Transmission,recombination and conversion of mitochondrial markers in relation to the mobility of a group I intron in Chlamydomonas

Mitochondrial DNA transmission has been analyzed in diploids produced from sexual crosses or artificial fusions between Chlamydomonas strains which differ by several genetic markers: a group I intron (Cs cob.1 or intron), three restriction sites (Nh, Nc and H markers) located 0.5–5 kb from the insertion site of the intron, and a MUD2 point mutation (27 bp from the insertion site) conferring resistance to myxothiazol. Recombination between mitochondrial markers is a general property of all crosses and fusions analyzed. In crosses between two intron-containing (⁺) strains or two intron-less (^–) strains, the transmission is preferentially paternal (mt ^-), with a preoponderance depending on the nature of the parental genomes. In crosses between (⁺) and (^–) strains, the conversion of intron-less molecules into intron⁺ is frequent when the (⁺) parent is maternal (mt ⁺) and nearly absolute when the (⁺) parent is paternal (mt ^-). In 94% of cases, the conversion is accompanied by the co-conversion of the MUD2 marker. In both crosses and artificial fusions, the conversion of (^–) into (⁺) also influences the transmission of the more distant Nh, Nc and H markers. It is hypothesized that the more frequent transmission of the genome containing the intron results from the elimination of (^–) molecules, as a result of a double-strand cut which is induced by an endonuclease encoded by the intron.     

Electrophoretic analysis of the nuclear and organellar genomes in the ultra-small alga Cyanidioschyzon merolae

Electrophoretic analysis reveals that the nucleus of the ultra-small eukaryotic alga Cyanidioschyzon merolae contains approximately 11.7×10⁶ base pairs (11.7 Mb) of DNA. This compact genome is fragmented into 15 small chromosomes ranging in size from 410 to 1700 kb. The migratory behaviour of chloroplast DNA is consistent with the presence of a circular plastid genome of about 170 kb. The conformation of mitochondrial DNA resembles that in yeasts and fungi and is predominantly linear and heterogenous in size.     

NIT2, the nitrogen regulatory protein of Neurospora crassa,binds upstream of nia,the tomato nitrate reductase gene,in vitro

Summary The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa,alter promoter element recognition

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.     

Isolation of mutants sensitive to 2-amiriopurine and alkylating agents and evidence for the role of DNA methylation in Penicillium chrysogenum

Summary Using high performance liquid chromatography, the presence of N6-methyladenine has been found at a level of 0.1 mol percent in DNA extracted from Penicillium chysogenum. No 5-methylcytosine was detected. A mutant strain HP547, which is sensitive to the lethal effects of N-methyl-N¹-nitro-N-nitrosoguanidine, methylmethane sulphonate and the base analogue 2-aminopurine shows an increased spontaneous mutation rate and no detectable DNA methylation. Comparison of restriction enzyme digests of wild type and undermethylated strains indicated that methylation was occurring at a different sequence to that of the Dam methylase system of E. coli.     

Physical evidence for recombination of chloroplast dna in hybrid progeny of Chlamydomonas eugametos and C. moewusii

Summary Differences in the restriction endonuclease fragmentation patterns of chloroplast DNA (cpDNA) from C. eugametos and C. moewusii have been used to study the inheritance of these DNAs in interspecific hybrids. Analysis of the cpDNAs from ten randomly selected F₁ hybrids, in each case revealed cpDNA to be recombinant for AvaI and BstEII restriction sites, although fragments characteristic of C. eugametos, the mt+ parent, were typically found in excess of those for C. moewusii, the mt– parent. In backcrosses between an F ₁ mt+ hybrid and C. moewusii mt–, seven randomly selected B₁ hybrids showed cpDNA restriction patterns either identical to or highly similar to that of the mt+ parent. We propose that cpDNA molecules are predominantly transmitted by the mt+ parent in both F₁ and B₁ generations but that selection favors survival of F₁ progeny with recombinant chloroplast genomes which avoid interspecific incompatibilities. On the surface, the inheritance of recombinant cpDNA contrasts with the simultaneous uniparental inheritance of two putative chloroplast markers (sr-2 and er-nM1 ⁺). However, it may be that these two markers are by chance associated with cpDNA sequences of the mt+ parent which were selected in all F₁ hybrids.     

The fate of mitochondrial DNAs of mt + and mt − origin in gametes and zygotes of Chlamydomonas

Summary In order to study the mechanism responsible for the uniparental transmission of the mitochondrial genome in crosses between Chlamydomonas reinhardtii and C. smithii, we have analyzed the fate of mitochondrial DNA during gametogenesis, zygospore differentiation and sporulation by hybridization experiments. Both mt ⁺ and mt ^– gametes contain the same amount of mitochondrial DNA and the two parental genomes persist for several days in the zygotes. The DNA of mt ⁺ origin is slowly eliminated during the period of zygote maturation. Light is required for total elimination of mt ⁺ mitochondrial DNA in the zygospores. Using appropriate restriction enzymes, we have been unable to detect methylation of the mitochondrial DNA during gametogenesis or zygospore formation. The possibility that the mt ⁺ mitochondria themselves are specifically eliminated in the course of zygote maturation is discussed.     

Detection of chloroplast DNA by using fluorescent monoclonal anti-bromodeoxyuridine antibody and analysis of its fate during zygote formation in Chlamydomonas reinhardtii

Summary A monoclonal anti-bromodeoxyuridine antibody conjugated to fluorescein was used to detect the chloroplast nucleoids after specific incorporation of bromodeoxyuridine (BUdR) into the chloroplast DNA of Chlamydomonas reinhardtii. The incorporation of BUdR was enhanced by simultaneous treatment with fluorodeoxyuridine (FUdR). The method was applied to analyze the fate of chloroplast DNA in zygotes resulting from mating between BUdR-treated gametes (mt ⁺ or mt ^-) and untreated gametes of opposite mating-type. In crosses between wild-type strains, the nucleoids of mt ⁺ origin remained in the large majority of zygotes whereas those of mt ^- origin most often disappeared within the first hours following copulation. In crosses of the type mat-3 mt ⁺xwild-type mt ^- (the mat-3 mutation permits a high transmission of chloroplast genes from the mt ^- parent), the nucleoids of mt ^- origin were generally not eliminated which indicates that the mat-3 mutation prevents the selective destruction of paternal chloroplast DNA in the zygote.     

Mutations in a nuclear gene of Chlamydomonas cause the loss of two chloroplast ribosomal proteins,one synthesized in the chloroplast and the other in the cytoplasm

Summary The allelic nuclear mutations of Chlamydomonas reinhardtii, cr-6 and cr-7, result in the loss of two proteins from the large subunit of the chloroplast ribosome. One of these proteins, L-13, is synthesized in the chloroplast and the other, L29, is made in the cytoplasm. The loss of these two proteins is correlated with the inability of the large subunits of the chloroplast ribosomes to form monomers which incorporate labeled phenylalanine at normal rates in response to a polyuridylic acid template. Using antisera raised against L13 and L29, we found that protein L-13 was synthesized in appreciable amounts in pulse labeled cells of cr-6 and cr-7, but protein L-29 was not. We conclude that the inability to synthesize protein L29 is a primary defect in both cr-6 and cr-7 and that this protein is required for the stable assembly of protein L-13 into chloroplast ribosomes. The absence of one or both of these proteins from the large subunit of chloroplast ribosomes of the mutants interferes with the ability of the small and large subunits to associate properly into normal 70S monomers.     

Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum

Summary A hybrid cefE gene was constructed by juxtaposing promoter sequences from the Penicillium chrysogenum pcbC gene to the open reading frame of the Streptomyces clavuligerus cefE gene. In S. clavuligerus the cefE gene codes for the enzyme penicillin N expandase [also known as deacetoxycephalosporin C synthetase (DAOCS)]. To insert the hybrid cefE gene into P. chrysogenum the vector pPS65 was constructed; pPS65 contains the hybrid cefE gene and the Aspergillus nidulans amdS gene. The amdS gene encodes acetamidase and provides for dominant selection in P. chrysogenum. Protoplasts of P. chrysogenum were transformed with pPS65 and selected for the ability to grow on acetamide medium. Extracts of cells cultivated in penicillin production medium were analyzed for penicillin N expandase activity. Penicillin N expandase activity was detected in approximately one-third of the transformants tested. Transformants WG9-69C-01 and WG9-61L-03 had the highest specific activities of penicillin N expandase: 4.3% and 10.3%, respectively, relative to the amount of penicillin N expandase in S. clavuligerus. Untrasformed P. chrysogenum exhibited no penicillin N expandase activity. Analysis of the penicillin V titer revealed that WG9-61L-03 produced titers equal to that of the recipient strain while the amount of penicillin V produced in WG9-69C-01 was reduced by five fold.     

Changes in chloroplast genome composition and recombination during the maturation of zygospores of Chlamydomonas reinhardtii

Summary In crosses of the unicellular green alga Chlamydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems.     

A model for the rapid vegetative segregation of multiple chloroplast genomes in Chlamydomonas: Assumptions and predictions of the model

Summary Physical evidence indicates that the chloroplast DNA of Chlamydomonas reinhardtii is composed of approximately 75 copies of a small unique sequence. Genetic analysis of zygotes biparental for chloroplast genes shows rapid vegetative segregation of parental chloroplast alleles. Zygote clones composed entirely of homoplasmic progeny cells predominate within 10–20 post-mating generations. A model is proposed here which reconciles the high multiplicity of chloroplast genes with their rapid vegetative segregation rates. Clustering of genomes into a small number of discrete areas (nucleoids) within the chloroplast reduces the effective number of segregating units. A non-random distribution of nucleoids to daughter cells, dictated solely by the spatial arrangement of parental nucleoids with respect to the plane of chloroplast division, further increases the rate of segregation from heteroplasmic cells. Recombination between parental chloroplast genomes is viewed as an indication of nucleoid fusion, and can account for differences in the patterns and rates of segregation at different gene loci. Within such fused nucleoids, clustering of parental genomes and a non-random distribution, again based solely on physical positioning of the genomes, to daughter nucleoids, could act to promote rapid genetic purification of heteroplasmic nucleoids. The effects of biased parental nucleoid ratios, and of potentially unequal nucleoid distributions to daughter chloroplasts are also discussed with respect to observed rates and patterns of chloroplast gene segregation.     

Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana

We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana. The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria. The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides. Experiments suggest there is a single copy of this gene in A. thaliana and multiple copies in Brassica species. The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.     

Organization of the psbE,psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome

Summary The genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE — 8 by spacer —psbF — 110 by spacer — orf38 — 87 by spacer — orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T > A > G > C in the mRNA-like strand and group II-like boundary sequences at their 3 and 5 ends having the consensus 5-GTGTG .. INTRON .. TTAATTTNAT-3. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE psbF orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.Abbreviations and notations DEP diethyl pyrocarbonate; gene names follow the convention of Hallick and Bottomley (1983) - psaA, psaB genes for the P₇₀₀ apoproteins - psbE and psbF genes for the subunits of cytochrome b ₅₅₉ - orfN open reading frame of N codons     

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum

A 1 380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.