Showing papers in "Current Genetics in 1997"

The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression.

Abstract: The goal of the fungal mitochondrial genome project (FMGP) is to sequence complete mitochondrial genomes for a representative sample of the major fungal lineages; to analyze the genome structure, gene content, and conserved sequence elements of these sequences; and to study the evolution of gene expression in fungal mitochondria. By using our new sequence data for evolutionary studies, we were able to construct phylogenetic trees that provide further solid evidence that animals and fungi share a common ancestor to the exclusion of chlorophytes and protists. With a database comprising multiple mitochondrial gene sequences, the level of support for our mitochondrial phylogenies is unprecedented, in comparison to trees inferred with nuclear ribosomal RNA sequences. We also found several new molecular features in the mitochondrial genomes of lower fungi, including: (1) tRNA editing, which is the same type as that found in the mitochondria of the amoeboid protozoan Acanthamoeba castellanii; (2) two novel types of putative mobile DNA elements, one encoding a site-specific endonuclease that confers mobility on the element, and the other constituting a class of highly compact, structured elements; and (3) a large number of introns, which provide insights into intron origins and evolution. Here, we present an overview of these results, and discuss examples of the diversity of structures found in the fungal mitochondrial genome.

The evolution of the Calvin cycle from prokaryotic to eukaryotic chromosomes: a case study of functional redundancy in ancient pathways through endosymbiosis

Abstract: The evolutionary histories of the 12 enzymes that catalyze the reactions of the Calvin cycle in higher-plant chloroplasts are summarized. They are shown to be encoded by a mixture of nuclear genes of cyanobacterial and proteobacterial origin. Moreover, where cytosolic isoforms of these enzymes are found they are almost invariably encoded by genes of clearly endosymbiont origin. We infer that endosymbiosis resulted in functional redundancy that was eliminated through differential gene loss, with intruding eubacterial genes repeatedly replacing pre-existing nuclear counterparts to which they were either functionally or structurally homologous. Our findings fail to support the `product-specificity corollary', which predicts re-targeting of nuclear-encoded gene products to the organelle from whose genome they originated. Rather it would appear that the enzymes of central carbohydrate metabolism have evolved novel targeting possibilities regardless of their origins. Our findings suggest a new hypothesis to explain organelle genome persistence, based on the testable idea that some organelle-encoded gene products might be toxic when present in the cytosol or other inappropriate cellular compartments.

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Abstract: Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66-100% for isolates of different subgroups within an AG, and 55-96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1

Abstract: Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested soil significantly reduced the disease caused by R. solani in cotton plants.

Laccase from the white-rot fungus Trametes versicolor : cDNA cloning of lcc1 and expression in Pichia pastoris

Abstract: A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter. Transformants were found to secrete active recombinant enzyme after induction with methanol. The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining activity in liquid cultures. The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae α-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9. The activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for the construct encoding the α-factor secretion signal. Utilisation of the P. pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P. pastoris GS115.

The highly rearranged chloroplast genome of Trachelium caeruleum (Campanulaceae): multiple inversions, inverted repeat expansion and contraction, transposition, insertions/deletions, and several repeat families

Abstract: Comprehensive gene mapping reveals that the chloroplast genome of Trachelium caeruleum is highly rearranged relative to those of other land plants. Evolutionary scenarios that consist of seven to ten inversions, one or two transpositions, both expansion and contraction of the typically size-conserved inverted repeat, a presumed gene loss, deletions within two large open reading frames and several insertions, are sufficient to derive the Trachelium arrangement from the ancestral angiosperm chloroplast DNA arrangement. Two of the rearrangements disrupt transcriptional units that are otherwise conserved among land plants. At least five families of small dispersed repeats exist in the Trachelium chloroplast genome. Most of the repeats are associated with inversion endpoints and may have facilitated inversions through recombination across homologous repeats.

Brassica nap cytoplasmic male sterility is associated with expression of a mtDNA region containing a chimeric gene similar to the pol CMS-associated orf224 gene

Abstract: Two different cytoplasmic male-sterility (CMS) systems, nap and pol, are found in the oilseed rape (canola) species Brassica napus. Physical mapping studies have previously shown that organizational differences between the sterile pol and fertile cam mitochondrial genomes are restricted to a relatively small region immediately upstream of the atp6 gene. An approximately 4.5-kb pol mtDNA segment containing a chimeric open reading frame (orf224) co-transcribed with atp6 is missing from cam mtDNA and located at a different site on nap mtDNA; expression of the orf224/atp6 gene region is highly correlated with the pol CMS trait. Sequence analysis now shows that the transposed nap segment contains an open reading frame (ORF) related to, but distinct from, pol orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino acids possessing 79% sequence similarity to the predicted product of the pol orf224 gene. nap orf222 is co-transcribed with the third exon of the trans-spliced nad5 gene and another ORF. orf222 transcripts are several times more abundant in nap CMS than in fertility restored nap-cytoplasm plants and qualitative transcript differences for the region between CMS and restored plants are found as well. Expression of the orf222/nad5c/orf139 region is specifically correlated with nap CMS: of 21 mitochondrial gene regions examined, including all the sites of rearrangement between the nap and fertile cam mitochondrial genomes and 22 known genes, only the orf222/nad5c/orf139 region detected transcript differences between maintainer cam cytoplasm, nap CMS- and fertility restored nap cytoplasm-plants. Our results suggest that expression of the orf222/nad5c/orf139 region may be associated with nap CMS, and, more generally, that different forms of CMS may be associated with genes encoding structurally similar proteins.

Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii.

Abstract: In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source and on other environmental factors. We have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1/ars) into a wall-less strain of C. reinhardtii. A new and sensitive method, based on the use of alpha-naphthylsulphate as a substrate and a diazonium salt as a chromogenic post-coupling agent, was developed to detect the activity of arylsulphatase (an enzyme which is almost completely secreted in the culture medium) both in vitro and in agar plates. The transformants carrying nit1/ars did not express arylsulphatase when grown in ammonium-sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitrite. The nit1/ars construct, however, was not expressed in the nit2 mutant lacking a specific transcription regulator controlling the expression of nit1. These results, together with the observation that the transcription of nit1/ars is initiated at the same sites as the nit1 endogenous gene, confirms the hypothesis that the regulation of nit1 expression takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direct measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure has permitted the analysis of the effects of nested deletions in the nit1 promoter region on the expression of the reporter gene. The results indicate that the -282 to -198 sequence is required for transcription to occur and that the -751 to -282 region contains several elements mediating nit1 expression.

Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis

Abstract: Experiments were designed to clone and identify genes of the fungal phytopathogen Colletotrichum gloeosporioides expressed at high levels during growth on the compatible host Stylosanthes guianensis when compared with expression in axenic culture. A cDNA clone (pCgGS) that hybridised preferentially to a cDNA probe prepared from infected leaves was isolated by the differential screening of a cDNA library from a nitrogen-starved axenic culture of C. gloeosporioides. The DNA sequence of pCgGS is highly homologous to genes for glutamine synthetase (GS) in other organisms. pCgGS contained all of the conserved regions assigned as catalytic domains in GS enzymes. Comparison with genomic sequences indicated that in C. gloeosporioides the GS gene is present as a single copy with three introns. To our knowledge this is the first report of the cloning of a GS from a filamentous fungus. A second clone (pCgRL1) was also isolated and represented a partial cDNA of the 25s rRNA of C. gloeosporioides. Because pCgRL1 did not hybridise to plant rRNA under high-stringency hybridisation conditions, it was used as a reference to quantify the expression of fungal GS mRNA during pathogenesis in S. guianensis compared to fungal growth in axenic culture. The results indicated that elevated expression of GS occurred during pathogenesis of C. gloeosporioides on S. guianensis, particularly at early stages of infection where expression was about six-times higher than during growth in rich culture media. This work also demonstrates that fungal-specific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expression in infected plants.

Self-fertility and uni-directional mating-type switching in Ceratocystis coerulescens, a filamentous ascomycete

Abstract: Individual perithecia from selfings of most Ceratocystis species produce both self-fertile and self-sterile progeny, apparently due to uni-directional mating-type switching. In C. coerulescens, male-only mutants of otherwise hermaphroditic and self-fertile strains were self-sterile and were used in crossings to demonstrate that this species has two mating-types. Only MAT-2 strains are capable of selfing, and half of the progeny from a MAT-2 selfing are MAT-1. Male-only, MAT-2 mutants are self-sterile and cross only with MAT-1 strains. Similarly, self-fertile strains generally cross with only MAT-1 strains. MAT-1 strains only cross with MAT-2 strains and never self. It is hypothesized that the switch in mating-type during selfing is associated with a deletion of the MAT-2 gene.

Isolation and characterization of rad51 orthologs from Coprinus cinereus and Lycopersicon esculentum, and phylogenetic analysis of eukaryotic recA homologs.

Abstract: In eubacteria, the recA gene has long been recognized as essential for homologous recombination and DNA repair. Recent work has identified recA homologs in archaebacteria and eukaryotes, thus emphasizing the universal role this gene plays in DNA metabolism. We have isolated and characterized two new recA homologs, one from the basidiomycete Coprinus cinereus and the other from the angiosperm Lycopersicon esculentum. Like the RAD51 gene of Saccharomyces cerevisiae, the Coprinus gene is highly induced by gamma irradiation and during meiosis. Phylogenetic analyses of eukarotic recA homologs reveal a gene duplication early in eukaryotic evolution which gave rise to two putatively monophyletic groups of recA-like genes. One group of 11 characterized genes, designated the rad51 group, is orthologous to the Saccharomyces RAD51 gene and also contains the Coprinus and Lycopersicon genes. The other group of seven genes, designated the dmc1 group, is orthologous to the Saccharomyces DMC1 gene. Sequence comparisons and phylogenetic analysis reveal extensive lineage- and gene-specific differences in rates of RecA protein evolution. Dmc1 consistently evolves faster than Rad51, and fungal proteins of both types, especially those of Saccharomyces, change rapidly, particularly in comparison to the slowly evolving vertebrate proteins. The Drosophila Rad51 protein has undergone remarkably rapid sequence divergence.

Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis

Abstract: The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other α-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial α-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creAd mutants and carbon catabolite repressed by D-glucose.

Two genes of the putative mitochondrial fatty acid synthase in the genome of Saccharomyces cerevisiae.

Abstract: In order to find further genes of the mitochondrial fatty acid synthase, we searched the genome of Saccharomyces cerevisiae for sequences that are homologous to conserved regions of bacterial fatty acid synthase genes. We found the gene products of ORF YKL055c (EMBL Accession No. X75781) and of YOR221C (EMBL Accession No. X92441) to be homologous to bacterial 3-oxoacyl-(acyl carrier protein) reductases and to malonyl-CoA:ACP-transferases, respectively. We disrupted these two genes which in both cases led to a respiratory deficient phenotype, as is the case for the genes encoding a mitochondrial acyl carrier protein and a β-ketoacyl-ACP synthase. We propose to call the above mentioned genes OAR1[3-oxo-acyl-(acyl carrier protein) reductase] and MCT1 (malonyl-CoA:ACP transferase). They are presumed to be part of a type-II mitochondrial fatty acid synthase, a relic of the endosymbiontic origin of mitochondria, delivering substrates for phospholipid re-modelling and/or repair.

Homologous recombination in the fission yeast Schizosaccharomyces pombe: different requirements for the rhp51+, rhp54+ and rad22+ genes.

Abstract: The Schizosaccharomyces pombe rhp51 + , rad22 + and rhp54 + genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Δ and rhp54Δ strains are extremely sensitive to ionizing radiation; the rad22Δ mutant turned out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Δ and rhp54Δ are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Δ mutant is only reduced by a factor of two. Blot-analysis demonstrated that the majority of the leu+ transformants of the wild-type and rad22Δ strains have integrated one or more copies of the vector. Gene conversion events were observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Δ and a rhp54Δ background. Meiotic recombination is hardly affected in the rad22Δ mutant. The rhp51Δ and rhp54Δ strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Δ strain and 27% in the rhp54Δ strain, as compared with wild-type cells. However, in the rhp51Δ mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe.

The actin gene of the glaucocystophyte Cyanophora paradoxa: Analysis of the coding region and introns, and an actin phylogeny of eukaryotes

Abstract: We isolated the actin gene of the glaucocystophyte alga Cyanophora paradoxa and analyzed the coding region and its introns. Phylogenetic analyses of the actin coding region and the inferred protein sequence in data sets containing 47 other actin sequences show Cyanophora to be a member of the eukaryotic crown-group radiation in agreement with ribosomal DNA sequence analyses. Four of the five Cyanophora actin introns are relatively short (55-59 nt) and occupy novel positions in a catalogue of actin introns containing 56 distinct sites. The fifth intron has a length of 171 nt and occurs also in actin genes from green algae and the crustacean Artemia.

The Neurospora crassa cya-5 nuclear gene encodes a protein with a region of homology to the Saccharomyces cerevisiae PET309 protein and is required in a post-transcriptional step for the expression of the mitochondrially encoded COXI protein

Abstract: The cya-5 nuclear mutant of Neurosopora crassa was previously shown to be deficient in cytochrome aa3, cytochrome c oxidase activity, and the immunologically detectable COXI protein. We have now demonstrated that the mitochondria of this mutant contain mRNA for the COXI protein and that COXI cannot be detected during pulse-chase labeling experiments of mitochondrial translation products. Cloning and analysis of the cya-5 gene reveal a long open reading frame capable of encoding a 1136 amino-acid protein. Sequence analysis suggests that the potential CYA-5 protein contains a mitochondrial targeting sequence at its amino-terminus. The long open reading frame also contains a 200 amino-acid region with homology to the PET309 protein, which is required for the production or stability of intron-containing coxI mRNAs, as well as the translation of mature coxI mRNAs, in the yeast Saccharomyces cerevisiae. These data suggest that the CYA-5 protein of N. crassa is required in a post-transcriptional step for COXI expression, most probably for the efficient translation of coxI mRNA.

Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli

Abstract: The extent of heterokaryon (also termed somatic or vegetative) incompatibility among black Aspergillus strains was examined using nitrate non-utilising mutants selected on chlorate medium. Pairings of complementary mutants showed that somatic compatibility between different strains is exceptional in natural populations of the asexual black Aspergilli. Mycoviruses are present in a considerable fraction of the sampled natural population, but surprisingly, horizontal transfer of mycoviruses only occurs-at least under laboratory conditions-between the (very rare) compatible combinations of strains. Thus, unlike other fungal species, somatic incompatibility in black Aspergilli efficiently blocks virus transfer. Viruses present in black Aspergillus isolates are highly efficiently transmitted to asexual progeny.

Carbon regulation of the cuticle-degrading enzyme PR1 from Metarhizium anisopliae may involve a trans-acting DNA-binding protein CRR1, a functional equivalent of the Aspergillus nidulans CREA protein

Abstract: The pr1 gene of the entomopathogenic fungus Metarhizium anisopliae encodes a serine protease that is highly active towards the insect cuticle and whose synthesis is subject to both carbon and nitrogen repression. The pr1 promoter region was sequenced revealing the presence of putative CREA- and AREA-binding sites. In vitro bandshift experiments demonstrated that an Aspergillus nidulans GST-CREA fusion protein was capable of binding to two of the three putative CREA sites. Using a PCR-based strategy the M. anisopliae crr1 gene was identified; it encodes a putative C2H2-type DNA-binding protein with significant sequence similarity to A. nidulans CREA. Complementation experiments with an A. nidulans strain carrying creA204 demonstrated that CRR1 can partially substitute for CREA function.

Characterization of the mitochondrial cytochrome b gene from venturia inaequalis

Abstract: A new class of agricultural fungicides derived from the group of antifungal strobilurins acts as specific respiration inhibitors by binding to mitochondrial cytochrome b. The cytochrome b gene was cloned and sequenced from the mitochondrial genome of Venturia inaequalis, the causal agent of apple scab. The gene was 10.65 kbp in size and contained seven exons and six introns. The exons encoded a protein of 393 amino acids. Comparison of the deduced amino-acid sequence with cytochrome b proteins from other fungi revealed highest homologies to the respective proteins of Aspergillus nidulans, Podospora anserina and Neurospora crassa. All amino acids of the V. inaequalis cytochrome b at positions altered in mutants of Saccharomyces cerevisiae resistant to strobilurins, and other fungi with reduced sensitivities to strobilurins, were identical to wild-type isolates of several fungi. The cloning and characterization of the V. inaequalis cytochrome b gene is the initial step in the assessment of resistance risks inherent to the strobilurin fungicides.

Multiple recruitment of class-I aldolase to chloroplasts and eubacterial origin of eukaryotic class-II aldolases revealed by cDNAs from Euglena gracilis.

Abstract: The photosynthetic protist Euglena gracilis is one of few organisms known to possess both class-I and class-II fructose-1,6-bisphosphate aldolases (FBA). We have isolated cDNA clones encoding the precursor of chloroplast class-I FBA and cytosolic class-II FBA from Euglena. Chloroplast class-I FBA is encoded as a single subunit rather than as a polyprotein, its deduced transit peptide of 139 amino acids possesses structural motifs neccessary for precursor import across Euglena's three outer chloroplast membranes. Evolutionary analyses reveal that the class-I FBA of Euglena was recruited to the chloroplast independently from the chloroplast class-I FBA of chlorophytes and may derive from the cytosolic homologue of the secondary chlorophytic endosymbiont. Two distinct subfamilies of class-II FBA genes are shown to exist in eubacteria, which can be traced to an ancient gene duplication which occurred in the common ancestor of contemporary gram-positive and proteobacterial lineages. Subsequent duplications involving eubacterial class-II FBA genes resulted in functional specialization of the encoded products for substrates other than fructose-1,6-bisphosphate. Class-II FBA genes of Euglena and ascomycetes are shown to be of eubacterial origin, having been acquired via endosymbiotic gene transfer, probably from the antecedants of mitochondria. The data provide evidence for the chimaeric nature of eukaryotic genomes.

Mutations in SPK1/RAD53 that specifically abolish checkpoint but not growth-related functions.

Abstract: SPK1/RAD53/SAD1/MEC2 encodes an essential protein kinase of Saccharomyces cerevisiae and is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We have isolated two mutant alleles of SPK1 (spk1K227A and spk1-1A208P) that are defective for checkpoint-arrest functions but retain wild-type levels of SPK1-associated growth activity. Both mutations occur within conserved regions of the kinase domain of SPK1 resulting in a substantial reduction in the catalytic activity of Spk1. Thus, while minimal levels of Spk1 kinase activity are capable of supporting normal rates of growth, higher levels are required for checkpoint functions. In addition, using deletional analysis we have identified a region within the N-terminus of Spk1 outside of the conserved kinase domain that is required for checkpoint functions. Interestingly, this region may be important in the regulation of Spk1 kinase activity.

Characterization of the saccharomyces cerevisiae fcy1 gene encoding cytosine deaminase and its homologue fca1 of candida albicans

Abstract: By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10(-2) M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected.

Variability of karyotypes and RAPD types in genetically related strains of Cryptococcus neoformans

Abstract: Variation in karyotypes and RAPD patterns of genetically related strains of Cryptococcus neoformans were analyzed. Capsular and filamentous mutants usually differ in their karyotypes from wild-types, but the RAPD patterns were found to be similar. Karyotype differences were observed in most heterothallic matings, but RAPD patterns remained identical. After self-sporulation of a diploid strain, minor chromosomal length polymorphism and minor changes in the RAPD types occurred. Three mechanisms, either alone or in combination, may in varying degrees contribute to the karyotype variation of C. neoformans: (1) mitotically induced changes; (2) karyotype changes as a result of meiotic recombination, and (3) mutagen-induced changes. The present data do not support the meiotic maintenance hypothesis, which claims that the amount of CLP generated is inversely proportional to the frequency of meiosis.

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination.

Abstract: The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea.

Cloning and sequencing of the Aa-Pri1 gene specifically expressed during fruiting initiation in the edible mushroom Agrocybe aegerita, and analysis of the predicted amino-acid sequence

Abstract: A gene (Aa-Pri1) specifically expressed during fruiting initiation of the basidiomycete Agrocybe aegerita was cloned. The total length of the Aa-Pri1 gene was 492 bp including a class-II intron of 54 bp size located at nt +125; the open reading frame encoded for a 145-aa protein of 16 093 Da. CCAAT (–156) and TATAAAT (– 83) boxes, and T(A)5T(A)2 (+593) and T(A)3T(A)4T(A)6T (+608) putative polyadenylation sequences were identified. The putative transcription start point was located at position 49. The Aa-Pri1 transcript was abundant only during fruiting initiation and was undetectable in the other stages of development. The Aa-Pri1 protein was hydrophilic, with a 20-aa hydrophobic motif in the NH2-terminal part, determining a putative α-helix. Two putative glycosylation sites were identified. Aa-Pri1 protein activity may be controlled by the phosphorylation of several residues by different protein kinases.

Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane

Abstract: We have previously isolated the yeast nuclear gene OXA1 and showed that Oxa1p is required for the formation of the cytochrome c oxidase and ATP synthase complexes. We have expressed Oxa1p in E. coli and shown that it is toxic and rapidly degraded. Nevertheless, a truncated protein was successfully expressed and antibodies have been raised against this truncated protein. These antibodies recognise a protein in mitochondrially enriched fractions. In vitro mitochondrial import experiments demonstrate that the import of Oxa1p is accompanied by the cleavage of a long pre-sequence. Osmotic swelling and alkaline carbonate extraction show that Oxa1p is an integral membrane protein located in the inner membrane of mitochondria. The relationships between the sub-mitochondrial location and the function of Oxa1p are discussed.

Intron sequences provide a tool for high-resolution phylogenetic analysis of volvocine algae

Abstract: Three nuclear spliceosomal introns in conserved locations were amplified and sequenced from 28 strains representing 14 species and 4 genera of volvocalean green algae Data derived from the three different introns yielded congruent results in nearly all cases In pairwise comparisons, a spectrum of taxon-specific sequence differences ranging from complete identity to no significant similarity was observed, with the most distantly related organisms lacking any conserved elements apart from exon-intron boundaries and a pyrimidine-rich stretch near the 3′ splice site A metric (SI50), providing a measure of the degree of similarity of any pair of intron sequences, was defined and used to calculate phylogenetic distances between organisms whose introns displayed statistically significant similarities The rate of sequences divergence in the introns was great enough to provide useful information about relationships among different geographical isolates of a single species, but in most cases was too great to provide reliable guides to relationships above the species level A substitution rate of approximately 3 × 10−8 per intron position per year was estimated, which is about 150-fold higher than in nuclear genes encoding rRNA and about 10-fold higher than the synonymous substitution rate in protein-coding regions Thus, these homologous introns not only provide useful information about intraspecific phylogenetic relationships, but also illustrate the concept that different parts of a gene may be subject to extremely different intensities of selection The intron data generated here (1) reliably resolve for the first time the relationships among the five most extensively studied strains of Volvox, (2) reveal that two other Volvox species may be more closely related than had previously been suspected, (3) confirm prior evidence that particular isolates of Eudorina elegans and Pleodorina illinoisensis appear to be sibling taxa, and (4) contribute to the resolution of several hitherto unsettled issues in Chlamydomonas taxonomy

The VPS4 gene is involved in protein transport out of a yeast pre-vacuolar endosome-like compartment

Abstract: Four yeast mutants were isolated in a screen for dominant-negative vacuolar protein-sorting mutants, secreting a carboxypeptidase Y-invertase hybrid protein. In addition to defects in the sorting/transport of soluble vacuolar hydrolases, the mutants accumulated a pre-vacuolar endosome-like compartment. The mutant alleles causing the defects were identified as the members of the VPS4 gene locus, each harbouring single-point mutations leading to amino-acid exchanges at positions 233 (E233Q), 211 (E211 K), and 178 (G178D). These mutations all reside within a 200 amino-acid-long ATPase module, common to members of the AAA-protein family. The VPS4 gene product shows homology to the yeast Sec18p (50% similarity and 25% identity), which is involved in several vesicle-mediated protein transport steps and homotypic membrane fusion events. Disruption of the VPS4 gene leads to a recessive vacuolar protein-sorting phenotype. About 40% of newly synthesized CPY is secreted as the Golgi-modified p2CPY precursor form. Transport of secretory proteins to the plasma membrane is normal as demonstrated by the secretion of invertase and α-factor. The α-factor, however, is secreted as a partially processed precursor, caused by defects in late Golgi function. The vps4 mutants also exhibit defects in fluid-phase endocytosis, as demonstrated by the accumulation of Lucifer Yellow in a pre-vacuolar endosome-like compartment. Based on the pleiotropic phenotype of the vps4 mutants and on the sequence homology to NSF/Sec18p, we propose that the VPS4 gene product is required for efficient transport out of the pre-vacuolar endosome-like compartment.

STP1, a gene involved in pre-tRNA processing in yeast, is important for amino-acid uptake and transcription of the permease gene BAP2

Abstract: The bap1 mutant of Saccharomyces cerevisiae was previously isolated by its reduced uptake of branched-chain amino acids. In the present study, the corresponding wild-type gene was cloned and partial sequencing and subsequent genetic analysis revealed identity to STP1, a gene involved in tRNA maturation. The decrease in amino-acid uptake caused by stp1 mutations is independent of GCN4. It was previously found that the BAP2 promoter can be activated by the presence of amino acids, notably leucine, in the medium. We found that this activation depends on STP1. As a simple hypothesis we propose that Stp1p is a transcription factor which activates BAP2, and probably other amino-acid permease genes.

Chromosomal reorganization during meiosis of Saccharomyces cerevisiae baker's yeasts

Abstract: The genomic constitution of two S. cerevisiae baker's yeasts and their meiotic products have been analyzed by pulsed-field gel-electrophoresis, hybridization with specific gene probes, marker segregation, and flow cytometry. The parental strains have chromosomal patterns substantially different from those of laboratory strains used as controls. This pattern is partly the result of there being more than one copy of homologous chromosomes of different size, as judged by Southern-blot hybridization carried out with specific gene probes. Flow cytometry indicated that the strains have a 2.7 C DNA content. Tetrad analysis showed disomy for some chromosomes and tetrasomy for others. When two complete tetrads were subjected to molecular analysis the results confirmed instances of segregation of homologous chromosomes of different size. However, the presence of chromosomal bands absent in the parentals and the disappearance of chromosomal bands present in the parental strains were frequently seen. This result was attributed to two different phenomena: (1) the presence of multiple Ty1 and Ty2 transposable elements which seem to undergo interchromosomal translocation together with amplification, giving rise to differences in chromosomal size; (2) the presence of multiple Y′ subtelomeric regions, giving rise to asymmetrical homologous recombination and, as a consequence, differences betwen the size of the recombinant chromosomes and the non-recombinant parental chromosomes. Chromosomal reorganization occurs with a very high frequency during meiosis. By contrast, mitosis is very stable, as judged by the reproducible electrophoretic karyotype shown by the parental strains in successive generations.