Abstract: The present invention provides An isolated polypeptide which has proline specific endoprotease activity, selected from the group consisting of:(a) a polypeptide which has an amino acid sequence which has at least 40% amino acid sequence identity with amino acids 1 to 526 of SEQ ID NO:2 or a fragment thereof;(b) a polypeptide which is encoded by a polynucleotide which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or a fragment thereof which is at least 80% or 90% identical over 60, preferably over 100 nucleotides, more preferably at least 90% identical over 200 nucleotides, or (ii) a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO:1.

Abstract: The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing filamentous fungal expressed sequenced tags. The present invention also relates to filamentous fungal expressed sequenced tags and to computer readable media and substrates containing such expressed sequenced tags for monitoring expression of a plurality of genes in filamentous fungal cells.

Abstract: The present invention provides for methods of screening agents for cancer therapeutic and prophylactic activity. In particular embodiments, cells of the cellular slime mold Dictyostelium discoideum are contacted with candidate agents and the expression of genes in the Nucleotide Excision Repair (NER) and Base Excision Repair (BER) pathways are examined. Such genes include the helicases repB and repD, and the apurinic-apyrmidinic endonuclease APE.

Abstract: The present invention relates to a method for facilitating site directed homologous recombination in an organism to produce mutants comprising: 1) providing a large insert vector library comprising one or more large insert vectors, each of said large insert vectors comprising a piece of DNA, said DNA piece comprising multiple genes from a target organism and a first selectable marker functional for selection in bacteria; 2) providing a second vector comprising a transposable element, said transposable element comprising a nucleotide sequence coding for a second selectable marker flanked on each side by an inverted repeat sequence, wherein said selectable marker is bifunctional for selection in bacteria and the target organism and wherein said inverted repeat sequences are functional as a binding site for a transposase; 3) incubating said library with said second vector in the presence of a transposase specific for the inverted repeat sequences on the plasmid vector, such that the transposable element is tr

Abstract: The object of this invention is a procedure for obtaining a genetically altered merodiploid strain of Penicillium chrysogenum in which the activity of a regulating gene that controls penicillin biosynthesis has been altered, so that this strain is an overproducer of penicillin. This invention represents the first case in which the biosynthesis of a metabolite of industrial interest has been increased by the manipulation of a regulating gene, and therefore is a considerable novelty compared with the previously used technologies.

Abstract: Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence.

Abstract: The present invention relates to methods for preparing variants of a nucleotide sequence, comprising: (a) introducing into a population of filamentous fungal host cells: (i) one or more circular plasmids comprising a DNA sequence and a plasmid replicator mediating autonomous replication, wherein the one or more circularized plasmids are linearized by digestion of the DNA sequence and removal of a portion of the DNA sequence; and (ii) a library of DNA fragments comprising one or more mutations of the DNA sequence, wherein the fragments comprise at least two regions, one or more regions which are homologous to the 5′ region or the 3′ region of the gap in the linearized DNA sequence and/or plasmid sequence and one or more second regions which are homologous to the 5′ region or the 3′ region of the DNA fragments of the library; wherein the linearized plasmids and the DNA fragments recombine by in vivo recombination to produce a plurality of autonomously replicating plasmids comprising one

Abstract: The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

Abstract: The present invention provides methods and compositions that enable the experimental determination as to whether any gene in the genome of a diploid pathogenic organism is essential, and whether it is required for virulence or pathogenicity. The methods involve the construction of genetic mutants in which one allele of a specific gene is inactivated while the other allele of the gene is placed under conditional expression. The identification of essential genes and those genes critical to the development of virulent infections, provides a basis for the development of screens for new drugs against such pathogenic organisms.

Abstract: The invention concerns the integration of a gene of interest into the genome of a Yarrowia strain devoid of zeta sequences, by transforming said strain using a vector bearing zeta sequences.

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

Abstract: Novel polyketides and novel methods of efficiently producing both new and known polyketides, using recombinant technology, are disclosed. In particular, a novel host-vector system is described which is used to produce polyketide synthases which in turn catalyze the production of a variety of polyketides.

Abstract: The present invention is directed to isolated transducing phages, methods of isolating transducing phages, and methods of using transducing phages including, for instance, transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe, and producing a secondary metabolite from a microbe. The transducing phages typically have a broad host range, and transduce microbes in the Order Actinomycetales, in particular in the Family Streptomycetaceae, including Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces avermitilis, and Saccharopolyspora erythraea. The transducing phages can be specialized transducing phages or generalized transducing phages.

Abstract: Mismatch Repair Detection (MRD), a novel method for DNA-variation detection, utilizes bacteria to detect mismatches by a change in expression of a marker gene. DNA fragments to be screened for variation are cloned into two MRD plasmids, and bacteria are transformed with heteroduplexes of these constructs. Resulting colonies express the marker gene in the absence of a mismatch, and-lack expression in the presence of a mismatch. MRD is capable of detecting a single mismatch within 10 kb of DNA. In addition, MRD can analyze many fragments simultaneously, offering a powerful method for high-throughput genotyping and mutation detection.

Abstract: A method for in vivo production of a library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element independently of the host chromosomal replication machinery. The genetic element comprises i) an origin of replication from which replication is initiated, ii) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic, iii) a gene encoding the polypeptide of interest. Also methods for the generation of a DNA sequence encoding a desired variant of a polypeptide of interest, and for the determination of such a DNA sequence are described.

Abstract: The present invention is directed to a vector for expressing a gene of interest in a Candida species. The vector of the present invention comprises promoter and terminator sequences from a native Candida gene. This vector can be used to complement deleted genes in a Candida species. The present invention is also directed to a method for identifying genes essential for growth of a Candida species. Additionally, the present invention is directed to a method for screening for anti-fungal compounds effective against Candida species.

Abstract: Methods are provided for isolation of DNA sequences encoding proteins with properties of interest by means of expression cloning in filamentous fungal host cells. The isolated DNA sequences are useful in processes for producing the proteins of interest.

Abstract: The present invention relates to a transcription factor found in filamentous fungi, especially in Aspergillii, DNA sequences coding for said factor, its transformation into and expression in fungal host organisms, and the use of said factor in such hosts for increasing the expression of a polypeptide of interest being produced by said host.

Abstract: The infection of a mammalian host by a microorganism can be prevented or treated through the alteration of the expression of hypha-specific gene. These compounds may be used in the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised or immunosuppressed humans, for example, those having AIDS or undergoing transplantation or anti-cancer therapy.

Abstract: A process for site-directed integration of multiple copies of a gene in a mould is provided, which comprises transforming a mould cell containing in its chromosomal DNA a restriction site for a rare-cutting endonuclease, e.g., I-Scel, preferably introduced at a desired locus, e.g., within a selectable marker gene or in the neighborhood thereof, with a piece of DNA comprising multiple copies of at least one expressible gene comprising at least one structural gene encoding a desired protein, surrounded by two DNA fragments homologous to part of the DNA upstream and downstream, and in the neighborhood, of said restriction site, while during the transformation of the mould the presence of the rare-cutting endonuclease is provided, followed by selecting or screening for a mould cell in which the multiple gene copies of said expressible gene are inserted into the chromosomal DNA of the mould.

Abstract: Disclosed herein are novel phenol oxidizing enzymes naturally-produced by strains of the species Stachybotrys which possess a pH optima in the alkaline range and which are useful in modifying the color associated with dyes and colored compounds, as well as in anti-dye transfer applications. Also disclosed herein are biologically-pure cultures of strains of the genus Stachybotrys, designated herein Stachybotrys parvispora MUCL 38996 and Stachybotrys chartarum MUCL 38898, which are capable of naturally-producing the novel phenol oxidizing enzymes. Disclosed herein is the amino acid and nucleic acid sequence for Stachybotrys phenol oxidizing enzymes as well as expression vectors and host cells comprising the nucleic acid. Disclosed herein are methods for producing the phenol oxidizing enzyme as well as methods for constructing expression hosts.

Abstract: The present invention relates to methods for facilitating site directed homologous recombination in a eukaryotic organism to produce genomic mutants using transposon mediated mutagenesis of cosmid vectors carrying large genomic inserts from the target eukaryotic organism. The transposon carries a bifunctional marker that can be used for selection in both bacteria and the target eukaryotic organism. Minimization of the length of the cosmid vector allows for maximization of the size of the genomic insert carried by the cosmid. Maximization of the size of the genomic insert increases the frequency of homologous recombination with the genome of the target eukaryotic organism.

Abstract: The use of a means to vary Ubc4p or Ubc5p activity in a fungal cell to control the copy number of a plasmid in the cell. The level of Ubc4p or Ubc5p activity may be reduced/abolished (for example by gene deletion, mutagenesis to provide a less active protein, production of antisense mRNA or production of competitive peptides) to raise the copy number and increase yield of a protein encoded by the plasmid.

Abstract: The present invention cloned a cDNA clone encoding isopentenyl diphosphate (hereafter “IPP”) isomerase (EC 5.3.3.2) from a cDNA library of Hevea brasiliensis latex. The clone has a continuous open reading frame encoding a peptide of 234 amino acids with a predicted molecular mass of 26.7 kDa. The deduced protein is acidic with an isoelectric point of 4.7 and shows high sequence identity with other IPP isomerases. The recombinant protein expressed in Escherichia coli showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particle (WRP) deprived of initiating allylic diphosphates were performed with the addition of IPP isomerase in the reaction mixture. Results revealed that the recombinant IPP isomerase is catalytically active in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in rubber biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree.

Abstract: The invention relates to promoters of the genes glutamate dehydrogenase, &bgr;-acetylhexosaminidase and &ggr;-actin and their use in systems of expression, secretion and anti-sense of filamentary fungi. The invention also relates to the use of the promoters of the genes which code: (I) glutamate dehydrogenase NADP depending (EC.1.4.1.4) of Penicillium chrysogenum, (II) &ggr;-N-actylhexosaminidase (EC.3.2.1.52) of Penicillium chrysogenum and (III) &ggr;-actin of Penicillium chrysogenum and Acrimonium chrysogenum, which can be used for the construction of potent vectors of expression and secretion useful both for P. chrysogenum and for A. chrysogenum and related species. These promoters can also be used for blocking the genic expression through anti-sense construction. Under the control of the above mentioned promoters, it is possible to conduct the expression of other genes in filamentary fungi, thereby increasing the production of antibiotics and/or proteins inherent to the same.

Abstract: The present invention relates to a filamentous fungus useful for the production of heterologous polypeptides, having been modified by recombinant DNA technology in a manner by which the expression of alkaline proteases have been completely or partially inactivated. The invention also encompasses processes for the production of proteins of interest in high yields by using the fungi of the invention. The invention furthermore relates to methods for producing such fungi and DNA constructs to be used in these methods.

Abstract: The present invention is directed to isolated transducing phages, methods of isolating transducing phages, and methods of using transducing phages including, for instance, transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe, and producing a secondary metabolite from a microbe. The transducing phages typically have a broad host range, and transduce microbes in the Order Actinomycetales, in particular in the Family Streptomycetaceae, including Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces avermitilis, and Saccharopolyspora erythraea. The transducing phages can be specialized transducing phages or generalized transducing phages.

Abstract: DNA encoding the gene for the synthetase enzyme capable of generating &dgr; (L-a-aminoadipyl)-L-crysteinyl-D-valine (ACV) from its constituent amino acids was obtained from several penicillin and cephalosporin producing organisms, e.g. Penicillium chrysogenum, cephalosporium and a Flavobacterium species. The DNA was used to prepare recombinant vectors comprising the ACV synthetase gene and hosts transformed with such vectors. The ACV synthetase gene can form part of a gene cluster comprising other genes involved in -&bgr;-lactam biosynthesis and the production of penicillin by expression of the entire biosynthetic gene cluster for the synthesis of penicillin from primary amino acids is described. Suitable hosts in which expression can take place include heterologous hosts which are naturally non-producers of penicillin.

Abstract: The invention relates to Agrobacterium mediated transformation of moulds comprising species of the fungal sub-divisions Ascomycotina, Basidiomycotina, Deuteromycotina, Mastigomycotina, and Zygomycotina. Examples demonstrate the transformation of Aspergillus awamori (both protoplasts and conidia), Aspergillus nidulans, Aspergillus niger, Colletotrichum gloeosporioides, Fusarium solani pisi, Neurospora crassa, Trichoderma reesei, Pleurotus ostreatus and Agaricus bisporus (all conidia), and Fusarium graminearum (both conidia and rehydrated freeze dried ATCC material). Especially for Aspergillus awamori the transformation frequency is much higher than with conventional mould transformation techniques. It has further been found that not only one expressable gene can be introduced into these moulds, but even multiple copies of such gene, which, moreover, can be targeted e.g. in the chromosomal pyrG locus, as exemplified for A. awamori.

Abstract: The present invention is directed to recombinant nucleic acids encoding lactoferrin variants and portions thereof, having modified iron-binding capacity, and to vectors comprising same recombinant nucleic acids. The present invention is further directed to methods of producing such vectors, and to transfected cells harboring the same. Methods for the production of lactoferrin variants and portions thereof, in various eukaryotic or prokaryotic cells are also provided. Finally, the invention is directed to lactoferrin variants and portions thereof encoded by the nucleic acids of the invention and produced by the processes of the invention. Thus, the invention provides an efficient and economical means for the production of recombinant lactoferrin variants and portions thereof.

Abstract: Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence.

Abstract: Hybrid and novel polyketide synthases and polyketides are produced by use of a multiple vector system. The combinatorial possibilities offered by placing the various catalytic activities of PKS systems on separate vectors permits the construction of improved libraries of PKS and polyketides. In addition, polyketides can be produced in hosts that ordinarily do not produce polyketides by supplying, along with an expression system for the desired PKS, an expression system for holo ACP synthase.

Abstract: A process for expressing extracellular .beta.-glucosidase in a filamentous fungus by expressing a fungal DNA sequence encoding enhanced, deleted or altered .beta.-glucosidase in a recombinant host microorganism is disclosed. Recombinant fungal cellulase compositions containing enhanced, deleted or altered expression of .beta.-glucosidase is also disclosed.

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

Abstract: The invention relates to the DNA and protein encoded by the GA4 locus. This protein is believed to be a member of the family of enzymes involved in the biosynthesis of the gibberellin family (GA) of plant growth hormones which promote various growth and developmental processes in higher plants, such as seed germination, stem elongation, flowering and fruiting. More specifically, the protein encoded by the GA4 locus is an hydroxylase. The invention also relates to vectors containing the DNA and the expression of the protein encoded by the DNA of the invention in a host cell. Additional aspects of the invention are drawn to host cells transformed with the DNA or antisense sequence of the invention, the use of such host cells for the maintenance, or expression or inhibition of expression of the DNA of the invention and to transgenic plants containing DNA of the invention. Finally, the invention also relates to the use of the protein encoded by the GA4 locus to alter aspects of plant growth.

Abstract: The present invention relates to novel host cells and to methods of producing proteins. More specifically the invention relates to a host cell useful for the expression of heterologous proteins, which host cell has been genetically modified in order to express significantly reduced levels of a metalloprotease. Moreover the invention relates to a method of producing a heterologous protein, which method comprises cultivating the host cell in a suitable growth medium, followed by recovery of the desired protein.

Abstract: The invention concerns genes encoding recombinases that can be used to promote homologous recombination in eukaryotic cells. The application teaches methods by which a recombinase of one species can be used to isolate a homologous recombinase of a different species and methods to identify the isolated homologs. Recombinases from Ustilago maydis, Saccharomyces cerevisiae and humans are specifically included in the invention.The invention encompasses the method of producing an isolated recombinase protein in a prokaryotic cell and recovering the product in an active form. The invention also encompasses a genetically engineered gene which encodes a non-naturally occurring recombinase that causes a greater rate of recombination than does the naturally occurring recombinase. The invention further encompasses the use of recombinase proteins and of recombinase genes to promote homologous recombination, including recombination between a host cell genome and a chimeric oligonucleotide, i.e.

Abstract: Strains of Trichoderma spp. that are improved in their capacity as biological control agents for phytopathogenic fungi and nematodes are obtained that are able to over-produce a proteinase, with the aid of a transformation method which involves introducing the gene prb1 of Trichoderma that codes for the proteinase PrB1 under the control of adequate means of regulating expression, producing multiple copies in a stable manner; this ensures that the control achieved of the disease that is caused by pathogenic fungi or nematodes is better in the transgenic strain than in the uncultivated strain that is used as a receptor of the genetic information.