCCATCTA CTCATCAACT CAGATCCTCC AGGAGACTTG TACACCATCT TTTGAGGCAC 1740

AGAAACCCAA TAGTCAACCG CGGACTGCGC ATCATG 1776 ( 2 ) INFORMATION FOR SEQ ID NO : 22 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 1776 base pairs

(B) TYPE : nucleic acid

( C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 22 :

CAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120

TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180

GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360

GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480

TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540

TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTTCTAAAT ATATATCTAG 720 TGGCCAGAAT GCCTAGGTCA CCTCTAAATG TGTAATTTGC CTGCTTGACC GATCTAAACT 780

GTTCGAAGCC CGAATGTAGG ATTGTTATCC GAACTCTGCT CGTAGAGGCA TGTTGTGAAT 840

CTGTGTCGGG CAGGACACGC CTCGAAGGTT CACGGCAAGG GAAACCACCG ATAGCAGTGT 900

CTAGTAGCAA CCTGTAAAGC CGCAATGCAG CATCACTGGA AAATACAAAC CAATGGCTAA 960

AAGTACATAA GTTAATGCCT AAAGAAGTCA TATACCAGCG GCTAATAATT GTACAATCAA 1020

GTGGCTAAAC GTACCGTAAT TTGCCAACGC GTTTCTAGAT TGCAGAAGCA CGGCAAAGCC 1080

CACTTACCCA CGTTTGTTTC TTCACTCAGT CCAATCTCAG CTGGTGATCC CCCAATTGGG 1140

TCGCTTGTTT GTTCCGGTGA AGTGAAAGAA GACAGAGGTA AGAATGTCTG ACTCGGAGCG 1200

TTTTGCATAC AACCAAGGGC AGTGATGGAA GACAGTGAAA TGTTGACATT CAAGGAGTAT 1260

TTAGCCAGGG ATGCTTGAGT GTATCGTGTA AGGAGGTTTG TCTGCCGATA CGACGAATAC 1320

TGTATAGTCA CTTCTGATGA AGTGGTCCAT ATTGAAATGT AAGTCGGCAC TGAACAGGCA 1380

AAAGATTGAG TTGAAACTGC CTAAGATCTC GGGCCCTCGG GCTTCGGCTT TGGGTGTACA 1440

TGTTTGTGCT CCGGGCAAAT GCAAAGTGTG GTAGGATCGA CACACTGCTG CCTTTACCAA 1500

GCAGCTGAGG GTATGTGATA GGCAAATGTT CAGGGGCCAC TGCATGGTTT CGAATAGAAA 1560

GAGAAGCTTA GCCAAGAACA ATAGCCGATA AAGATAGCCT CATTAAACGA AATGAGCTAG 1620

TAGGCAAAGT CAGCGAATGT GTATATATAA AGGTTCGAGG TCCGTGCCTC CCTCATGCTC 1680

TCCCCATCTA CTCATCAACT CAGATCCTCC AGGAGACTTG TACACCATCT TTTGAGGCAC 1740

AGAAACCCAA TAGTCAACCG CGGACTGCGC ATCATG 1776 (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 745 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GGACCTACCC AGTCTCACTA CGGCCAGTGC GGCGGTATTG GCTACAGCGG CCCCACGGTC 60

TGCGCCAGCG GCACAACTTG CCAGGTCCTG AACCCTTACT ACTCTCAGTG CCTGTAAAGC 120

TCCGTGCGAA AGCCTGACGC ACCGGTAGAT TCTTGGTGAG CCCGTATCAT GACGGCGGCG 180

GGAGCTACAT GGCCCCGGGT GATTTATTTT TTTTGTATCT ACTTCTGACC CTTTTCAAAT 240

ATACGGTCAA CTCATCTTTC ACTGGAGATG CGGCCTGCTT GGTATTGCGA TGTTGTCAGC 300

TTGGCAAATT GTGGCTTTCG AAAACACAAA ACGATTCCTT AGTAGCCATG CATTTTAAGA 360

TAACGGAATA GAAGAAAGAG GAAATTAAAA AAAAAAAAAA AACAAACATC CCGTTCATAA 420

CCCGTAGAAT CGCCGCTCTT CGTGTATCCC AGTACCACGT CAAAGGTATT CATGATCGTT 480

CAATGTTGAT ATTGTTCCGC CAGTATGGCT CCACCCCCAT CTCCGCGAAT CTCCTCTTCT 540

CGAACGCGGT AGTGGCTGCT GCCAATTGGT AATGACCATA GGGAGACAAA CAGCATAATA 600

GCAACAGTGG AAATTAGTGG CGCAATAATT GAGAACACAG TGAGACCATA GCTGGCGGCC 660

TGGAAAGCAC TGTTGGAGAC CAACTTGTCC GTTGCGAGGC CAACTTGCAT TGCTGTCAAG 720 ACGATGACAA CGTAGCCGAG GACCC 745 (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1627 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

GGCGGTATTG GCTACAGCGG CCCCACGGTC TGCGCCAGCG GCACAACTTG CCAGGTCCTG 60

AACCCTTACT ACTCTCAGTG CCTGTAAAGC TCCGTGCGAA AGCCTGACGC ACCGGTAGAT 120

TCTTGGTGAG CCCGTATCAT GACGGCGGCG GGAGCTACAT GGCCCCGGGT GATTTATTTT 180

TTTTGTATCT ACTTCTGACC CTTTTCAAAT ATACGGTCAA CTCATCTTTC ACTGGAGATG 240

CGGCCTGCTT GGTATTGCGA TGTTGTCAGC TTGGCAAATT GTGGCTTTCG AAAACACAAA 300

ACGATTCCTT AGTAGCCATG CATCGGGATC CTTTAAGATA ACGGAATAGA AGAAAGAGGA 360

AATTAAAAAA AAAAAAAAAA CAAACATCCC GTTCATAACC CGTAGAATCG CCGCTCTTCG 420

TGTATCCCAG TACCACGGCA AAGGTATTTC ATGATCGTTC AATGTTGATA TTGTTCCCGC 480

CAGTATGGCT GCACCCCCAT CTCCGCGAAT CTCCTCTTCT CGAACGCGGT AGTGGCGCGC 540

CAATTGGTAA TGACCATAGG GAGACAAACA GCATAATAGC AACAGTGGAA ATTAGTGGCG 600

CAATAATTGA GAACACAGTG AGACCATAGC TGGCGGCCTG GAAAGCACTG TTGGAGACCA 660

ACTTGTCCGT TGCGAGGCCA ACTTGCATTG CTGTCAAGAC GATGACAACG TAGCCGAGGA 720

CCGTCACAAG GGACGCAAAG TTGTCGCGGA TGAGGTCTCC GTAGATGGCA TAGCCGGCAA 780

TCCGAGAGTA GCCTCTCAAC AGGTGGCCTT TTCGAAACCG GTAAACCTTG TTCAGACGTC 840

CTAGCCGCAG CTCACCGTAC CAGTATCGAG GATTGACGGC AGAATAGCAG TGGCTCTCCA 900

GGATTTGACT GGACAAAATC TTCCAGTATT CCCAGGTCAC AGTGTCTGGC AGAAGTCCCT 960

TCTCGCGTGC ANTCGAAAGT CGCTATAGTG CGCAATGAGA GCACAGTAGG AGAATAGGAA 1020

CCCGCGAGCA CATTGTTCAA TCTCCACATG AATTGGATGA CTGCTGGGCA GAATGTGCTG 1080

CCTCCAAAAT CCTGCGTCCA ACAGATACTC TGGCAGGGGC TTCAGATGAA TGCCTCTGGG 1140

CCCCCAGATA AGATGCAGCT CTGGATTCTC GGTTACNATG ATATCGCGAG AGAGCACGAG 1200

TTGGTGATGG AGGGACAGGA GGCATAGGTC GCGCAGGCCC ATAACCAGTC TTGCACAGCA 1260

TTGATCTTAC CTCACGAGGA GCTCCTGATG CAGAAACTCC TCCATGTTGC TGATTGGGTT 1320

GAGAATTTCA TCGCTCCTGG ATCGTATGGT TGCTGGCAAG ACCCTGCTTA ACCGTGCCGT 1380

GTCATGGTCA TCTCTGGTGG CTTCGTCGCT GGCCTGTCTT TGCAATTCGA CAGCAAATGG 1440

TGGAGATCTC TCTATCGTGA CAGTCATGGT AGCGATAGCT AGGTGTCGTT GCACGCACAT 1500

AGGCCGAAAT GCGAAGTGGA AAGAATTTCC CGGNTGCGGA ATGAAGTCTC GTCATTTTGT 1560

ACTCGTACTC GACACCTCCA CCGAAGTGTT AATAATGGAT CCACGATGCC AAAAAGCTTG 1620

TGCATGC 1627 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 91 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

GGACTGGCAT CATGGCGCCC TCAGTTACAC TGCCGTTGAC CACGGCCATC CTGGCCATTG 60

CCCGGCTCGT CGCCGCCCAG CAACCGGGTA C 91 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 18..95

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

AACCGCGGAC TGGCATC ATG GCG CCC TCA GTT ACA CTG CCG TTG ACC ACG 50

Met Ala Pro Ser Val Thr Leu Pro Leu Thr Thr 1 5 10

GCC ATC CTG GCC ATT GCC CGG CTC GTC GCC GCC CAG CAA CCG GGT 95

Ala lie Leu Ala lie Ala Arg Leu Val Ala Ala Gin Gin Pro Gly 15 20 25

AC 97

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Met Ala Pro Ser Val Thr Leu Pro Leu Thr Thr Ala lie Leu Ala lie 1 5 10 15

Ala Arg Leu Val Ala Ala Gin Gin Pro Gly 20 25

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ACT ACG TAG TCG ACT 15

Claims

WHAT IS CLAIMED IS:

1. A method for cloning a promoter that is active in a desired environmental condition, said method comprising: a. exposing a host to said environmental condition; b. extracting mRNA from said host; c. preparing a cDNA bank from said mRNA; d. detectably labelling a sample of said cDNA; e. hybridizing said labelled labelled cDNA to said cDNA bank; f. selecting clones from said hybridization of step (e) on the basis of the intensity of the hybridization; g. determining the relative abundancy of said selected clones in the cDNA bank of step (c); h. identifying the most abundant clones of step (g); and i. using the inserts of the clones of step (h) to identify and clone the host promoter that was responsible for expression of the corresponding mRNA under said environmental condition.

2. The method of claim 1 , wherein said condition is growth in glucose-containing medium.

3. The method of claim 1 , wherein the host is a filamentous fungi.

4. The method of claim 1, wherein the host is selected from the group consisting of Trichoderma, Aspergillus, Claviceps purpurea, Penicillium chrysogenum, Magnaporthe grisea, Neurospora, Mycosphaerella spp. , Collectotrichum trifolii, the dimorphic fungus Histoplasmia capsulatum, Nectia haematococca (anamorph:F#søπ«m solani f. sp. phaseoli and f. sp. pisi), Ustilago violacea, Ustilago maydis, Cephalosporium acremonium, Schizophyllum commune, Podospora anserina, Sordaria macrospora, Mucor circinelloides, and Collectotrichum capsici.

5. The method of claim 4, wherein the host is Trichoderma.

6. The method of claim 5, wherein the host is T. reesei.

1. An isolated promoter capable of expression of an operably- linked coding sequence in a fungal host grown on glucose.

8. The promoter of claim 7, wherein said promoter is cloned by a method comprising: a. exposing a host to said environmental condition; b. extracting mRNA from said host; c. preparing a cDNA bank from a first sample of said mRNA; d. detectably labelling a sample of said cDNA; e. hybridizing said labelled labelled cDNA to said cDNA bank; f. selecting clones from said hybridization of step (e) on the basis of the intensity of the hybridization; g. determining the relative abundancy of said selected clones in the cDNA bank of step (c); h. identifying the most abundant clones of step (g); and i. using the inserts of the clones of step (h) to identify and clone the host promoter that was responsible for expression of the corresponding mRNA under said environmental condition.

9. The promoter of claim 7, wherein said host is a filamentous fungi.

10. The promoter of claim 9, wherein said host is selected from the group consisting of Trichoderma, Aspergillus, Claviceps purpurea, Penicillium chrysogenum, Magnaporthe grisea,

Neurospora, Mycosphaerella spp. , Collectotrichum trifolii, the dimorphic fungus Histoplasmia capsulatum, Nectia haematococca (zniimoτph'.Fusarium solani f. sp. phaseoli and f. sp. pisi), Ustilago violacea, Ustilago maydis, Cephalosporium acremonium, Schizophyllum commune,

Podospora anserina, Sordaria macrospora, Mucor circinelloides, and Collectotrichum capsici.

11. The promoter of claim 10, wherein said host is Trichoderma.

12. The promoter of claim 11 , wherein said host is selected from the group consisting of T. reesei, T. harzianum,

T. longibrachiatum, T. viride, and T. koningii.

13. The promoter of claim 12, wherein said host is T. reesei.

14. The promoter of claim 13, wherein said promoter is the te 7 promoter.

15. The promoter of claim 14, wherein said tefl promoter contains promoter elements of the 1.2 kb sequence adjacent to the translational start site of SEQ ID 1.

16. The promoter of claim 13, wherein said promoter is the promoter of SEQ ID 2.

17. The promoter of claim 13, wherein said promoter is the promoter of SEQ ID 3.

18. The promoter of claim 13, wherein said promoter is the promoter of SEQ ID 4.

19. The promoter of claim 13, wherein said promoter is the promoter of SEQ ID 5.

20. The promoter of claim 13, wherein said promoter is the promoter of SEQ ID 6.

21. The promoter of claim 7, wherein said promoter is an altered cbhl promoter, such alteration decreasing the ability of glucose to repress said cbhl promoter.

22. The promoter of claim 21, wherein said native cbhl promoter has an altered mig-like sequence at approximately position -720 to -715.

23. The promoter of claim 22, wherein said mig-like sequence is

5 '-GTGGGG.

24. The promoter of claim 22, wherein said altered mig-like sequence 5 '-TCTAGA.

25. The promoter of claim 24, wherein said promoter is the cbhl promoter of pMI-24.

26. The promoter of claim 21, wherein said native cbhl promoter has the sequence TCTAAA at position -1505 to -1500 and the sequence TCTAGA at position -720 to -715.

27. The promoter of claim 22, wherein said native cbhl promoter has the sequence TCTAAA at position -1505 to -1500 and the sequence TCTAAA at position -1001 to -996 and the sequence

TCTAGA at position -720 to -715.

28. A promoter, wherein said promoter is selected from the cbhl promoter of the group consistin of pML016del5(ll), pMI-24, pMI-27, pMI-28, pMLO16del5(l l), SEQ ID 19, SEQ ID 20, SEQ ID 21 and SEQ ID 22.

29. A vector comprising the promoter of claim 7.

30. The vector of claim 29, wherein said promoter is operably linked to a coding sequence.

31. The vector of claim 30, wherein said coding sequence encodes an enzyme hydrolysing lignocellulose.

32. A host cell transformed with the vector of claim 31.

33. The vector of claim 32, wherein said vector is selected from the group consisting of pTHNlOOB, pML016del5(ll), pMI-24, pMI-27, pMI-28.

34. A host cell transformed with the vector of claim 33.

35. A host cell transformed with the vector of claim 30.

36. The host cell of claim 35, wherein said cell is a fungal cell.

37. The host cell of claim 36, wherein said fungal cell is that of a fungus selected from the group consisting of Trichoderma, Aspergillus, Claviceps purpurea, Peniάllium chrysogenum, Magnaporthe grisea, Neurospora, Mycosphaerella spp. ,

Collectotrichum trifolii, the dimorphic fungus Histoplasmia capsulatum, Nectia haematococca (_mamoτp :Fusarium solani f. sp. phaseoli and f. sp. pisi), Ustilago violacea, Ustilago maydis, Cephalosporium acremonium, Schizophyllum commune, Podospora anserina, Sordaria macrospora, Mucor drcinelloides, and Collectotrichum capsici.

38. The host cell of claim 37, wherein said fungus is Trichoderma.

39. The host cell of claim 38, wherein said fungus is selected from the group consisting of T. reesei, T. harzianum, T. longibrachiatum, T. viride, and T. koningii.

40. The host cell of claim 39, wherein said fungus is T. reesei.

41. An enzyme composition produced by a method comprising: a. growing the host cell of claim 35 in the presence of glucose; b. separating the host cell from the growth medium; and c. using said growth medium of step (b) as the source of the enzymes in said enzyme composition.