A mitochondrial reading frame which may code for a second form of ATPase subunit 9 in Aspergillus nidulans

Summary The nucleotide sequence of a 74 codon reading frame from the Aspergillus nidulans mitochondrial genome is presented. The derived amino acid sequence displays typical features of dicyclohexylcarbodiimide (DCCD) binding proteins and is 84% homologous with a mitochondrial reading frame that potentially encodes an ATPase subunit 9 polypeptide in Neurospora crassa. However, in A. nidulans, as in N. crassa, there is strong biochemical and genetic evidence that this subunit is in fact nuclearly-encoded. In both organisms the DCCD-binding protein found in the F₀ complexes of mitochondria from actively-growing cultures is almost certainly the product of this nuclear gene, and definitely not that of the mitochondrial reading frame. The discovery of an intact open reading frame than can code for a DCCD-binding protein in the mitochondrial genome of a second species of filamentous fungus strenghthens the possibility that the presence of a mitochondrial version of this gene has some biological significance.     

Resistance to cadmium is under the control of the CAD2 gene in the yeast Saccharomyces cerevisiae

Summary A cadmium-resistant strain, X3382-3A, which is able to grow in a medium containing 0.2 mM cadmium sulfate, was picked out from our laboratory stock strains of Saccharomyces cerevisiae. The cadmium resistance of this strain is controlled by a single dominant nuclear gene, denoted as CAD2. The locus of CAD2 was mapped by gene linkage to a site 15.5 centimorgans to the right of the his7 locus on the right arm of chromosome II. The cadmium resistance of the strain carrying CAD2 was evaluated for its properties of cadmium uptake, cadmium distribution and cadmium-metallothionein formation, in comparison with those of some other strains. The results suggest that the novel type of cadmium resistance controlled by CAD2 does not involve production of a cadmiumm-metallothionein.     

Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome

Summary The nuclear gene coding for the mitochondrial subunit 9 of the F₀F₁-ATP synthase complex was isolated from a genomic library of Podospora anserina. Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid. The P. anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger. In contrast to the situation in Saccharomyces cerevisiae, N. crassa and A. nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P. anserina could be detected. Thus, in P. anserina this gene appears to be exclusively encoded by the nuclear genome.     

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

Chimeric organization of two genes for the soybean mitochondrial ATPase subunit 6

Summary There are two copies of the ATPase subunit 6 (atp6) gene in the soybean mitochondrial genome which differ in their gene organization but share extensive homology with the maize atp6 gene except at their 5 ends. The two soybean genes are chimeric, containing regions with homology to other known mitochondrial genes at their 5 ends. Sequences homologous to the cytochrome oxidase subunit II (coxII) are located in one copy and sequences homologous to the ATPase subunit 9 (atp9) gene are located in the other copy, both of which contain methionine (ATG) codons that are in-frame with the remainder of the atp6 open reading frame. At least the copy of atp6 that contains the coxII sequence at its 5 end is abundantly transcribed to give an RNA of approximately 1,200 nucleotides.     

Chromium resistant mutants of the yeast Saccharomyces cerevisiae

Summary Many strains of Saccharomyces cerevisiae do not grow on YPD agar containing 750 g/ml CrO₃. Mutants able to grow in the presence of 850 g/ml CrO₃ were obtained from such strains after UV mutagenesis. All of the mutants grew even in the presence of 1,000 /ml CrO₃. Chromium resistance was dominant or partial dominant over normal response, therefore it was impossible to determine the number of genetic loci by complementation analysis. However, the segregation of representative mutants strongly indicated that resistance was determined by single mutations. In addition, a limited analysis of recombination suggested that the chromium resistant mutations were located on a certain region of the yeast genome. Although it was determined that the mutants had slightly reduced rates of Cr⁶⁺ uptake, the exact mechanism of resistance was not discovered. According to the studies of interactions between resistant mutations and sensitive mutations, however, we have proposed a preliminary pathway of Cr⁶⁺ detoxification.     

Mutational studies of the major tRNA region of the S. cerevisiae mitochondrial genome

Summary The major tRNA genes in S. cerevisiae mitochondria are contained within a 20 kb segment of the mitochondrial DNA. In order to analyze the functional role of this region we have isolated several mitochondrial mutations, which are temperature-sensitive for growth on non-fermentable carbon sources. These mutations, localized in the major tRNA cluster region, can be classified in different groups according to their (a) genetic and physical localization, (b) spectrum of suppression and (c) biochemical characteristics. Some of these are mutations in tRNA genes which affect tRNA function; others alter the synthesis of the gene product. Finally, we found two mutations localized in, or in the vicinity of, the open reading frame RF2. RF2 has been postulated to be a maturase-like protein (Michel 1984) but no function for it has yet been demonstrated. The existence of defective mutants may confirm that RF2 is indeed necessary for mitochondrial biogenesis and so allow for a study of the expression of this gene.     

Physical map of the COB region in mitochondrial DNA of Saccharomyces cerevisiae

Summary A physical map of the COB region in mtDNA of yeast has been established. This region harbours a split gene coding for apocytochrome b. It includes restriction sites of seven endonucleases (EcoRI, HindII, HindIII, HaeIII, HpaII, AluI and BamHI). Various mtDNA sequences of this region, retained in a series of genetically characterized rho^– clones have been allocated to this map. The combination of this physical map with a genetic map of the rho^– clones revealed that 1) cob^– mutational sites spread over 8,400 bp, 2) mutations in sequences coding for apocytochrome b map in five distinct segments which are separated by intervening sequences with minimum lengths from 350 to 1,900 bp.Abbreviations mtDNA mitochondrial DNA - b bases - bp basepairs     

Biosynthetic and regulatory role of lys9 mutants of Saccharomyces cerevisiae

Summary Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity. Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, -aminoadipate aminotransferase, -aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells. Levels of these enzymes in lys2, lys14, and lys15 S mutants were the same or lower than those in wild-type cells. The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids. Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants. Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed. These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.     

Palindrome content of the yeast Saccharomyces cerevisiae genome

Palindromic sequences are important DNA motifs involved in the regulation of different cellular processes, but are also a potential source of genetic instability. In order to initiate a systematic study of palindromes at the whole genome level, we developed a computer program that can identify, locate and count palindromes in a given sequence in a strictly defined way. All palindromes, defined as identical inverted repeats without spacer DNA, can be analyzed and sorted according to their size, frequency, GC content or alphabetically. This program was then used to prepare a catalog of all palindromes present in the chromosomal DNA of the yeast Saccharomyces cerevisiae. For each palindrome size, the observed palindrome counts were significantly different from those in the randomly generated equivalents of the yeast genome. However, while the short palindromes (2–12 bp) were under-represented, the palindromes longer than 12 bp were over-represented, AT-rich and preferentially located in the intergenic regions. The 44-bp palindrome found between the genes CDC53 and LYS21 on chromosome IV was the longest palindrome identified and contained only two C-G base pairs. Avoidance of coding regions was also observed for palindromes of 4–12 bp, but was less pronounced. Dinucleotide analysis indicated a strong bias against palindromic dinucleotides that could explain the observed short palindrome avoidance. We discuss some possible mechanisms that may influence the evolutionary dynamics of palindromic sequences in the yeast genome.     

Linear mitochondrial genome organization in vivo in the genus Pythium

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.     

Control of the expression of the ADE2 gene of the yeast Saccharomyces cerevisiae

The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae. We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene. Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA. The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4 ⁺ strains and in an adenine-supplemented medium. In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression. Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system. The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.     

The regulation of mitochondrial DNA levels in Saccharomyces cerevisiae

Summary Mitochondrial DNA (mtDNA) synthesis can continue under conditions which block cell division and nuclear DNA (nDNA) synthesis, producing cells with several times the normal level of mtDNA. We have examined mtDNA synthesis in cultures recovering from such cell cycle blocks. Our results show that the rate of mtDNA synthesis is not affected either during a block of the cell cycle with -factor or during recovery from a perturbation in the amount of mtDNA/cell induced by blocking the cell cycle with -factor or cdc4. The normal mtDNA content was restored a period of several generations when permissive conditions were restored. These results suggest that mtDNA synthesis is coupled to cell growth.     

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase

Summary The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5kkb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51 600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.     

Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae

In this paper we report the cloning, sequencing and functional characterization of CEN12 and an associated autonomously replicating sequence (ARS) from the budding yeast Saccharomyces cerevisiae. In the course of studying a dynamin-related gene, DNM1, we previously physically mapped the gene to chromosome 12. Genetic mapping showed that the gene was tightly linked (0.35 cM) to the centromere. Subcloning experiments revealed that a centromerelike activity was included in a small segment of DNA immediately downstream from the DNM1 gene. Mitotic centromere activity was discerned by the ability of the region to de-stabilize a centromere-containing plasmid, and to stabilize an ARS-containing plasmid. Meiotic centromere activity was determined by the first-division segregation in crosses of ARS plasmids containing this region. The DNA sequence of this region revealed a sequence with strong homology to the consensus for yeast centromeres.