Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3′ end-processing

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA^Asp gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3 end-processing of the tRNA^Asp. Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3 end-processing. One such suppressor mutation was further characterized: it restores tRNA^Asp maturation and growth at 36°C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.     

The oliC3 gene of Aspergillus niger: isolation,sequence and use as a selectable marker for transformation

Summary The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycinresistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5 untranslated sequences with those from other fungi.     

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli -glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.     

Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase α: evidence for a multifunctional role of Mcl1 in DNA replication and repair

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2 and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1⁺, which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase . Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.     

Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.     

Polymorphism of the mitochondrial L-RNA gene of the basidiomycete Coprinus cinereus

Summary Two unexpectedly small mitochondrial (mt) genomes of Coprinus cinereus, P and S, were compared with the H and J genomes we have described previously. H and J are 42 kb in size and differ in having alternative 1.23 kb insertions in or adjacent to the co-1 gene. P and S DNAs lacked both insertions and had an identical 4.4 kb deletion between the co-1 and L-RNA gene. P DNA contained a 700 by insertion and S DNA a 300 by deletion within a sequence coding the L-RNA gene. This was shown by Southern blot analysis using probes containing the 5 or the 3 exon sequences of the L-RNA gene of Neurospora crassa. These hybridisations showed also that the L-RNA gene and co-1 gene in the C. cinereus mt genome are oppositely orientated and must be transcribed from different DNA strands. No DNA homology was detected using probes containing intron sequences from the L-RNA genes of Saccharomyces cerevisiae or N. crassa. There was no evidence of respiratory deficiency in P and S strains and transfer of nuclei by dikaryon formation made it possible to recombine H nuclei with P and S mitochondria, S nuclei with H and P mitochondria and P nuclei with H mitochondria with no apparent detrimental effect on growth. We conclude that P and S mtDNAs represent naturally occurring variants of the C. cinereus mt genome.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.     

The cytoplasmic male-sterility (CMS) determinant of common bean is widespread in Phaseolus coccineus L. and Phaseolus vulgaris L.

To identify regions of the mitochondrial genome that potentially could specify cytoplasmic male sterility (CMS) in Phaseolus coccineus (including P. polyanthus), and to define differences amongst P. coccineus lines, mitochondrial (mt)DNA restriction patterns and Southern blots of total DNA from sterile and fertile lines were analysed. By restriction endonuclease mapping we isolated a region which was specific to CMS lines flanking an F1-ATPase -subunit (atpA) gene. DNA sequence analysis of this region showed 99.9% homology to the region previously isolated from P. vulgaris CMS Sprite. A high frequency of plants carrying the CMS-fragment was observed in a wild Phaseolus population, perhaps explaining the occurrence of inter- and intraspecific gene flow observed in the autogamous species P. vulgaris.     

An yeast nuclear mutation conferring temperature-sensitivity to the mitochondrial tryptophanyl-tRNA synthetase

Summary The conditional respiratory-deficient Saccharomyces cerevisiae mutant pet-ts2281 was complemented by an yeast genomic DNA library. The gene thus isolated was sequenced and proved to be identical to the known MSW1 sequence encoding mitochondrial tryptophanyl-tRNA synthetase (Myers and Tzagoloff 1985). Compared to the wild-type, the ts2281 mutant allele of MSW1 contained a single TC transition leading to a LeuSer replacement at position 294 of the protein sequence. In addition to this mutational alteration, our sequence data for the wild-type gene differ from the originally published MSW1 sequence at five other DNA positions which affect two locally restricted regions of the polypeptide chain. As expected, at the non-permissive temperature ts2281 cells are specifically defective in mitochondrial trp-tRNA formation and, thus, in overall mitochondrial protein synthesis. In addition, the patterns of cytochrome b mRNA maturation intermediates were distinctly different in ts2281 and wild-type yeast cells. The mutational effect of the observed amino-acid substitution in ts2281 is discussed in terms of weakened hydrogen bonding in the C-terminal half of the MSW1-encoded protein.     

The mitochondrial genome of fertile maize (Zea mays L.) contains two copies of the gene encoding the α-subunit of the F1-ATPase

Summary In contrast to the situation in animals and fungi the -subunit of the mitochondrial F₁-ATPase is encoded by two identical mitochondrial genes (ATP A) in male fertile maize (Zea mays L.). Cytoplasmic male sterile (T, C and S) maize mitochondrial genomes only contain a single copy of the gene. Sequence analysis reveals that the uninterrupted coding region of both copies of the gene is 1,524 by long and encodes a polypeptide of 508 amino acids with a molecular weight of 55,117. The predicted amino acid sequence shares over 60% homology with the nuclear encoded -subunit from yeast and bovine ATPases and approx. 50% with the corresponding chloroplast and bacterial polypeptides.     

Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Summary The endo--1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5 to the initiation codon for the B. subtilis -glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of -glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

Genetic and molecular analysis of REC114, an early meiotic recombination gene in yeast

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3 end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed.     

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris,B. trigyna and B. webbiana: evolutionary aspects

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5 region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.     

Molecular cloning of the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans

Summary The Aspergillus nidulans 3-phosphoglycerate kinase gene (PGK) has been isolated from a phage genomic library, using the equivalent yeast gene as a hybridization probe. The location of the PGK gene within the cloned DNA has been physically mapped. The DNA sequence of a small region of the putative PGK has been determined and found to code for amino acids corresponding to the N-terminal end of the PGK protein. In contrast to the yeast PGK gene the Aspergillus gene contains a 57 base pair intron occurring between the coding sequences for amino acid 22 and 23.A DNA fragment encompassing the PGK gene was shown to hybridize a 1,700 base poly(A) mRNA, sufficient to encode the PGK polypeptide.