Terminal protein association and sequence homology in linear mitochondrial plasmid-like DNAs of sorghum and maize

The linear extrachromosomal mitochondrial plasmid-like DNAs from the Ru cytoplasm of maize, and M35-1 and IS1112C cytoplasms of sorghum, possess 5 terminally-attached proteins. These molecules required proteinase K treatment for mobility in agarose gels and were susceptible to exonuclease III but not lambda exonuclease cleavage. Hybridizations, under stringent conditions, indicated that the sorghum plasmid-like DNAs, N1 and N2, did not possess DNA sequence homology to cloned central regions of S1 and S2, the linear mitochondrial plasmid-like DNAs present in S cytoplasm of maize. In addition, a novel 4.2kb, DNAase sensitive, RNAase insensitive band, exhibiting homology to internal sequences from maize S2, was observed in the sorghum IS1112C cytoplasm only.     

Extrachromosomal plasmid-like DNA in the obligate parasitic fungus Erysiphe graminis f. sp. hordei

Summary The obligate parasitic fungus, Erysiphe graminis f. sp. hordei, was found to harbour plasmid-like extrachromosomal DNA. A 1.35-kb fragment of this 9kb plasmid was cloned into the pUC12 vector. No homology was detected to nuclear or mitochondrial DNA. As only about half of the 27 isolates examined contained plasmid-like DNA, this appears to be inessential for fungal survival. The plasmid is frequent in European isolates and is found in both newly collected isolates and in isolates kept under laboratory conditions for many years. No correlation between presence of plasmid and specific avirulence/virulence genes was found. The plasmid appear to be located in the mitochondria.     

Biogenesis and replication of small plasmid-like derivatives of the mitochondrial DNA in Neurospora crassa

For reasons that are not obvious, sets of related, small, plasmid-like elements appear spontaneously and become amplified in the mitochondria of some cytochrome-deficient and/or UV-sensitive mutants of Neurospora crassa. These plasmid-like DNAs are multimeric series of circular molecules, each consisting of a finite number of identical tandem repeats of a relatively short mtDNA-derived nucleotide sequence (monomer). The plasmid-like elements that have been characterized in this study consist of monomers that vary in length from 125 to 296 base pairs, depending on the strain of origin. Each monomer includes a GC-rich palindrome that is followed by the promoter and a short section of the 5' terminal region of the mitochondrial large-subunit rRNA gene (rnl). Analyses of the nucleotide sequences of variants of this group of elements indicates that they are not generated by intra-molecular recombination, but are the result of single- or double-strand DNA breaks that are produced by a mismatch or base excision repair process. These elements do not appear to contain a defined origin of replication, but replicate by a recombination-dependent rolling-circle mechanism. One- and two-dimensional gel electrophoresis of the plasmid-like element derived Hind III and Pst I fragments combined with S1 nuclease treatments suggest that the intergenic GC-rich palindromes, which are ubiquitous in the mtDNA Neurospora, could be replication fork pausing points.     

Two linear plasmids in mitochondria of Fusarium solani f. sp. cucurbitae

Two linear plasmid-like DNAs designated pFSC1 (9.2 kbp) and pFSC2 (8.3 kbp) were found in an isolate of the plant pathogenic fungus Fusarium solani f. sp. cucurbitae race 1. The plasmids were maternally inherited and copurified with mitochondrial DNA obtained from a mitochondria-enriched cell fraction suggesting that they are located in mitochondria. The plasmids did not share extensive sequence similarity. No homology was detected between either plasmid and the nuclear or mitochondrial genome when cloned plasmids were used as probes in Southern hybridization analyses. The fungus was cured of plasmids by ethidium bromide treatment. Compared to the plasmid-containing isolate, plasmid-cured derivatives had reduced pathogenicity on a susceptible plant host, Cucurbita maxima "Pink Banana."     

Identification of mitochondrial plasmid-like DNAs in Picea abies (L.) Karst.

The Norway spruce (Picea abies (L.) Karst.) mitochondrial DNA has been extracted from embryonal suspensor masses. In addition to a master chromosome, a family of plasmid-like DNAs were identified. These latter shared cross homologies but had no evident sequence homology with the master chromosome. The occurrence of mitochondrial plasmid-like DNAs was investigated in trees from different provenances. A vast majority of trees displayed extrachromosomal DNA elements of variable stoechiometry. For some trees, the sequences homologous to the extrachromosomal DNA elements were found associated with high molecular weight DNA. Received: 9 July 1997 / Revision received: 29 January 1998 / Accepted: 29 November 1998     

Asexual transmission, non-suppressiveness and meiotic extinction of small plasmid-like derivatives of the mitochondrial DNA in Neurospora crassa

For reasons that are not obvious, sets of related plasmid-like elements that consist of short segments of DNA that overlap the 5' terminal region of the mitochondrial large-subunit rRNA gene sometimes appear spontaneously and become amplified in the mitochondria of some cytochrome-deficient and/or UV-sensitive mutants of Neurospora crassa. These elements are transmitted efficiently through hyphal anastomoses and appear to invade the mitochondria of recipient strains, but they do not cause senescence and at best cause only slight deficiencies in cytochromes a and b even though they are transcribed copiously. Hence, the small elements are not suppressive and, unlike large deletion derivatives of the mitochondrial chromosome, do not displace normal mtDNA molecules in vegetatively propagated mycelia. Unlike the mitochondrial chromosome, large plasmid-like mtDNA derivatives and true mitochondrial plasmids, the small plasmid-like mtDNA derivatives are rarely transmitted sexually even though they persist without selection in very high copy numbers in vegetative cells. The high copy numbers and high stability of these elements in vegetatively propagated cultures suggests that their monomers contain all the features required for their replication and transmission in the hyphae and conidia of Neurospora. However, the mt-rnl-derived molecules appear to lack a sequence or attribute required for the maintenance or transmission of mitochondrial genetic elements at some stage of the sexual reproductive cycle, including ascospore maturation and germination.     

黄瓜线粒体类质粒pC1,pC4在品种间的分布及同源性研究

在黄瓜（津研四号）线粒体中存在4种线粒体类质粒（pCl,pC2,pC3,pC4),对14个黄瓜品种的线粒体类质分布情况进行了研究,发现在津春二号,津春五号,津新密刺,津绿四号和津研四吃5个品种中存在线粒体类质粒,其余9个品种无线粒体类质粒,类质粒的存在有一定随机性,不同品种中的同一种类质粒间具有同源性,pC4类质粒不仅与自身的核DNA同源,也与其他品种的核基因组有同源性,pC4同源序列在黄瓜核基因组中为多拷贝的,拷贝之间是不连续的,在丝瓜和西葫芦的核基因组中也有pC4的同源序列,因此,推测pC4可能在葫芦科分化的早期就已存在并整合于核中,某些品种缺乏类质粒是由于在进化过程中发生的类质粒的丢失。     

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

Purification and characterization of plasmid-like DNA from the antimycotic producing fungus, Scytalidium flavo-brunneum

The azasterol producing strain of Scytalidium flavo-brunneum (ATCC 28804) was examined for the presence of a plasmid-like DNA. Several different plasmid preparation procedures yielded DNA which migrated as single bands of equivalent molecular weight when analyzed by gel electrophoresis. Electron microscopy and λ exonuclease digestion data were consistent with a covalently closed circular structure. A complete restriction map for a circular 9.1-kb plasmid-like DNA was deduced from analysis of restriction enzyme digests and Southern blot hybridizations of restriction fragments. Visualization of the plasmid by electron microscopy revealed a measured contour length of 8.9 kb, using pBR322 as a standard. Southern hybridization analysis using plasmid-like DNA as the probe detected no homology to the non-azasterol producing strains of Scytalidium flavo-brunneum or mitochondrial DNA from azasterol producing strain.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5-flanking sequence contains heat shock elements and other potential regulatory sequences.     

Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.     

Variable Bacteriocin Production in the Commercial Starter Lactococcus lactis DPC4275 Is Linked to the Formation of the Cointegrate Plasmid pMRC02

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763

Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt+). The 6.2 kb HindIII-PstI fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the PstI site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.