The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

Summary Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.     

Homologous recombination and allele replacement in transformants of Fusarium fujikuroi

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others. Received: 5 November 1999 / Accepted: 16 March 2000     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

Extragenic suppressor mutations of a β-tubulin mutation in the basidiomyceteCoprinus cinereus: Isolation and genetic and biochemical analyses

In total, 111 revertants were isolated from the oidia of a heat-sensitive, -tubulin mutant BEN193 (benA193) of the basidiomyceteCoprinus cinereus after mutagenesis by ultraviolet. Of the 111 revertants, 48 were genetically analyzed. In 15 of the 48 revertants, reversion was due to mutations at loci unlinked tobenA, whereas in the remaining 33 revertants, reversion was due to mutations withinbenA or at loci closely linked tobenA. The 15 extragenic suppressor mutations comprised three groups in terms of genetic linkage; two of them were designated asmipA andmipB. Suppressor mutations in the third group were found to bebenC, one of the four loci we have previously identified as genes conferring benomyl resistance. Biochemical analysis revealed thatbenC ⁺ is a structural gene for a major -tubulin in vegetative hyphae.     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR region gene

Summary A phage lambda gene that gives a 100-fold increase in recombinant frequencies for RecABC pathway-mediated, phage-plasmid homologous recombination (Shen and Huang 1986) maps to ninG (orf-204) of lambda. We call this gene rap, for recombination adept with plasmid. A similar determinant exists in Charon 4A and maps in 80-derived sequences, between nin5 and the Rz homology with lambda. The absence of the Rap⁺ phenotype from certain lambda vectors explains the inefficiency of screening the resulting phage libraries using phage-plasmid homologous recombination. The mapping of rap permits the construction of lambda vectors more suitable for this screening technique.     

Mutations in {beta}-Tubulin Block Transhyphal Migration of Nuclei in Dikaryosis in the Homobasidiomycete Coprinus cinereus

ß-Tubulins from fourteen benomyl-resistant strainsof the homobasidiomycete Coprinus cinereus, which carry thebenA, benB, benC or benD mutations, were analyzed by urea SDS-PAGEor isoelectric focusing and subsequent immunoblot analysis.Electrophoretic aberrations in a major ß-tubulin isotype,denoted ß1 were found in two strains, BEN154 and BEN215,both of which carry benomyl resistance mutations in benA ⁺ Theaberrations of ß1 in BEN154 and BEN215 cosegregatedwith benomyl resistance among the progeny of outcrosses of BEN154 and BEN215 to wild type, indicating that the ß1aberrations were caused by the benA mutations. Both the mutantand wild-type ß1 tubulins were present in the heterozygousdikaryons, BEN 154/wild-type and BEN215/wild-type, ruling outpost-translational modification as a possible cause for theaberrations in ß1. Thus, we conclude that benA isa structural gene for ß1. Transhyphal migration ofnuclei in dikaryosis was blocked in the mycelia of BEN 154 andin its progeny that carried benA (ß1 mutation), demonstratingthat microtubules are involved in the migration process. Nuclearmigration in dikaryosis seems to differ in terms of mechanism,at least in part, from the migration of tetrad nuclei from basidiainto prespores during formation of basidiospores and from themigration of nuclei from basidiospores into hyphae during germination,because a benA mutation blocked the former without affectingthe latter two processes. (Received May 19, 1989; Accepted August 30, 1989)     

Effects of methyl benzimidazoIe-2-yl carbamate on microtubule and actin cytoskeleton inAspergillus nidulans

Summary The effects of methyl benzimidazole-2-yl carbamate (MBC) on microtubule and actin cytoskeleton were analyzed by indirect immunofluorescence and transmission electron microscopy in a wild-type strain and a benomyl-resistant mutant (benA ₁₀) ofAspergillus nidulans. The treatment of the wild-type strain with sublethal doses of MBC not only caused depolymerization of cytoplasmic microtubules (MTs), but also changed the pattern of actin at the hyphal tips. In the MBC-treated hyphae, the actin fluorescence was concentrated at the very tip region of the hypha, whereas in the control hyphae, the actin fluorescence was weak at the very tip and strong below the tip. The dose of MBC used for the wild-type strain did not depolymerize the MTs or modify the actin organization at the apex in the mutant strain, which confirmed that the change in actin distribution in the wild-type strain was due to the disruption of MTs. In the mutant strain, a seven times higher concentration of MBC than in the wild-type strain was required to depolymerize MTs and to alter the actin organization at the apex. The ultrastructural study of the MBC-treated hyphae revealed that the area containing apical vesicles was larger and the number of microvesicles was higher than in control hyphae. These changes probably resulted from the disassembly of MTs and the reorientation of actin cytoskeleton in MBC-treated apexes and suggested that MTs would organize the actin at the apex, which in turn would restrict the vesicle fusion to a narrow area at the hyphal tip. In treated hyphae of both strains without cytoplasmic MTs, mitotic spindles were detected although in lower number and with slightly modified morphology.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - EM electron microscopy - ER endoplasmic reticulum - IIP indirect immunofluorescence - MBC methyl benzimidazole-2-yl carbamate - MTs microtubules     

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

Cloning and characterization of a DNA repair gene inHaemophilus influenzae

Using a high-efficiency DNA cloning vector pJ1–8, a DNA repair geneuvr1 has been self-cloned in bacteriumHaemophilus influenzae. Chimeric plasmid pKuvrl, carrying wild type allele ofuvr1 gene and flanking DNA sequences, specifically complements auvr1 gene mutation in the bacterial chromosome. Auvr1_} mutation could be transferred from chromosome byin vivo recombination to pKuvr1 and isolated and designated as plasmid pKuvrl₋. Plasmid pKuvrl carries a 11.3 kb chromosomal DNA insert which was scanned for the presence of any other DNA repair genes by a novel method of directed mutagenesis. Preliminary analysis of the 3 new mutants isolated by this method supports the notion that the insert contains more than one gene concerned with ultraviolet radiation-sensitivity.     

Cloning of the sacS gene encoding a positive regulator of the sucrose regulon in Bacillus subtilis

The sacS gene controls the expression of 2 saccharolytic enzymes in Bacillus subtilis (sucrase and levansucrase).This paper describes a recombinant plasmid containing a mutant allele, sacS^c. The plasmid was isolated from a B. subtilis DNA bank established in Escherichia coli. Moreover, it was shown that the sacS^c allele, placed on a high-copy plasmid, is dominant over the wild-type chromosomal sacS⁺ allele. This result strongly suggests that the sacS gene encodes a positive regulatory protein.     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Cloning and characterization of the pH 2.5 acid phosphatase gene, appA: Cyclic AMP mediated negative regulation

Summary The structural gene for an acid phosphatase coded for by the gene appA of Escherichia coli K12 was cloned from a cosmid library into pBR322 and the restriction map determined. Several appA deletion plasmids and a smaller appA ⁺ plasmid were constructed by in vitro recombination techniques and tested for their ability to complement an appA1 mutation. The appA gene was localized within a 2.1 kb segment. Its orientation was determined by construction of a hybrid plasmid carrying an appA-lacZ fusion. -galactosidase synthesized from the appA promoter was negatively regulated by cyclic AMP.     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

Spontaneous amplification of yeast CEN ARS plasmids

Summary Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-4 allele (as well as the URA3 and TRP1 markers) yielded His⁺ transformants at 0.1%–50% the frequency of Ura⁺ Trp⁺ transformants. Additional His⁺ derivatives arose on continuous growth of transformants originally scored as His^- Ura⁺ Trp⁺. In all cases, the His⁺ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His⁺ phenotype was lost more readily than the Ura⁺ Trp⁺ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura⁺ Trp⁺ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.     

Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae

Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a γ-aminobutyric acid (GABA) receptor subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Alanine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, this same alanine residue is replaced either by a serine, or, in some resistant strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of the green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other alleles with the following amino acid replacements: A302 → Glycine (allele G), A302 → Serine^TCG (allele S) and A302 → Serine^AGT (allele S′). Evidence from direct nucleotide sequencing and Single Stranded Conformational Polymorphism (SSCP) analysis shows that at least three of these different Rdl alleles (i.e. A, G and S) are commonly present in individual aphids or aphid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutation confirm the presence of two independent Rdl-like loci in M. persicae. One locus carries the susceptible alanine (A) and/or resistant glycine (G) allele while the other carries the two serine alleles (S or S′). Whereas resistance levels are correlated with the glycine replacement, the S allele was present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacements at the first (A/G) but not the second (S/S′) locus. Phylogenetic analysis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen through a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit diversity are discussed. Received: 10 March 1998 / Accepted: 21 July 1998     

Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae

Summary Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.     

Effects of amino-acid substitutions in β tubulin on benomyl sensitivity and microtubule functions inCoprinus cinereus

The sensitivity of the homobasidiomyceteCoprinus cinereus to the benzimidazole fungicide benomyl allowed us to isolate β-tubulin mutants as strains resistant to benomyl. To understand the molecular basis for the interaction between benomyl and β tubulin and for cellular defects in the β-tubulin mutants, we first analyzed the wild-type β1-tubulin gene (benA) ofC. cinereus, revealing thatbenA contains eight introns and encodes a 445 amino-acid protein. We then characterized 16 β1-tubulin mutants. The 16 mutations involved 11 different amino-acid substitutions at 10 different residues in β1 tubulin. The mutated residues were widely distributed along the primary sequence of β1 tubulin, from residue 3 in the N-terminal domain to residue 350 in the intermediate domain, but half of them appeared to be close to the αβ intradimer interface in an atomic model determined by electron crystallography. The benomyl resistant strain BEN 193, which exhibits clear heat sensitivity for hyphal growth and defects in various cellular processes, had a novel mutation, i.e., the Leu to Phe substitution at residue 350. Benomyl resistance and the heat sensitivity in BEN 193 were suppressed by additional amino-acid substitutions at various residues in β1 tubulin, suggesting that conformational changes of β1 tubulin are involved in the alterations. The DDBJ/GeneBank/EMBL accession number for the sequence reported in this paper is AB000116.     

On the mechanism of mitotic recombination in Aspergillus nidulans. I. Intragenic recombination and DNA replication

Summary Adenine or pABA starvation induce mitotic recombination within the ad9 and paba1 cistrons respectively. Adenine concentrations in the plating medium as low as 1×10^-8 M increase recombination frequency; the concentration optimal in respect to induced recombination frequency is 5×10^-7 M.Recombination within the paba1 cistron is stimulated by low pABA concentrations, or caseine hydrolysate, or methionine.Aminopterin applied for one or two hours before conidia of pABA-requiring diploid are plated on proper selective media, induces recombination within the pro1, ad9 and paba1 cistrons. Conclusion is drawn that it is adenine or thymine starvation which induce mitotic recombination.The implications of this and other similar evidence are discussed.