Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo

Mitochondrial RNA was isolated from the morel strain Morchella conica 3 harbouring the linear plasmid pMC3-2 and subjected to gel electrophoresis followed by a Northern analysis using cloned fragments of the plasmid pMC3-2 as probes. Hybridization was obtained only with central parts of pMC3-2 and specific bands of mtRNA. The hybridization bands (2.8 kb and 1.0 kb) correspond in size to the length of the two ORFs of pMC3-2 which were deduced from nucleotide-sequence data. Thus, both ORFs, one encoding a DNA polymerase and the other a yet unknown protein, are transcribed in the mitochondria of the plasmid-bearing Morchella conica strain.     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5′ ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3′-CCCCC-5′, similar to those of Escherichia coli phage PRD1. The pMLP1 plasmid harbors two long open reading frames (ORF1 and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase. Received: 9 February 2000 / Accepted: 18 July 2000     

Cryptic DNA plasmids of the heterothallic yeast Saccharomycopsis crataegensis

Summary Three DNA plasmids, designated pScrl-1, pScrl-2, and pScrl-3 have been found in a strain of the heterothallic yeast Saccharomycopsis crataegensis (NRRL Y-5902). pScrl-l, -2 and -3 are, respectively, 15, 7, and 5 kilobase pairs (kbp) in size. Based on the results of exonuclease digestions, all three plasmids appear to be linear molecules with blocked 5 ends. All three plasmids also have a lower buoyant density than does nuclear DNA of S. crataegensis. The two lower molecular weight plasmids hybridize strongly with one another, but only weakly to the higher molecular weight plasmid. Two of four related S. crataegensis strains surveyed were found to contain two plasmids that are of the same size as the two larger plasmids of Y-5902. Evidence is presented indicating that the plasmids in strain Y-5902 reside in the cytosol since they were found not to be located within the major organelles (mitochondria and nuclei).     

Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector

Summary The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSCI and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5 termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent M_r of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [¹²⁵I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 by (pFSC1) and 1,027bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of the these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSCI conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.     

R-type plasmids in mitochondria from a single source of Zea luxurians teosinte

Two linear DNA plasmids resembling the R1 and R2 plasmids that are present in the mitochondria of several South American strains of maize were found in mitochondria from a single source of Zea luxurians collected by L. Mazoti. The Mazoti mtDNA is closely related to mtDNAs of other Z. luxurians, but mitochondria derived from the other Z. luxurians sources lack the plasmids. The larger plasmid from Mazoti mitochondria, M1, was cloned and large portions of it were sequenced. Restriction mapping and sequence comparisons showed that approximately 4.9 kb is similar to the S1 plasmid of maize and an additional 2.6 kb is related to R1 sequences integrated into the main mitochondrial genome of N cytoplasm. Therefore, the M1 plasmid appears to be very similar to the R1 plasmid. The inverted repeats at the ends of the M1 plasmid are not identitical. The left end IR is similar to the S-TIRs found at the termini of the S plasmids. The right end IR more closely resembles the integrated R1 sequences, including the variant region of the TIR. Whereas the variant region contains 13 bp in the S-TIRs and 15 bp in an integrated version of R1, it is 16 bp long in M1. The region of M1 that has no homology to the S1 plasmid is expressed at very low levels in Mazoti and RU cytoplasms, but at much higher levels in CMS-S mitochondria, where part of it is present in the main mitochondrial genome.     

Linear,non-mitochondrial plasmids of Alternaria alternata

Summary Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5 ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.     

Organization and structure of plastome psbF,psbL, petG and ORF712 genes in Chlamydomonas reinhardtii

Summary We have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement. psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b₅₅₉ locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5-and 3-ends to the ribosomal protein rps3 gene, but contains, a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.     

Kluyveromyces lactis killer system: ORF1 of pGKL2 has no function in immunity expression and is dispensable for killer plasmid replication and maintenance

Summary To functionally characterize the genes encoded by the larger killer plasmid pGKL2 from Kluyveromyces lactis a previously developed in-vivo recombination system was exploited. An in-vitro modified version of the cytoplasmically expressible LEU2 gene cartridge (LEU2 ^*) flanked by appropriate pGKL2 segments was used to replace the central part of the ORF1 region of pGKL2. Transformation of a Leu^- killer strain resulted in the expected disruption of ORF1 in the resident pGKL2. The Leu⁺ transformants obtained can be assigned to three classes. Class I carries both killer plasmids, pGKL1/2, and the recombinant pGKL2 derivative termed pRKL2. Class II and III additionally harbor palindrome and hairpin-like plasmids, respectively. Upon subculturing of class I transformants under selective pressure, segregation of the native pGKL2 and the recombinant pRKL2 eventually occurs resulting in total loss of pGKL2. No differences concerning killer and immunity phenotype between a pRKL2-harboring strain and the native pGKL2-carrying recipient could be detected. Thus pGKL2 ORF1 is dispensable for both expression of killer/immunity phenotypes and for the replication and maintenance of the K. lactis killer plasmids.     

Proteins are attached to the ends of a linear plasmid in the filamentous fungus Ascobolus immersus

Summary In seven out of eleven wild strains of the Ascomycete Ascobolus immersus plasmid DNA was found. There was great variability with respect to size and number of the plasmids in the strains concerned. For a further analysis two plasmids originating from one wild strain were submitted to restriction analysis and electron microscopy. Both turned out to be linear having different molecular weights (pAIl = 7.9 kb, pAI2 = 5.6 kb). Denaturation of pAI2 and subsequent renaturation revealed the presence of inverted repeats (0.7 kb) at both ends. After treatment with proteinase K and 5 and 3 specific exonucleases it became evident that the 5 ends of pAI2 are linked with proteins. In this respect it is similar in structure to other linear genetic elements such as the linear plasmids found in Zea mays and the genomes of adenoviruses.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

DNA sequence and organization of the mitochondrial ND1 gene from Podospora anserina: analysis of alternate splice sites

Summary Earlier, we reported that the ND1 mitochondrial gene of Podospora anserina is mosaic, containing at least three class I introns. We have now completed the sequence of the ND1 gene and have determined that it contains four class I introns of 1,820, 2,631, 2,256 and 2,597 by with the entire gene complex containing 10,505 bp, only 1,101 of which are exon sequences. Introns 1 and 3 appear to be related in that their open reading frames (ORFS) exhibit extensive amino acid sequence similarity and like the URFN sequence from Neurospora crassa have multiple sequence repetitions. Introns 2 and 4 are similar in that both appear to be mosaic introns. Where intron 2 has many short ORFS, intron 4 has two, 391 and 262 as respectively. The first ORF has some patch work sequence similarity with one of the intron 2 ORFs but the second ORF is strikingly similar to the single intron ORF in the ND1 gene of N. crassa. Just upstream of the sequences necessary to form the central core of the P. anserina intron 4 secondary structure, there is a 17 bp sequence which is an exact replica of the exon sequence abutting the 5 flank of the 1,118 by N. crassa ND1 intron. Secondary structure analysis suggests that the 2,597 by intron 4 can fold as an entity but a similar structure can be constructed just from an 1,130 bp portion by utilizing the 17 bp element as an alternate splice site. Detailed structural analysis suggests that intron 4 (as well as the single ND1 intron from N. crassa) can utilize helical configurations which bring the downstream open reading frame into juxtaposition with the exon sequences.     

The complete DNA sequence of the mitochondrial genome of Podospora anserina

Summary The complete 94,192 bp sequence of the mitochondrial genome from race s of Podospora anserina is presented (1 kb=10³ base pairs). Three regions unique to race A are also presented bringing the size of this genome to 100,314 bp. Race s contains 31 group I introns (33 in race A) and 2 group II introns (3 in race A). Analysis shows that the group I introns can be categorized according to families both with regard to secondary structure and their open reading frames. All identified genes are transcribed from the same strand. Except for the lack of ATPase 9, the Podospora genome contains the same genes as its fungal counterprts, N. crassa and A. nidulans. About 20% of the genome has not yet been identified. DNA sequence studies of several excision-amplification plasmids demonstrate a common feature to be the presence of short repeated sequences at both termini with a prevalence of GGCGCAAGCTC.     

Interaction between mitochondrial DNA and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the IrRNA-gene

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pC1K1 and pClT5, to the upstream region of the large ribosomal RNA gene in the mtDNA of three strains (W3, T5 and K) has been investigated in detail to explore the widespread phenomenon of homology between mt plasmids and mtDNA in C. purpurea. Sequence comparison indicates that recombination between free plasmids and mtDNA is the cause of the observed homology. The process is similar to the integration of the structurally related adenoviruses into the mammalian genome. As in other fungi, palindromic sequences seem to be involved in this mitochondrial recombination process.     

Sequencing and Characterisation of p74 Gene in Two Isolates of Anticarsia Gemmatalis MNPV

P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern.     

Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

Complete Nucleotide Sequence and Phylogenetic Analyses of Hepatitis B Virus Isolated from Two Pileated Gibbons

Virus-like particles (VLPs, named HmTV1-17), about 40nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5 located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the –1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.     

Genome Organization and Expression of the <Emphasis Type="Italic">Penicillium stoloniferum</Emphasis> Virus F

The complete sequences of three double-stranded (ds) RNAs (referred to F1, F2 and F3) of Penicillium stoloniferum virus F (PsV-F) were established. The F1 dsRNA was 1677 bp in length, and it contained one open reading frame (ORF) of 538 amino acids (molecular weight of 63 kDa, referred to P63), The F2 dsRNA was 1500 by in length, and also it contained one ORF of 420 amino acids (molecular weight of 46 kDa, referred to P46). The F3 dsRNA was 677 bp in length, but contained a small ORF with unknown function. A sequence motif of (5′-CGTAAAA-3′) was found only at the 5′ termini of the F1 and F2 dsRNAs, and a sequence motif of (5′-TAAAAAAAAA-3′) was found at the 3′ termini of all three dsRNA segments. The predicted amino acid sequence of F1 showed 38–48% sequence homology with the putative dsRNA-dependent RNA polymerases (RdRp) of dsRNA viruses, but the predicted amino acid of F2 showed no homology. Phylogenetic analysis using the RdRp sequences of the various Partitiviruses and Alphacryptoviruses revealed that PsV-F clustered well with Partitiviruses, but showed remote relationship with PsV-S. Near full-length and positive-sense single-stranded (ss) RNAs derived from the Fl, F2 and F3 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P63 and P46 showed a positive reaction against PsV-F antiserum, indicating P63 and P46 as RdRp and capsid protein, respectively. These results suggest that PsV-F can be a member of Partitivirus, but it is quite distinct from PsV-S electrophoretically, serologically and genetically, though both viruses coexist in the same cell.     

The Saccharomyces cerevisiae HIS3 and LYS2 genes complement the Schizosaccharomyces pombe his5-303 and lys1-131 mutations,respectively: new selectable markers and new multi-purpose multicopy shuttle vectors,pSP3 and pSP4

Three new S. pombe plasmids are described. Plasmids pSP3 and pSP4 are two Schizosaccharomyces pombe ars1 multicopy vectors with the Saccharomyces cerevisiae HIS3 or LYS2 genes as selectable markers. They complement the S. pombe his5-303 or lys1-131 mutations, respectively. Plasmid pSPars1 is a vector carrying the S. pombe ars1 and a unique NdeI site which allows the introduction of any selectable marker therefore bringing a unified vector backbone for the construction of new S. pombe/S. cerevisiae/E. coli shuttle vectors. These plasmids permit classical molecular genetic techniques to be performed directly.