Cloning of an Aspergillus niger invertase gene by expression in Trichoderma reesei

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5^- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.     

Isolation of uridine auxotrophs from Trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes

Summary Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (>10⁴ transformants per g DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.     

Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformants

Summary To develop a transformation system in the filamentous fungus Podospora anserina we have selected ura5 mutants deficient in orotidylic acid pyrophosphorylase using a positive screening. These mutants could be transformed to prototrophy by an hybrid vector carrying the ura5 gene of this organism. The properties of the transformants have been analysed. In most cases integration of the transforming vector occurred outside the ura5 locus and frequently repeated tandem copies of the vector were found. Reversion of the transformants could also be selected and we found that it can occur by exact or only partial excision of the integrated vector.Abbreviations bp base pairs - kb 1,000 bp - EtBr ethidium bromide - PEG polyethyleneglycol     

Homologous transformation of the edible basidiomycete Agrocybe aegerita with the URA1 gene: characterization of integrative events and of rearranged free plasmids in transformants

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per g of DNA. The phenotype of the stable Ura+transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUra1-1. Integration of pUra1-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.     

Use of a <Emphasis Type="Italic">ura5</Emphasis><Superscript>+</Superscript>–<Emphasis Type="Italic">lys7</Emphasis><Superscript>+</Superscript> cassette to construct unmarked gene knock-ins in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

While the counterselectable Schizosaccharomyces pombe ura4 ⁺ gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 ⁺ confers 5FOA-resistant (5FOA^R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA^R mutants. Relative to the same approach using the homologous URA3 ⁺ gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOA^R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA^R strains carry mutations in either of two genes; ura4 ⁺ and ura5 ⁺. We then cloned the S. pombe ura5 ⁺ orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 ⁺ together with the S. pombe lys7 ⁺ gene. Using this doubly marked cassette to disrupt the sck1 ⁺ kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ⁻ and ura5 ⁻ mutants by screening 5FOA^R colonies for the loss of the lys7 ⁺ marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOA^R colonies in our studies arose from background ura4 ⁻ and ura5 ⁻ mutations.     

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP ⁺ transformants). Plasmids isolated from the transformants (URA ⁺ TRP ⁺) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.     

Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the URA3 gene of the yeast Saccharomyces cerevisiae

The mutational specificity of the base analog 6-N-hydroxylaminopurine(HAP) was studied in the URA3 gene of the yeast Saccharomycescerevisiae. Twenty-nine independent HAP-induced ura3 mutationswere sequenced. GC     

Constitutive mutants for orotidine 5 phosphate decarboxylase and dihydroorotic acid dehydrogenase in Saccharomyces cerevisiae

Summary A mutant yeast constitutive for synthesis of both dihydroorotic acid dehydrogenase and orotidine 5 phosphate decarboxylase has been isolated. This phenotype is due to a single gene unlinked to any of the five genes of the pyrimidine pathway. This gene, called ppr1, induces the unlinked genes ural and ura3. Non-chromosomal cloned ura3 is also under the control of ppr1.     

Effect of ochre nonsense mutations on yeast URA1 mRNA stability

Summary The genetic map of 27 mutants of the URA1 yeast gene has been established by meiotic recombination and 16 nonsense mutations characterized. The half life of URA1 mRNA was determined by two independent methods in the wild-type and in two ochre mutants localized at each extremity of the genetic map. A halflife of 15 min was found for the wild-type and for one of the ochre mutants. This half-life was radically reduced in the other ochre mutant whereas the instantaneous rate of its mRNA synthesis remained constant. After subcloning various endonucleolytic fragments the coding sequence of the URA1 gene was restricted to a 1.65 kb fragment within a 5.7 kb yeast DNA segment. Direct visualization of the URA1 mRNA by Northern hybridization of denatured RNA with a URA1 specific DNA probe revealed a length of 1.5 kb.     

Diverged 5s rRNA sequences adjacent to 5s rRNA genes in the rDNA of Pythium pachycaule

The non-transcribed spacer of the ribosomal DNA repeat ofPythium pachycaule was found to exist in two versions, one about 200 by longer than the other. Both versions contained a conserved 5s rRNA gene in inverted orientation and a diverged copy of the 5s sequence as a tandem repeat, with a spacer of about 180 by between them. This is the first report of multiple 5s-like sequences in the non-transcribed spacer of the rDNA of any organism. The two diverged 5s sequences differ from each other at nine positions in the 5 half of the sequence, but they have the same central 10-bp deletion and their 3 halves are identical. The diverged sequences, 5s and 5s, when aligned with the 5s sequence, have the same base at 63.9 and 58.8% of the positions respectively. The spacers between 5s sequences in both versions were highly conserved. The secondary structures of potential rRNA products of the diverged 5s sequences indicate that they may be pseudogenes. The relationships between the functional 5s gene and the diverged gene copies suggest that the 5s sequence family ofP. pachycaule originated by duplication of an ancestral gene, followed by divergence and the introduction of heterogeneity. This work shows the potential for tandemization of 5s genes in the NTS, and may shed light on the evolutionary events that led to the transition from the rDNA-linked 5s genes found inPythium species with filamentous zoosporangia to the unlinked tandem arrays found inPythium species with globose zoosporangia.     

High efficiency transformation of Kluyveromyces marxianus by a replicative plasmid

Kluyveromyces marxianus can be transformed with an efficiency of 10⁵ transformants/g of DNA by a replicative plasmid using electroporation. In order to obtain this efficiency, we isolated ura^- mutants cells which can be complemented by the URA3 gene from Saccharomyces cerevisiae. The URA3 gene and KARS2, a replicative origin from Kluyveromyces lactis which functions in K. marxianus, were ligated together in a plasmid which can be used as a vector to transform this strain.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Molecular genetic properties of the yeast Torulaspora pretoriensis: Characterization of chromosomal DNA and genetic transformation by Saccharomyces cerevisiae-based plasmids

Chromosomal DNA banding patterns were obtained for three strains of Torulaspora pretoriensis by contour-clamped homogenous-electric-field gel electrophoresis. Chromosomes were resolved into six or seven bands in the range of 800 to 2000 kb, and a polymorphism of these lengths was observed. By Southern-blot analysis, the three strains were shown to lack the DNA sequences homologous to the URA3, LEU2, TRP1, and HO genes of Saccharomyces cerevisiae. A uracil auxotrophic mutant derived from T. pretoriensis was transformed with three plasmids (YEp24, YRpHI, and YCp50) carrying the URA3 gene of S. cerevisiae by the lithium acetate method.     

Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast

A disruption cassette has been constructed containing the LEU2 gene flanked by directly repeated sitespecific recombination sites of the yeast plasmid, pSB3, which resembles the 2 m DNA of Saccharomyces cerevisiae. A disruption constructed by inserting this DNA fragment acquires a Leu⁺ phenotype, which can be easily removed by expressing the FLP-PSB3 gene encoding the site-specific recombinase of pSB3. A test was made using a Schizosaccharomyces pombe host. The ura4 ⁺ gene of S. pombe was replaced with the ura4::LEU2 gene constructed by inserting the disruption cassette into the ura4 ⁺ gene. Then, the FLP-pSB3 gene driven by the nmt1 ⁺ promoter was introduced into this disruptant. Upon de-repression of the nmt1 promoter by removing thiamine from the medium, the rate of appearance of Leu^- was increased. As expected the ura4 ⁺ locus underwent a structural change. Thus, the FLP-pSB3 protein and its target site can function adequately in S. pombe.     

Isolation,sequencing, and characterization of crp-5, a gene encoding a Neurospora ribosomal protein

A Neurospora crassa cytoplasmic ribosomal protein gene, crp-5, has been isolated and characterized. The cDNA was isolated by a differential screening of a cDNA library for glucose-inducible genes. The cDNA was subsequently used to identify and isolate crp-5 genomic sequences. Computer analysis of the DNA sequences showed that they contain an open reading frame which encodes a protein homologous to the rat ribosomal protein S26. The crp-5 mRNA levels are regulated in a carbon-source-dependent manner. The organization of the gene and the region upstream of the coding sequences are discussed.     

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences

Summary Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho^–) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho^– mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).Abbreviations mtDNA mitochondrial DNA - bp base pairs - kbp kilobase pairs     

Two family G xylanase genes from Chaetomium gracile and their expression in Aspergillus nidulans

With oligonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into Aspergillus nidulans and found to be expressed with their own promoters.     

Construction of a complete URA5 deletion strain of a human pathogenic yeast Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human pathogen, which infects the central nervous system causing the fatal disease, meningitis. In order to understand the genetic background of this human pathogen, the basic molecular manipulation techniques of deletion, overexpression, and so on have been developed. URA5, a gene encoding orotate phosphoribosyltransferase, has frequently been used to introduce foreign gene fragments by complementing ura5 mutant strains, which are not, however, stable; reversion to uracil prototroph is thus frequently observed on selective condition. The high possibility of reversion makes it inconvenient to use this mutation to identify appropriate transformants and thus, manipulation in molecular genetics. We report here the isolation of a stable ura5 mutant of C. neoformans, designated as TAD1, by eliminating the URA5 gene by homologous recombination using the biolistic DNA delivery system. The availability of the stable ura5 mutant offers the advantage that no spontaneous reversion occurs so that a satisfactory rate of homologous recombination can be achieved. The strain will allow efficient genomic analysis in C. neoformans.