Reorientation of the distal region in linkage group IIR of fission yeast

The genetic map of the fission yeast Schizosaccharomyces pombe has been revised in the distal region of chromosome arm IIR. The spo4 locus, hitherto considered the outermost marker, has been moved to an intermediate position. As a result, and in accordance with recent physical mapping data, the order of the entire distal subgroup of some 12 genetic markers is reversed relative to previously published gene maps.     

Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase

Summary The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the homologous (i.e. am) site as well as non-nomologous sites. Partial nucleotide analysis of the fragment has revealed the 5 end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.Abbreviations bp base pairs - kb 1,000 bp - GDH NADP-Iinked glutamate dehydrogenase - NADP nicotinamide adenine dinuc leotide phosphate - SDS sodium dodecyl sulfate - phage lambda - DTT dithiothreitol - SSC 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0     

Analysis of conversion tracts associated with recombination events at the am locus of Neurospora crassa

In cross B163, heteroallelic am ¹ am ⁶ and heterozygous for both conventional genetic flanking markers and closer molecular markers, we previously found that the majority on flanker exchanges were remote from events that generated prototrophic recombinants. We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am. Segregation of these markers in B163 prototrophs confirms that the majority of meiotic recombination events at am resulted from gene conversion. Conversion of am ⁶, the distal allele, is more frequent than conversion of am ¹. Twelve percent of tracts in am ⁶ convertants were discontinuous while 30% of continuous tracts converting am ⁶ extend less than 741 bp. Received: 8 September 1997 / 15 April 1998     

Generation of new functional mutant alleles by premeiotic disruption of the Neurospora crassa am gene

Summary In the further analysis of a cross in which the mis-sense allele, am ³, of the Neurospora crassa am (glutamate dehydrogenase) gene was present in one parent together with two ectopic wild-type gene copies, one ascus was identified in which the two ectopic copies had been inactivated by the RIP process whereas the am ³ allele continued to produce its characteristic enzyme variety in active, but heat-sensitive, form. The am ³ allele had also acquired a new HindIII restriction site. It had no detectable methylation. The mutations responsible respectively for the new restriction site and the modified enzyme properties were separated from each other, and from the original am ³ mutation, by selecting for intragenic recombination on either side of the am ³ site. In this way two new effectively wild-type alleles were generated, one characterised by its heat-sensitive and kinetically modified enzyme product and the other by a new HindIII site. These results demonstrate that the RIP phenomenon can be a source of new functional alleles.     

Premeiotic disruption of duplicated and triplicated copies of the Neurospora crassa am (glutamate dehydrogenase) gene

Summary Premeiotic inactivation of duplicated sequences (the RIP phenomenon of Selker et al.) was studied by tetrad analysis using ectopic copies of am ⁺ (coding for NADP-specific glutamate dehydrogenase) and a missense allele am ³, coding for a distinctive form of the enzyme, at the normal locus. In duplication crosses either both gene copies were inactivated or neither. Two inactivated am ³ derivatives were shown to have undergone methylation and numerous base-pair changes, reflected in losses and gains of restriction sites, but without sequence rearrangement. Cutting at restriction sites within the disrupted sequences was incomplete but became almost complete following growth in the presence of 5-azacytidine. In a triplication cross in which one parent carried two unlinked ectopic gene copies together with am ³ at the normal locus, premeiotic inactivation, when it occurred, tended to affect two of the three copies in any one ascus, but there were a few asci in which all three were inactivated.     

A novel (TG) n (GA) m repeat polymorphism 254 bp downstream of the mast cell chymase (CMA1) gene is associated with atopic asthma and total serum IgE levels

The gene for mast cell chymase (CMA1) is an ideal candidate for investigating genetic predisposition to atopic asthma, as it is an important mediator of inflammation and remodeling in the asthmatic lung. Various studies have examined the association between –1903 G/A polymorphism and allergic phenotypes, but inconsistent results have been obtained. We investigated the association of this SNP and a novel (TG)_n(GA)_m repeat polymorphism (accession no. BV210164) 254 bp downstream of the gene with asthma and its associated traits in a case-control study in two independent cohorts recruited from the Indian population. A significant association was observed for the (TG)_n(GA)_m repeat with asthma (p<0.05) in both the cohorts. Although no association was observed for the –1903 G/A SNP with asthma, a significant association was observed between the genotypes and serum IgE levels (p=0.003 and 0.0004 for cohort A and B). When haplotypes were compared between patients and controls, the haplotype G_43 was found at higher frequency in controls (p=0.05). Also, on comparing major haplotypes (>5%) with respect to log total serum IgE levels, a significant difference was obtained (p=0.018 and p=0.046 for cohorts A and B). These results suggest that the CMA1 gene contributes to asthma susceptibility and may be involved in regulating IgE levels in atopic asthma.     

Sterile UGA nonsense mutants of fission yeast

Summary Eight sterile mutants, which regain their fertility upon reactivation of an inactivated UGA suppressor allele of the serine tRNA gene sup3, are shown to carry UGA nonsense alleles of two established ste genes, ste1 (one mutant) and ste6 (two mutants), and of two novel genes, ste9 (four mutants) and ste10 (one leaky mutant of ras1 ^-/ste5-like cell morphology). The mutant alleles of ste1 and ste9 lead to a defect in both conjugation and meiosis, whereas those of ste6 and ste10 affect mating only. Two of the four genes map to chromosome I, ste1 in the left arm 6 cM distal of ura1, and ste9 in the right arm 3 cM distal of ade2. The ste10 and ste6 genes are located in the right arms of chromosomes II and III, respectively, the former 4 cM distal of trp1 and the latter 1 cM proximal or distal of trp3.     

The rice psb-A chloroplast gene has a standard location

Summary The chloroplast DNA-encoded gene psbA codes for the 32 kD protein of photosystem II. It has been mapped in several monocots to a location in the large single copy segment very near to the end of one of the inverted repeats present in the chloroplast genome of most land plants. The psbA gene of rice has been located much further from the inverted repeat, suggesting that rice differs from related species in the location of this gene. Indirect evidence reported here suggests that this gene actually maps to the usual cereal location, at least in the seven varieties tested, which include representatives of three subspecies.     

Haplotype-based analysis of alpha 2A, 2B, and 2C adrenergic receptor genes captures information on common functional loci at each gene

The alpha 2-adrenergic receptors (₂-AR) mediate physiological effects of epinephrine and norepinephrine. Three genes encode ₂-AR subtypes carrying common functional polymorphisms (ADRA2A Asn251Lys, ADRA2B Ins/Del301-303 and ADRA2C Ins/Del322-325). We genotyped these functional markers plus a panel of single nucleotide polymorphisms evenly spaced over the gene regions to identify gene haplotype block structure. A total of 24 markers were genotyped in 96 Caucasians and 96 African Americans. ADRA2A and ADRA2B each had a single haplotype block at least 11 and 16 kb in size, respectively, in both populations. ADRA2C had one haplotype block of 10 kb in Caucasians only. For the three genes, haplotype diversity and the number of common haplotypes were highest in African Americans, but a similar number of markers (3–6) per block was sufficient to capture maximum diversity in either population. For each of the three genes, the haplotype was capable of capturing the information content of the known functional locus even when that locus was not genotyped. The ₂-AR haplotype maps and marker panels are useful tools for genetic linkage studies to detect effects of known and unknown ₂-AR functional loci.Supported by National Institutes of Health (NIH) Intramural Grants Z01 DE00366 and Z01 AA000301 and the Comprehensive Neuroscience Program Grant USUHS G192BR-C4 (Henry Jackson Foundation)     

Extrachromosomal genetics of Claviceps purpurea

Summary Claviceps purpurea strain K1 contains several linear mitochondrial plasmids comparable to those of higher plants (Tudzynski et al. 1983). Screening of ten Claviceps wild strains from different geographic origin revealed the presence of similar plasmids in at least five strains, indicating a widespread occurrence of these genetic traits. Whereas in strain K1 plasmids have no homology to high-molecular-weight mtDNA, in two of the other wild strains (W3 and W7) sequences homologous to plasmids of strain K1 are integrated in the mt genomic DNA. Physical and genetic maps of strains W3, W7 and K1 were established. Despite completely different physical maps, the relative orientation of 6 mt genes is identical. A DNA sequence homologous to plasmids of strain K1 (termed ) was found to be integrated in both W3 and W7 at the same site: at the 5-part of the 1rRNA gene. A second region () was localized near the srRNA gene of W7 only. These findings are discussed with respect to possible transposon-like properties of the Claviceps purpurea plasmids.     

Structure of the cloned Locusta migratoria mitochondrial genome: restriction mapping and sequence of its ND-1 (URF-1) gene

Summary We have cloned the entire mitochondrial genome of Locusta migratoria in four fragments and characterised by restriction mapping. In addition, we have sequenced a 1,095 kb region containing the ND-1 (URF-1) gene. The inferred primary structure of the protein is highly homologous to its Drosophila counterpart (68%). The gene is flanked at the 5 end by the tRNA _CUN ^leu gene, interrupted by the sequence TTG. The 3 end is flanked by the tRNA _ser ^UCN gene, followed by a sequence homologous to the 3 end of D. yakuba cytochrome b. The relative position of the genes is conserved between Locusta and Drosophila, thus indicating conservation of mitochondrial gene order in insects.     

MRG1-1, a dominant allele that confers methomyl resistance in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize

We have previously described a eukaryotic heterologous expression system, with the urf13TW gene in yeast, which mimics the disease susceptibility associated with the Texas cytoplasmic male sterility in maize. This yeast model was used to isolate yeast nuclear mutants conferring methomyl resistance. The genetic strategy we have developed focused on screening for nuclear dominant yeast mutations which restore methomyl resistance. MRG1-1, a yeast nuclear dominant allele, was identified as a methomyl-resistance restorer. We have shown that methomyl resistance co-segregated with a pleiotropic phenotype in the heterozygous MRG1-1/MRG1 diploids, detectable even in the absence of the maize-derived mitochondrial protein and/or methomyl. We observed an increase in oxygen uptake, a significant decrease of the levels of cytochrome aa₃, and a decrease in the growth yield. This phenotype is influenced by the carbon source and the results suggest a defect in the adaptation to the respiratory pathway in MRG1-1 yeast cells.     

Mitochondrial DNAs of the fungus Armillaria ostoyae: restriction map and length variation

A restriction-enzyme-site map is presented for the 147-kb mtDNA of North American Armillaria ostoyae. The locations of five structural genes, atp6, atp8, coxI, coxIII, and cob, along with the location and orientation of the large and small ribosomal RNA genes, were determined through Southern hybridizations with cloned genes from other fungal mtDNAs. Based on this map, the variation in mtDNA suggested geographic structure at two different levels. On a large geographic scale, 17 mtDNA types from North America were distinct, with respect to both size and restriction maps, from three mtDNA types from Europe. At the local scale, identical mtDNA types were evident among several different genetic individuals located no more than 1 km apart at a site in Michigan. No mtDNA type occurred more than once among genetic individuals from different regions of North America, although the occurrence of similar mtDNAs in isolates from distant regions suggested that this may occur at a low frequency with large sample sizes. Among the North American mtDNA types, analysis of discrete length variants was inconsistent with the hypothesis that the mtDNA of A. ostoyae evolves as a clonal lineage in which each length mutation represents a unique event. The two remaining hypotheses, that similar mutational events have occurred independently and that genetic exchange and recombination occurs among mtDNAs in natural populations of this species, remain to be tested.     

The STA2 and MEL1 genes of Saccharomyces cerevisiae are idiomorphic

Summary The STA2 (glucoamylase) gene of Saccharomyces cerevisiae has been mapped close to the end of the left arm of chromosome II. Meiotic analysis of a cross between a haploid strain containing STA2, and another strain carrying the melibiase gene MEL1 (which is known to be at the end of the left arm of chromosome II) produced parental ditype tetrads only. Since there is no significant DNA sequence similarity between the STA2 and MEL1 genes, or their respective flanking regions, we conclude that these two genes are carried by separate non-hybridizing sequences of chromosomal DNA, either of which can reside at the end of the left arm of chromosome II. By analogy with the mating-type locus of Neurospora crassa, we suggest that the STA2 and MEL1 genes are idiomorphs with respect to one another.     

Functional nuclear suppressor of mitochondrialoxi2 mutations in yeast

Summary A semidominant nuclear suppressor, callednam6, ofoxi2-V276 mitochondrial mutation has been isolated and characterized. The nuclear character ofnam6 was proved by its retention inrho° strains, lack of mitotic segregation in diploids and meiotic 2:2 segregation in tetrads. The specificity ofnam6 was tested on 315mit ^– mutations of four mitochondrial genes (oxi1, oxi2, oxi3, andcob-box). It suppresses clearly only three mutations in theoxi2 gene, restoring partially or completely cytochrome aa₃ formation. The results suggest a functional character of the suppression.     

Genomic sequence analysis of a nucleopolyhedrovirus isolated from the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>

The CL3 plaque isolate of Plutella xylostella multiple nucleopolyhedrovirus (PlxyMNPV-CL3) exhibits a high degree of genetic similarity with the Autographa californica MNPV (AcMNPV) but is significantly more virulent against the diamondback moth, P. xylostella, than AcMNPV. To identify genetic differences between PlxyMNPV-CL3 and AcMNPV that may account for the difference in virulence against P. xylostella, the genome sequence of the CL3 plaque isolate of PlxyMNPV was determined and compared to the genome sequence of AcMNPV isolate C6. The PlxyMNPV genome is 134,417 bp, 523 bp larger than the AcMNPV-C6 genome, and the nucleotide sequence is almost completely co-linear with that of AcMNPV-C6. Of the 153 open reading frames (ORFs) identified in PlxyMNPV, 151 had homologues in AcMNPV-C6, with a mean amino acid sequence identity of 98.5%. The PlxyMNPV genome possessed two features previously reported for other variants of AcMNPV: (1) an extra baculovirus repeated orf (bro) sequence located between the plxy29/ac30 and sod ORFs, and (2) the deletion of the AcMNPV pnk/pnl polynucleotide kinase/ligase gene. In addition, an 817 bp insert of unknown origin located between the fp25K and lef-9 genes was discovered. This insert contained two small ORFs and was detected in both tissue culture- and larvae-derived PlxyMNPV DNA by PCR. Finally, the PlxyMNPV-CL3 ie-2 gene encodes a product with a low level (37.3%) of amino acid sequence identity with the AcMNPV-C6 ie-2 product. PlxyMNPV-CL3 apparently acquired this variant ie2 gene by recombination with an undescribed nucleopolyhedrovirus. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ457003 (PlxyMNPV-CL3), DQ482644 (AcMNPV-C6 ac17), DQ482645 (AcMNPV-C6 ac52), DQ482646 (AcMNPV-C6 ac58/ac59), DQ482647 (AcMNPV-C6 ac106/ac107), DQ482648 (AcMNPV-C6 ac112/ac113), DQ482649 (AcMNPV-C6 odv-e18), DQ482650 (AcMNPV-C6 ac145), DQ482651 (AcMNPV-C6 arif-1), and DQ482652 (AcMNPV-C6 pep).     

Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 ⁺ gene, which is homologous to Saccharomyces cerevisiae LYS4 ⁺. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 ⁺-based cloning vector pRH3. In addition, we deleted the S. pombe his7 ⁺ gene with a lys2 ⁺-marked polymerase chain reaction (PCR) product and the S. pombe lys2 ⁺ gene with a his7 ⁺-marked PCR product. Strains carrying these deletions of lys2 ⁺ or his7 ⁺ serve as relatively efficient hosts for the deletion of the ade6 ⁺ gene by lys2 ⁺- or his7 ⁺-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 ⁺ as a selectable marker, along with improved strains for the use of his7 ⁺ to mark gene deletions.     

Genetic structure of a population of the fungus Leptosphaeria maculans in a disease nursery of Brassica napus in Australia

Microsatellite, minisatellite and mating type markers were used to determine the genetic structure of the fungus Leptosphaeria maculans within a disease nursery, where Brassica napus lines were screened for resistance to blackleg disease under high inoculum pressure. Fungal isolates were collected from pseudothecia in infected stubble and pycnidia within cotyledon lesions on seedlings within the nursery. Genetic diversity was high with gene diversity at H=0.700 across four polymorphic loci, and genotypic diversity at D=0.993. Among the 159 isolates analysed, 102 multilocus genotypes were identified. The even distribution of mating type idiomorphs MAT1-1 and MAT1-2 and gametic equilibrium within the population provided further evidence of random mating. Genetic diversity was distributed on a very fine scale in the disease nursery. The majority of genetic diversity (67%) was distributed among conidia within a lesion or among ascospores from a piece of stubble, while the remainder (33%) was distributed within lesions on seedlings or different stubble pieces. There were no among-group differences between samples from stubble and seedlings. This is consistent with the low level of genetic differentiation between the ascospore and conidia samples (F _ST=0.017) indicating that all isolates of L. maculans from the disease nursery most likely belong to one population, and that ascospores form the primary inoculum in the disease nursery.     

Evidence for outcrossing in Phytophthora sojae and linkage of a DNA marker to two avirulence genes

Two genetically different isolates of the homothallic Oomycete, Phytophthora sojae, were demonstrated to outcross and form hybrid oospores after co-culturing in vitro. Random amplified polymorphic DNA (RAPD) markers revealed ten hybrids among 354 oospores analysed. One F₁ hybrid was allowed to self fertilise and produce an F₂ population of 247 individuals. Among 53 F₂ individuals, selected at random, 18 polymorphic RAPD markers were observed to segregate at near 3:1 Mendelian ratios, consistent with segregation for dominant alleles at single loci. Segregation of virulence against soybean resistance genes Rps1a, 3a, and 5 revealed that the avirulence genes Avr1a, 3a and 5 were dominant to virulence. Avirulence against these three resistance genes appeared to be conditioned by one locus for Avr1a and two independent, complementary dominant loci for both Avr3a and Avr5. Segregation of virulence against Rps6 was in the ratio of 1:2:1 (avirulent:mixed reaction:virulent), suggesting a semi-dominant allele at a single locus. Two avirulence genes and one RAPD marker formed one linkage group, in the order Avr3a, OPH4-1, Avr5, each separated by approximately 5 cM. Our results confirm that outcrossing occurred between the parental isolates, and that sexual recombination under field conditions may play an important role in generating and maintaining genetic diversity in populations of P. sojae.