Highly efficient homologous integration via tandem exo-β-1,3-glucanase genes in the common mushroom,Agaricus bisporus

  Homologous integration was studied in the common mushroom, Agaricus bisporus, using a plasmid (pHAG3-1) carrying the hygromycin-resistance gene and a 3.2-kb genomic fragment from A. bisporus. Homologous integration was found in 30–60% of the transformants obtained with pHAG3-1 linearized at three different positions within the homologous sequence, generating either blunt, 5′- or 3′-protruding ends. The genomic fragment was found to contain two homologous open reading frames in tandem, which showed 60% similarity to exo-β-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The level of the corresponding mRNA is low in the vegetative mycelium and relatively high in fruiting bodies. In the vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position, exoglucanase mRNA was strongly increased without any apparent effect on growth rate or morphology. Received: 6 February 1996 / 27 March 1996     

Contribution of various classes of defective mitochondrial DNA molecules to senescence in Podospora anserina

The unavoidable arrest of vegetative growth in Podospora anserina (senescence process) is always correlated with rearrangements of the mitochondrial chromosome, mainly consisting in the amplification of particular regions as tandemly repeated circular molecules (senDNAs). One sequence systematically amplified in senescent cultures corresponds precisely to the first intron (intron α) of the cox1 gene; nevertheless, other regions (called β and γ) are also frequently amplified. The experiments presented in this paper show that cellular death is in some cases associated with the sole presence of large amounts of senDNA β. In addition, we provide evidence that senDNA β and senDNA α accumulate by different mechanisms, as previously proposed. This suggests that β senDNAs have a lethal effect on the mycelium on their own and most likely have replicative properties independent of the presence of sequence α. These data do not fit well with the current opinion that gives an essential role to intron α in the senescence of P. anserina. Received: 10 May / 11 November 1996     

Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola

A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 10⁵ spores. When assayed by PCR and DNA blot analyses, more than 50% of the transformants recovered had integrated in the targeted Mfcut1 locus. Both target-gene-specific and non-specific integrations carried direct (head-to-tail) repeat T-DNA integrations. Sequence analysis of these T-DNA integrations revealed that 26 bp of the T-DNA right border were missing at the junctions between direct repeats in all cases. The recombination event during non-specific T-DNA integration in this fungus was unlike that reported in Agrobacterium-mediated transformation in plants.Note: Nucleotide sequence data reported are available in GenBank under the accession number–DQ173196.     

Introduction of large DNA inserts into the barley pathogenic fungus, <Emphasis Type="Italic">Ustilago hordei</Emphasis>, via recombined binary BAC vectors and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Genetic transformation of organisms with large genome fragments containing complete genes, with regulatory elements or clusters of genes, can contribute to the functional analysis of such genes. However, large inserts, such as those found on bacterial artificial chromosome (BAC) clones, are often not easy to transfer. We exploited an existing technique to convert BAC clones, containing genomic DNA fragments from the barley-covered smut fungus Ustilago hordei to binary BACs (BIBACs) to make them transferable by the Agrobacterium tumefaciens T-DNA transfer machinery. Genetic transformation of U. hordei with BAC clones using polyethylene glycol or electroporation is difficult. As a proof of concept, two BAC clones were successfully converted into BIBAC vectors and transferred by A. tumefaciens into U. hordei and U. maydis, the related corn smut fungi. Molecular analysis of the transformants showed that the T-DNA containing the BAC clones with their inserts was stably integrated into the U. hordei genome. A transformation frequency of approximately 10⁻⁴ was achieved both for U. hordei sporidia and protoplasts; the efficiencies were 25–30 times higher for U. maydis. The combination of in vivo recombineering technology for BAC clones and A. tumefaciens-mediated transformation of Ustilago species should pave the way for functional genomics studies.     

Resistance to arsenite modulates levels of α-tubulin and sensitivity to paclitaxel in <Emphasis Type="Italic">Leishmania donovani</Emphasis>

Tubulin expression is known to alter due to drug resistance. Differentiation of Leishmania promastigotes into infectious amastigotes has been reported to be accompanied by differential tubulin gene expression. In this study, α-tubulin expression under various stages of differentiation was measured in an in vitro generated arsenite-resistant L. donovani strain. While levels of expression of α-tubulin were similar in wild type and resistant promastigotes, during conversion into axenic amastigotes the changes in the expression levels of α-tubulin varied widely between the two strains. Sensitivity of the two strains to paclitaxel (known to promote tubulin assembly) differed, with the resistant strain being two-fold more sensitive than the wild type strain. Paclitaxel was also seen to cause differential effects on α-tubulin levels in the two strains. Received: 5 January 2000 / Accepted: 15 March 2000     

An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-β-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species. Received: 5 February / 20 March 2000     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

Tubulin is hyperphosphorylated on serine and tyrosine residues in arsenite-resistant Leishmania donovani promastigotes

An arsenite-resistant strain of Leishmania donovani was generated in vitro by the sequential exposure of a wild type strain to increasing concentrations of sodium m-arsenite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole cell lysates of the two strains revealed that a protein band at the 55 kDa position showed slower migration in the resistant samples. This band was identified as tubulin by immunoblotting, with both α- and β-tubulin showing retarded migration in the resistant strain. Investigations into the reason for the observed slower migration revealed that phosphorylation of tubulin on both serine and tyrosine residues was enhanced in the resistant strain when compared to the wild type strain. Received: 5 January 2000 / Accepted: 15 March 2000     

<Emphasis Type="Italic">In Vivo</Emphasis> Dynamic Deformation of the Mitral Valve Annulus

Though mitral valve (MV) repair surgical procedures have increased in the United States [Gammie, J. S., et al. Ann. Thorac. Surg. 87(5):1431–1437, 2009; Nowicki, E. R., et al. Am. Heart J. 145(6):1058–1062, 2003], studies suggest that altering MV stress states may have an effect on tissue homeostasis, which could impact the long-term outcome [Accola, K. D., et al. Ann. Thorac. Surg. 79(4):1276–1283, 2005; Fasol, R., et al. Ann. Thorac. Surg. 77(6):1985–1988, 2004; Flameng, W., P. Herijgers, and K. Bogaerts. Circulation 107(12):1609–1613, 2003; Gillinov, A. M., et al. Ann. Thorac. Surg. 69(3):717–721, 2000]. Improved computational modeling that incorporates structural and geometrical data as well as cellular components has the potential to predict such changes; however, the absence of important boundary condition information limits current efforts. In this study, novel high definition in vivo annular kinematic data collected from surgically implanted sonocrystals in sheep was fit to a contiguous 3D spline based on quintic-order hermite shape functions with C² continuity. From the interpolated displacements, the annular axial strain and strain rate, bending, and twist along the entire annulus were calculated over the cardiac cycle. Axial strain was shown to be regionally and temporally variant with minimum and maximum values of −10 and 4%, respectively, observed. Similarly, regionally and temporally variant strain rate values, up to 100%/s contraction and 120%/s elongation, were observed. Both annular bend and twist data showed little deviation from unity with limited regional variations, indicating that most of the energy for deformation was associated with annular axial strain. The regionally and temporally variant strain/strain rate behavior of the annulus are related to the varied fibrous-muscle structure and contractile behavior of the annulus and surrounding ventricular structures, although specific details are still unavailable. With the high resolution shape and displacement information described in this work, high fidelity boundary conditions can be prescribed in future MV finite element models, leading to new insights into MV function and strategies for repair. Chad Eckert and Brett Zubiate contributed equally to this work.     

Investigation of the parasitic nematode Ascaridia galli (Shrank 1788) as a potential vector for Salmonella enterica dissemination in poultry

During recent years, the level of organically farmed poultry in Denmark has increased. Subsequent investigations have demonstrated an incidence of 64% of Ascaridia galli infections in layers established in organic farming systems. Studies to determine the interaction of Salmonella enterica with the parasitic nematode A. galli associated with poultry were undertaken to establish the significance of A. galli in the dissemination of S. enterica. A. galli was isolated from 40-week-old Lohmann Brown Salmonella-free layers. Worms were subsequently maintained in vitro and exposed to S. e. serovar Typhimurium at concentrations of 10⁵–10⁶ colony forming units/ml for varying times (24–144 h). Eggs were harvested aseptically from the worms and the associations of S. e. Typhimurium in relation both to the eggs and to structures on the surface of the worm were studied, using immunofluorescence, viable counts and in situ hybridisation. Results show attachment of S. e. Typhimurium to the outer coating of the eggs and possible internalisation. Evidence of association of the bacteria with the nematode eggs was further substantiated by establishing Salmonella infection in day-old chicks after dosing them with eggs harvested from parasitic worms infected in vitro with Salmonella. Received: 11 September 2000 / Accepted: 14 September 2000     

The mitochondrial DNAs of Agaricus: heterogeneity in A. bitorquis and homogeneity in A. brunnescens

Summary Mitochondrial DNAs were isolated from four cultivated strains of the commercial two-spored mushroom Agaricus brunnescens (bisporus) and from ten isolates of the four spored mushroom Agaricus bitorquis. Digestion of the fungal mitochondrial DNA with restriction endonucleases yielded numerous fragments. Summation of the fragment sizes gave a mitochondrial DNA size of 98.3 ± 2.4 kilobases (kb) (64.9 x 10⁶ daltons) for A. brunnescens. The size of the mitochondrial DNA ranged from 148.5 ± 10.8 kb (98.0 x 10⁶ daltons) to 176.3 ± 12.0 kb (116.4 x 10⁶ daltons) for A. bitorquis. The restriction patterns, produced by a variety of endonucleases, were identical for all four isolates of A. brunnescens. The ten isolates of A. bitorquis demonstrated extensive restriction pattern heterogeneity and have been tentatively assigned into four groups. Approximately 60% of the A. bitorquis mitochondrial DNA restriction fragments show sequence homology with A. brunnescens mitochondrial DNA based on DNA — DNA hybridizations.     

A novel `two-component' protein containing histidine kinase and response regulator domains required for sporulation in Aspergillus nidulans

We have characterised a novel Aspergillus nidulans gene encoding a `two-component' signalling protein (tcsA). tcsA encodes both a histidine kinase domain and a response regulator domain similar to those found in bacterial, lower eukaryotic and plant members of the two-component family of proteins, while two PAS domains in the amino-terminal region of the predicted tcsA product may monitor the signal which regulates a tcsA histidine kinase-response regulator phosphorelay. While tcsA is nonessential for vegetative growth, cells lacking the gene are unable to produce conidia on standard Aspergillus growth media. However, tcsA is not absolutely required for production since this defect is suppressed by growth on 1 M sorbitol. Received: 23 December 1999 / 1 March 2000     

A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae

We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Δ) strain of the yeast Saccharomyces cerevisiae. The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency. However, the fidelity of rejoining was lower for the rad52Δ extract than for normal wild-type. Received: 2 September /2 November 1997     

A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short (∼250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5′ and 3′ flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.     

A screening method for autoregulators of anthracycline-producing streptomycetes

A novel method was proposed for screening of autoregulators by anthracycline-producing strains of Streptomyces griseus. By means of a modified cosynthesis procedure, an indicator strain was detected capable of producing both leukaemomycin and aerial mycelium only in the presence yielding organisms and their mutants, but also by a streptomycin-producing strain of Streptomyces griseus as well as its non-differentiation derivative.     

Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker

The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae. Received: 16 March 2000 / Accepted: 18 July 2000     

Extended-spectrum β-lactamase-producing Shigella strains in Israel, 2000–2004

Routine susceptibility testing of 5,616 Shigella isolates at the National Shigella Reference Centre in Israel over a 5-year period (2000–2004) revealed resistance to ceftriaxone in one strain of Shigella boydii 2 and in two strains each of Shigella flexneri 2a, S. flexneri 6, and Shigella sonnei. All seven isolates were confirmed as producers of extended-spectrum β-lactamase (ESBL) by the combination disk method, the Vitek 1 system, and a modification of the double-disk synergy test, which is based on the inhibitory properties of clavulanic acid, tazobactam, and sulbactam. Tazobactam had the strongest effect in all seven strains. Molecular characterization of the ESBLs identified CTX-M-type enzymes, consisting of the CTX-M-9 group (n = 3), CTX-M-3 (n = 2), CTX-M-39 (n = 1), and CTX-M-2 group (n = 1). Three of the strains also carried bla-_OXA genes and a bla-_TEM gene. Although the prevalence of ESBLs in this study was low, further research is needed on the spread and transfer of resistance genes, both in hospitals and in the community.     

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family

Summary.  A universal primer (Sprimer: 5^′-GGX AAY AAY AGY GGX CAZ CC-3^′, X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3′-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province). Received June 30, 2000 Accepted September 28, 2000     

Gender-associated mitochondrial DNA heteroplasmy in the venerid clam Tapes philippinarum (Mollusca Bivalvia)

This paper provides evidence of a gender-associated mitochondrial DNA (mtDNA) heteroplasmy in the clam Tapes philippinarum. The pattern of tissue distribution of the two observed mtDNA types (referred to as M and F) parallels that of the doubly uniparental inheritance system of Mytilus. The mitochondrial genetic features of the clam are related to a different rate of evolution of the M- and F-type mtDNAs. Since clams are known to be phylogenetically very distant from mussels, the present findings support the hypothesis that the mechanism producing gender-associated heteroplasmy should be considered an ancestral character in the Bivalvia. Received: 2 August 2000 / Accepted: 3 December 2000