Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Summary Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35–36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.     

Genome analysis of imperfect fungi: electrophoretic karyotyping and characterization of the nuclear gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Curvularia lunata

Summary The gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes.     

Transformation of Aspergillus giganteus to hygromycin B resistance

Summary A wild strain of A. giganteus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of A. nidulans sequences. Stable transformants arose by heterogenous integration, mainly of tandem repeats of vector DNA at various sites in the host genome. Between 6 and 30 resistant colonies were obtained per g DNA per 3×10³ viable protoplasts. Vector DNA could be recovered by transformation of Escherichia coli with undigested genomic DNA from Aspergillus giganteus transformants.     

Transformation of the phytopathogenic fungus Septoria nodorum to hygromycin B resistance

Summary The phytopathogenic fungus Septoria nodorum has been transformed using a plasmid (pAN7-1) containing the Escherichia coli hygromycin phosphotransferase gene (hph). Large, stable hygromycin-resistant transformant colonies appeared at frequencies between 2 and 25 per g DNA when wheat-adapted and barley-adapted wild type strains were used as recipients. These transformants grew at hygromycin concentrations up to ten times that which inhibits the wild types. A second type of colony also developed on transformation plates. These appeared at higher frequencies, grew less vigorously and could not be subcultured in the presence of hygromycin. They are believed to be abortive transformants. Southern hybridization analyses indicated that transformation takes place via the integration of plasmid DNA into the fungal chromosomal DNA. Multiple integrations occur producing tandemly iterated arrays of plasmid molecules. Some transformants arose as heterokaryons. These could be resolved by propagation through a single spore and transformants purified in this way remained mitotically stable. All of 1,025 transformants tested were unchanged in pathogenicity. Reisolates from leaves retained their hygromycin-resistance, indicating that transformants remain stable during growth in plant tissue. Cotransformation of an unselected plasmid (p3SR2) carrying the Aspergillus nidulans amdS gene occurred at a high frequency.     

Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals

Trichoderma reesei was transformed to hygromycin B resistance using a novel vector, which contains the E. coli hygromycin B phosphotransferase gene (hph) fused between promoter and terminator elements of the homologous Trichoderma pkil (coding for pyruvate kinase) and cbh2 (coding for cellobiohydrolase II) genes, respectively. Transformation frequencies of over 1800–2500 transformants/g DNA were obtained, which is a 15–20-fold increase over that with pAN7-1, which contains hph between A. nidulans expression signals. Mitotically-stable transformants contained the hph gene and the regulatory sequences of the pkil promoter and the cbh2 terminator integrated into the genome. Evidence for preferentially ectopic integration is given.     

Transformation of Fulvia fulva,a fungal pathogen of tomato,to hygromycin B resistance

Summary A transformation system for the tomato pathogen Fulvia fulva has been developed. Hygromycin B resistant colonies were obtained after treatment of protoplasts with a plasmid containing an E. coli hygromycin B phosphotransferase gene fused to an Aspergillus nidulans promoter. The DNA was stably integrated into the genome. The number and sites of integrations varied among transformants. The demonstration of transformation opens the way for the molecular genetic analysis of the interaction of Fulvia with tomato.     

An electrophoretic karyotype of the cultivated mushroom —Agaricus bisporus

Thirteen chromosomal-sized DNA bands of the cultivated mushroom Agaricus bisporus have been resolved using the method of clamped homogeneous electric field (CHEF) electrophoresis. Using chromosome size standards from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans, the estimated size of the chromosomal DNAs ranged from 3.5 to 1.2 megabase pairs (Mb). By Southern hybridization with homologous gene probes, the chromosomal location of cellulase and laccase genes have been mapped. In addition, rDNA has been assigned to chromosomal bands using a heterologous gene probe. Genomic rearrangement is suggested in the commercial heterokaryon, as indicated by the presence of non-comigrating homologous chromosomes, identified by a number of probes for particular DNA sequences.     

Transformation of Botrytis cinerea with the hygromycin B resistance gene,hph

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), confering hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2–10 transformants per g of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.     

Investigation of plant organellar DNAs by pulsed-field gel electrophoresis

Mitochondrial (mt) DNAs from several higher-plant species (Arabidopsis thaliana, Beta vulgaris, Brassica hirta, Chenopodium album, Oenothera berteriana, Zea mays) were separated by pulsed-field gel electrophoresis (PFGE). Hybridization of the separated DNA with mtDNA-specific probes revealed an identical distribution of mtDNA sequences in all cases: part of the DNA formed a smear of linear molecules migrating into the gel, the rest remained in the well. Hybridization signals in the compression zone of the gels disappeared after RNase or alkaline treatment. It was shown that the linear molecules are not products of unspecific degradation by nucleases. All plastid (pt) DNA from leaves of Nicotiana tabacum remained in the well after PFGE. Separation of linear monomers and oligomers of the chloroplast chromosomes of N. tabacum was achieved by mild DNase treatment of the well-bound DNA. DNase treatment of well-bound mtDNA, however, generated a smear of linear molecules. PtDNA from cultured cells of C. album was found after PFGE to be partly well-bound, and partly separated into linear molecules with sizes of monomeric and oligomeric chromosomes. The ease with which it was possible to detect large linear molecules of plastid DNA indicates that shearing forces alone can not explain the smear of linear molecules obtained after PFGE of mtDNA. The results are discussed in relation to the structural organization of the mt genome of higher plants.     

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio – Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n = 20), Typhimurium (n = 4), Zanzibar (n = 2), Manhattan (n = 1), Bovismorbificans (n = 1), Amsterdam (n = 1), Saint Paul (n = 1), Kentucky (n = 1) and Muenster (n = 1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1–5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.     

The transformation of protoplasts of Leptosphaeria maculans to hygromycin B resistance

Summary Conditions are described for the efficient isolation and regeneration of protoplasts of a fungal pathogen of brassicas, Leptosphaeria maculans. Treatment of the protoplasts with DNA of the plasmid pAN7-1 (containing an E. coli hygromycin phosphotransferase gene with Aspergillus nidulans expression signals) and plating under selective conditions resulted in the formation of hygromycin 13-resistant colonies. Southern blot analysis of resistant colonies indicated that single copies of the plasmid had integrated into different sites in the genome. In twelve of the transformants analysed so far, the integration is stable through mitosis. The demonstration of efficient transformation is an essential first step in the molecular analysis of pathogenicity of this commercially important pathogen.     

Transformation of Penicillium roqueforti to phleomycin- and to hygromycin B-resistance

Summary A strain of Penicillium roqueforti was transformed to hygromycin B and phleomycin resistance using resistance genes under the control of A. nidulans sequences. The transformation efficiency ranged from 0.15 to 1 transformant per g DNA per 10⁶ viable protoplasts when transformants were selected on medium containing a high antibiotic concentration (7–10 times the minimum inhibitory concentration). Transformation resulted from either single copy or tandem integration of the phleomycin vector while the hygromycin vector was modified during integration. The transformed antibiotic-resistant phenotypes were mitotically stable with or without selective pressure.     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Identification,characterization and sequence of Candida albicans repetitive DNAs Rel-1 and Rel-2

Two moderately repetitive DNA elements, Rel-1 and Rel-2, were identified in a screen for clones that hybridized to a Candida albicans minichromosome. Rel-1, a 223-bp sequence, is C. albicans-specific. The 2789-bp Rel-2 sequence hybridizes weakly to C. stellatoidia DNA but not to DNA from several other yeast species. Genomic Southern-blot analysis indicated that Rel-1 and Rel-2 are often closely associated in the genome, suggesting that they may be subsequences of a larger repetitive element. Small subrepeats are located in the nucleotide sequence of both clones. Hybridization demonstrated that Rel-2 contains both repetitive and unique DNA sequences. The repetitive DNA is present on most, and perhaps all, C. albicans chromosomes. The unique sequence maps to chromosome 7; however, in some strains, it is also present on additional chromosomes.     

Electrophoretic karyotypes and gene mapping in eight species of the Fusarium sections Arthrosporiella and Sporotrichiella

Pulsed-field gel electrophoresis was used to identify karyotypes for eight species of the Fusarium sections Arthrosporiella and Sporotrichiella. The total number of chromosome-sized DNA molecules varied from six to nine, depending on the species. The sizes of chromosomes ranged from 0.4 to approximately 6.5 Mb which gave estimates of genome size of between 27.0 and 29.9 Mb. When fractionated chromosomes of the eight species were probed with Tox5, a gene coding for the key-enzyme of trichothecene biosynthesis, strong hybridization signals developed in F. poae and F. sporotrichioides, suggesting that of the eight species examined only these two have the genetic potentiality to produce trichothecene mycotoxins. By using heterologous probes from Aspergillus different rRNA loci have also been mapped on Fusarium chromosomes.     

Chromosomal polymorphism of the yeast Yarrowia lipolytica and related species: electrophoretic karyotyping and hybridization with cloned genes

Significant differences in electrophoretic karyotyping patterns were found among 27 strains of Y. lipolytica. Twenty-one of these strains were classified into four groups of similar karyotypes while six strains showed unique karyotypes. Chromosomal DNAs of different strains were hybridized with cloned genes of Y. lipolytica (URA3, LEU2, ARS18 and ARS68), which revealed four different bands in most strains. We conclude that the haploid chromosome number of Y. lipolytica is at least four, and possibly five or six. Electrophoretic karyotyping and hybridization with cloned genes of Y. lipolytica provided evidence of a large divergence between Y. lipolytica and related species of Saccharomycopsis, Endomycopsella and Endomyces.     

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.     

Molecular analysis and antimicrobial resistance of Salmonella isolates recovered from raw meat marketed in the area of "Grand Tunis", Tunisia

A total of 315 samples of chicken (60), beef (144), minced meat (56), lamb meat (33), merguez (10) and fish (12) were collected from various local outlet stores in the area of "Grand Tunis", Tunisia between 2006 and 2008. Salmonella was recovered from 80 samples with the highest occurrence in chicken (48.3%) followed by beef (29.8%), minced meat (10.7%) and lamb (6.0%). No Salmonella were isolated from 12 fish and 10 merguez samples (typical Tunisian sausages). Nine serovars were identified among the isolates with the predominance of Salmonella Typhimurium (n=25) followed by Salmonella Kentucky (n=14), Salmonella Suberu (n=12) and Salmonella Zanzibar (n=11). Isolated Salmonella were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) analysis, plasmid content and antimicrobial resistance profiling. Sixteen (20.0%) Salmonella isolates displayed resistance to ampicillin (13 isolates), streptomycin (five isolates), cefoperazone (two isolates), furazolodine (two isolates), with seven of these isolates displaying multiple resistance to at least two of these antimicriobal agents. PFGE analysis showed homogenous restriction patterns in each serovar. Compiled serotyping, PFGE analysis, plasmid profiling and antimicrobial resistance data provided additional discrimination.