MboI,ThaI and HinfI endonuclease cleavage maps of the yeast mitochondrial DNA

Summary MboI, HinfI and ThaI cleavage maps have been constructed for the region of the mitochondrial DNA from S. cerevisiae where transfer RNA genes are principally located. About 40 cleavage sites have been localized between the C and P genetic markers. The MboI map covers about 50% of the total mitochondrial genome. For constructing maps we have used a series of rho^– deletion mutants whose mitochondrial DNAs have a typical single deletion structure as judged by previous genetic and physical analyses. The mutant DNAs carry known transfer RNA genes and genetic markers and, therefore, the comparison between genetic and restriction maps has allowed us to localize individual transfer RNA genes within defined physical segments.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA - ThaI formerly TacI     

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences

Summary Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho^–) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho^– mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).Abbreviations mtDNA mitochondrial DNA - bp base pairs - kbp kilobase pairs     

Mitochondrial mutagenesis in Saccharomyces cerevisiae

Summary Four types of mit ^– mutations induced with manganese are found in the following relative proportions: oxi3 ^– > cob-box ^– > oxi2 ^– oxi1 ^–1. The frequences of loss of their respective mit ⁺ alleles in manganese-induced rho ^–] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.     

Genetic modification of the spontaneous rho − mutability in Saccharomyces cerevisiaee

Summary The phenotypic trait starry colony in Saccharomyces is associated with a high spontaneous rho ^– petite mutability. Genetic analysis of this trait has shown the high rho ^– mutability to be caused by several modifying genes present together in the strains studied. Every single modifying gene produces only a relatively small enhancement of the rho ^– mutability.     

The disomy for chromosome IV and the spontaneous rho- mutability in Saccharomyces cerevisiae

Summary The disomy for chromosome IV in the strains studied led to: i) a reduction in the red pigmentation of ade1 mutant colonies; ii) a decrease of the spontaneous rho ^– mutant frequency, and iii) an impairment of sporulation in hybrids descended from disomic parents. The nuclear srm1 mutation decreasing the spontaneous rho ^– mutability promoted the spontaneous extra chromosome loss in the disomes for chromosome IV. This result suggests a close connexion between the spontaneous rho ^– mutability and mitotic chromosome stability.     

A controversy concerning the structure of the mitochondrial genome of a yeast petite mutant: resolution by sequence analysis

Summary Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho ^–) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 by segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron

Summary The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8 826 bp and contains five exons (termed E1–E5) totalling 1 602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1–1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5a, and 1.5b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5a and K1 cox1.4/Sc cox1.5b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.EM BL Accession Number X57546     

Physical mapping of the mitochondrial genome of the cultivated mushroom Agaricus brunnescens (= A. bisporus)

Summary Mitochondrial (mt) DNA from the commercial mushroom Agaricus brunnescens Peck [= A. bisporus (Lange) Imbach] was purified by cesium chloride/bisbenzimide gradient centrifugation. A physical map of the mtDNA fragments produced by BamHI, EcoRl, and PvuII digestion was generated by filter hybridizations with radiolabelled BamHI mtDNA probes. The A. brunnescens mtDNA was a circular molecule 136 kilo-basepairs (kbp) in length and contained an inverted repeat between 4.6 and 9.2 kbp in size. Orientational isomers of the mitochondrial genome were not detected. The positions of six genes were located on the A. brunnescens mtDNA map by heterologous hybridization. No coding function has yet been ascribed to the inverted repeat. The large rRNA gene was located on the smaller single copy region. The genes for cytochrome b, cytochrome oxidase (subunit III), ATPase (subunits 8 and 6) and the small rRNA were located on different regions of the larger single copy region.     

The <Emphasis Type="Italic">rpl5</Emphasis>–<Emphasis Type="Italic">rps14</Emphasis> mitochondrial region: a hot spot for DNA rearrangements in <Emphasis Type="Italic">Solanum</Emphasis> spp. somatic hybrids

Southern analysis with rpl5 and rps14 mtDNA gene probes of Solanum tuberosum, S. commersonii and a sample of somatic hybrids detected polymorphisms between parents and the appearance of a novel restriction fragment in various hybrids. In one of them, detailed mtDNA analyses revealed various configurations of the rpl5–rps14 region present at different stoichiometries. Multiple inter-parental recombination events across homologous sequences were assumed to have caused these rearrangements. Sequence similarity searches detected one sequence putatively involved in the recombination upstream of the rpl5 gene. The presence of a second recombinogenic sequence was inferred. We propose two models to explain the mechanism responsible for obtaining the different rpl5–rps14 arrangements shown after somatic hybridization. Variability in the rpl5–rps14 region observed in both the parental species and their somatic hybrids suggests this region is a hot spot for mtDNA rearrangements in Solanum spp.Contribution no. 39 from the Institute of Plant Genetics, Research Division of Portici.Communicated by A. Brennicke     

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.     

Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides

Two overlapping cosmid clones (Cos1-1 and Cos9-1) carrying the Tox5 gene were isolated from a library of F. sporotrichioides strain NRRL 3299 genomic DNA. These cosmids were used to transform three T-2 toxin-deficient mutants that are blocked at different steps in the trichothecene pathway. Both cosmids restored T-2 toxin production to Tox3-1 ^– or Tox4-1 ^– mutants but neither restored T-2 toxin production to a Tox1–2 ^– mutant. The production of T-2 toxin by the complemented Tox3-1 ^– and Tox4-1 ^– mutants, as well as the production of diacetoxyscirpenol by the cosmid-transformed Tox1-2 ^– mutant, were 2- to 10- fold higher than in strain NRRL 3299. In addition, those transformants carrying Cos9-1 produced significantly higher levels of trichothecenes than transformants carrying Cos1-1. Two different DNA fragments (FSC13-9 and FSC14-5), representing the region of overlap between the cosmid clones, were isolated. These fragments specifically complemented either the Tox3-1 ^– mutant (FSC14-5) or the Tox4-1 ^– mutant (FSC13-9). The trichothecene-production phenotype of these transformants was similar to NRRL 3299. These results suggest that two or more genes involved in the biosynthesis of trichothecenes are closely linked to Tox5.     

Complete absence of mitochondrial DNA in the petite-negative yeastSchizosaccharomyces pombe leads to resistance towards the alkaloid lycorine

The petite-positive yeastSaccharomyces cerevisiae can be efficiently and completely converted to respiratory-deficient cytoplasmic petite mutants by intercalating drugs.Rho ^o petites fromSchizosaccharomyces pombe could only be obtained in strains carrying a nuclear mutation. In this paper we report the efficient isolation ofrho ^o mutants in aSch. pombe strain containing a mitochondrial mutator mutation. We also show that the alkaloid lycorine is able to differentiate between cells containing defective mitochondrial DNA (mit ^–) and those lacking mitochondrial DNA completely (rho ^o).Rho ^o cells are resistant to the alkaloid whereasmit ^– and wild-type cells show the same sensitivity.     

Sequences of two rbcS cDNA clones of Batophora oerstedii: structural and evolutionary considerations

Summary Two cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU) of Batophora oerstedii were isolated and sequenced. One clone contains the coding information for the complete SSU precursor protein. As in two other species of Dasycladaceae (Acetabularia mediterranea and A. cliftonii), the rbcS cDNA sequences of B. oerstedii display the codons TAA or TAG, which seem to code for glutamine, in the reading frame. The amino acid substitution rate for the SSU protein was calculated to be 0.35–0.41 amino acids per 10⁹ years per site based on the substitutions observed in the SSU amino acid sequences of Acetabularia and Batophora.     

Rearrangements in the Physarum polycephalum mitochondrial genome associated with a transition from linear mF-mtDNA recombinants to circular molecules

Although mitochondrial DNA (mtDNA) is transmitted to progeny from one parent only in Physarum polycephalum, the mtDNAs of progeny of mF⁺ plasmodia vary in structure. To clarify the mechanisms associated with the mitochondrial plasmid mF that generate mtDNA polymorphisms, 91 progeny of four strains (KM88 × JE8, KM88 × TU111, KM88 × NG111, Je90) were investigated using RFLP analysis, PCR, and pulse-field gel electrophoresis (PFGE). Nine mtDNA rearrangement types were found, with rearrangements occurring exclusively in the mF regions. PFGE revealed that, in the groups containing rearranged mtDNA, the linear mF–mtDNA recombinants had recircularized. Sequencing the rearranged region of one of the progeny suggested that the mF plasmid and the mtDNA recombine primarily at the ID sequences, linearizing the circular mtDNA. Recombination between the terminal region of the mF plasmid and a region about 1 kbp upstream of the mitochondrial/plasmid ID sequence results in a rearranged circular mtDNA, with variations caused by differences in the secondary recombination region.     

Human chromosome 3: high-resolution fluorescencein situ hybridization mapping of 40 uniqueNotl linking clones homologous to genes and cDNAs

Forty newNotl linking clones representing sequence tagged sites (STSs) were mapped by fluorescencein situ hybridization (FISH) to different regions of human chromosome 3 (HSA3). Clone NL1-245, containing human aminoacylase 1, was localized to 3p21.2–p21.1. Our previous localization of the CLC-2 chloride channel protein gene was refined to 3q27. Clone NL2-316 most likely contains a translocon-associated protein -subunit gene and was mapped to 3q23–q24. To our knowledge, this is the first time this gene has been mapped. OneNoti linking clone (NL1-229) probably contains a new protein phosphatase gene. This clone was mapped to 3p25. FiveNoti linking clones probably contain human expressed sequence tags (ESTs), as they possess sequences with a high level of identity (>90%) to cDNA clones. Other clones show 56–85% homology to known mammalian and human genes with various functions, including oncogenes and tumour-suppressor genes. These clones might represent new genes.accepted for publication by M. Schmid     

Dispersed discrete length polymorphism of mitochondrial DNA in the scallop Placopecten magellanicus (Gmelin)

Three separate regions exhibiting incremental length polymorphism have been found in the mitochondrial genome of the scallop Placopecten magellanicus. Each locus has a discrete and different unit of variation, measuring 1450 bp at locus I, 250 bp at locus II, and less than 100 bp at locus III. At least six size classes were observed at each locus, and individual variation can account for the intraspecific mtDNA size range of 31–42 kb, but not for the unusually large base size of the genome. Intramolecular hybridization patterns with clones of two of the variable regions indicate that there is a dispersed sequence similarity with the 1,450-bp locus-I repeat and its flanks, and with some part of locus II.     

The Lymantria dispar Nucleopolyhedrovirus Contains the Capsid-Associated p24 Protein Gene

During the course of investigations on a wild-type strain of Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), a region of the viral genome was analyzed and found to contain 697 bp that is lacking in the sequenced strain (5–6) of LdMNPV (Kuzio et al., Virology 253, 17–34, 1999). The sequenced strain of LdMNPV contains a mutation in the 25 K few polyhedra (FP) gene, and exhibits the phenotype of a FP mutant. The additional sequence was located at approximately 81.4 map units within the viral genome, and was found in 10 different wild-type LdMNPV genotypic variants analyzed. Since the additional sequence was found in all wild-type virus strains analyzed, this sequence should be included in the representative LdMNPV genome. Sequence analysis of the genomic region containing the additional sequences revealed the presence of a homologue of the Autographa californica MNPV capsid-associated p24 gene (ORF 129). This gene, absent in LdMNPV isolate 5–6, is also present in the Orgyia pseudotsugata MNPV, Bombyx mori NPV, Spodoptera exigua MNPV, S. litura MNPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, Buzura suppressaria SNPV, Xestia c-nigrum granulovirus, Plutella xylostella GV, and Cydia pomonella GV.     

Nuclear mutations affecting the stability of the mitochondrial genome inS. cerevisiae

Summary We have studied a pleiotropic mutationpetD inS. cerevisiae which both confers the inability to grow on glycerol (Gly^–) and greatly increases the frequency of cytoplasmic petites (Het). The first phenotype, Gly^–, is recessive, whereas the second, Het, is dominant. Genetic and biochemical analysis showed that the majority of the petites inpetD strains are not of therho° type (completely lacking mit-DNA),but of therho ^– type (containing partially deleted mit-DNA). This finding and the fact that the phenotype Het is dominant argue in favour of the involvement of thepetD product in the excision process of the mit-DNA. Another nuclear mutation,mod, was shown to exhibit a dominant epistasy with respect to the Het phenotype of the mutationpetD. Two types of Gly⁺ revertants frompetD mutants were isolated:rpa revertants, which restore completely the wild-type phenotype, andrpb revertants, which restore only the growth on glycerol, but still allow the production of high frequencies of cytoplasmic petites. Thus the mutationsmod andrpb permit the genetic uncoupling of two phenotypes induced by the mutationpetD.