Bacteriophage ?29 infection of Bacillus subtilis minicells

Summary Labelled chloroplast rRNAs from Spinacia oleracea were hybridized to restriction endonuclease digests of chloroplast DNA from Oenothera hookeri and Euglena gracilis, to mitochondrial DNA of Acanthamoeba castellanii, and to DNA of the E. coli rrn B operon in the transducing phage lambda rif^d18. The degree of homology is greatest for the 16S rRNA gene. Greater than 90% occurs between the two higher plant genes, 80% homology to the lower plant gene, 60%–70% homology to the bacterial gene, and 20% homology to the mitochondrial gene. The degree of hybridization varied considerably for the 23S and the 5S rRNA genes. Very high homology exists between the two higher plant genes, only about 50% homology for both the Euglena and bacterial genes, and no significant homology for the mitochondrial genes. These results show that any chloroplast (or E. coli) rRNA may be used as a probe to identify rRNA genes in other ctDNAs.Two RNA populations, each enriched for a different ctDNA-encoded mRNA, proved useful in the location of these genes on both higher plant ctDNAs. No significant hybridization was obtained using these probes to the Euglena ctDNA which seems to be too distantly related.Abbreviations Md megadalton, 10⁶ dalton - bp, kbp base pair, kilo base pair - SSC Standard saline citrate, 1 times SSC is 0.15M sodium, chloride, 0.015 M trisodium citrate, pH, 6.8 - mtDNA mitochondrial DNA - ctDNA chloroplast DNA - ctrRNA chloroplast ribosomal RNA     

Sequence analysis of the rDNA spacer ofParacentrotus lividus and observations about pre-rRNA processing

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchinParacentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor which has the same 5 terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.Abbreviations bp base pair(s) - Kb Kilobase(s) or 1000 bp - nt nucleotide(s) - rDNA DNA encoding rRNA - rRNA ribosomal RNA - S sedimentation constant     

Organization of the 5S RNA genes in macro- and micronuclei of Tetrahymena pyriformis

The organization of the 5S genes in macro- and micronuclei of Tetrahymena pyriformis was studied using restriction endonucleases. After complete digestion of macronuclear DNA with BamH-I or Hpa I, 5S RNA hybridized to a DNA fragment of approximately 280 base pairs (bp). When macronuclear DNA was only partially digested with these enzymes, hybridization with ³²P-5S RNA demonstrated an oligomeric series with a spacing of 280 bp. These results indicate that the 5S genes are tandemly repeated in macronuclei and that the repeating unit is 280 bp (or 180,000 daltons). Since 5S RNA is 120 nucleotides, we conclude that the 5S repeat units contain a 120 bp transcribed region and a 160 bp spacer region. When macronuclear DNA was digested with Eco RI, Bgl I, or Eco RI + Bgl I, 5S RNA hybridized to DNA of molecular weight 3–4×10⁶, suggesting that these enzymes do not cleave within a 5S repeat. These 3–4×10⁶ dalton fragments define the maximum size of an average cluster of 5S repeated units. Assuming the size of the 5S repeat to be 0.18×10⁶ daltons, there are about 15–20 5S repeats per average tandem cluster, and since there are 350 5S-genes per haploid genome, there must be approximately 15–20 tandem arrays. Results obtained using micronuclear DNA suggest that organization of the 5S-genes is very similar in macro- and micronuclei. Macronuclear rRNA genes are extracnromosomal palindromic dimers. In contrast, 5S genes in Tetrahymena were found to be integrated within the genomes of both macro- and micronuclei and not linked to the rRNA genes. Moreover, it is unlikely that they are palindromes; rather they appear to be tandemly repeated in head-to-tail linkages. Thus, the organization of the 5S genes in Tetrahymena is similar to that of higher eukaryotes.     

Suppression of mitochondrially-determined resistance to chloramphenicol and paromomycin by nuclear genes in Saccharomyces cerevisiae.

Summary Phenotypic revertants of a drug resistant strain of Saccharomyces cerevisiae were induced by mutgenesis with manganese. Several of these drug sensitive mutants have been shown to result from mutations in the nuclear genome that cause phenotypic modification (suppression) of the mitochondrially-determined drug resistant genotype.Four mutants carrying a single recessive nuclear gene capable of modifying mitochondrial chloramphenicol resistance are described; these may be assigned to three complementation groups. Chloramphenicol resistant mutants mapping at five separate mitochondrial loci are described. At least two of the nuclear genes cause modification of mitochondrial chloramphenicol resistance determined by mutations at three of these loci, but the other two loci are apparently non-suppressible by these nuclear alleles. This indicates that these modifiers do not act by causing a general decrease in cellular or mitochondrial permeability to the drug.A single dominant nuclear modifier of mitochondrial paromomycin resistance has been identified. It is non-allelic to and does not interact with the genes modifying mitochondrial chloramphenicol resistance.     

Biogenesis of mitochondria 51

Summary An examination of the effect of the aminoglycoside antibiotics paromomycin and neomycin on mitochondrial ribosome function in yeast has been made. Both antibiotics are potent inhibitors of protein synthesis in isolated mitochondria. With isolated mitochondrial ribosomes programmed with polyuridylic acid (poly U), the drugs are shown to inhibit polyphenylalanine synthesis at moderately high concentrations (above 100 g/ml). At lower concentrations (about 10 g/ml), paromomycin and neomycin cause a 2–3 fold stimulation in the extent of misreading of the UUU codons in poly U, over and above the significant level of misreading catalyzed by the ribosomes in the absence of drugs.Comparative studies have been made between a paromomycin sensitive strain D585-11C and a mutant strain 4810P carrying the parl-r mutation in mtDNA, which leads tohigh resistance to both paromomycin and neomycin in vivo. A high level of resistance to these antibiotics is observed in strain 4810P at the level of mitochondrial protein synthesis in vitro. Whilst the degree of resistance of isolated mitochondrial ribosomes from strain 4810P judged by the inhibition of polyphenylalanine synthesis by paromomycin and neomycin is not extensive, studies on misreading of the poly U message promoted by these drugs demonstrate convincingly the altered properties of mitochondrial ribosomes from the mutant strain 4810P. These ribosomes show resistance to the stimulation of misreading of the codon UUU brought about by paromomycin and neomycin in wild-type mitochondrial ribosomes. Although strain 4810P was originally isolated as being resistant to paromomycin, in all the in vitro amino acid incorporation systems tested here, the 4810P mitochondrial ribosomes show a higher degree of resistance to neomycin than to paromomycin.It is concluded that the parl-r mutation in strain 4810P affects a component of the mitochondrial ribosome, possibly by altering the 15S rRNA or a protein of the small ribosomal subunit. The further elucidation of the functions in the ribosomes that are modified by the parl-r mutation was hampered by the inability of current preparations of yeast mitochondrial ribosomes to translate efficiently natural messenger RNAs from the several sources tested.     

Purification of mitochondria and mitochondrial nucleic acids from embryogenic suspension cultures of a gymnosperm,Larix x leptoeuropaea

After a number of attempts to isolate mitochondria from different conifer tissues, embryogenic suspension cultures of hybrid larch (Larix x leptoeuropaea) were developed which enabled the purification of mitochondria using slight modifications to standard techniques. The mitochondrial purity was verified by analysis of the mitochondrial RNA, DNA and proteins. The larch mitochondrial genome size is surprisingly large (> 1000 kbp) and the polypeptide pattern differs greatly from those of wheat or potato mitochondria, suggesting that valuable evolutionary insights will be gained from comparisons between gymnosperm and angiosperm mitochondria. The ease with which embryogenic conifer suspensions can be initiated and used for mitochondrial purification implies that they will be the material of choice for future studies of this type.Abbreviations PCR polymerase chain reaction - mtDNA mitochondrial DNA - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediaminetetraacetic acid - BSA bovine serum albumin - EGTA ethylene glycol-bis(ß-aminoethyl ether) N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulphate - DEPC diethyl pyrocarbonate - PAGE polyacrylamide gel electrophoresis - IEF iso-electric focusing     

The Mitochondrial Genome of an Aquatic Plant,Spirodela polyrhiza

Background

Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.

Methods

Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method.

Conclusions

This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.     

Physical mapping of genes on yeast mitochondrial DNA: Localization of antibiotic resistance loci, and rRNA and tRNA genes

Summary We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA)¹ generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases.The antibiotic resistance loci (ant ^R) were mapped by hybridization of ³H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)-P(27-36.Ou)-O_II (58.3-62u)-O_I (80-84u)-E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with ¹²⁵I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70-76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.Supported by USPHS Training Grant T32-GM-07197.Supported by USPHS Training Grant 5-T01-GM-0090-19.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U. S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Structure and expression of the overlapping ND4L and ND5 genes of Neurospora crassa mitochondria

Summary Genes homologous to the mammalian mitochondrial NADH dehydrogenase subunit genes ND4L and ND5 were identified in the mitochondrial genome of the filamentous fungus Neurospora crassa, and the structure and expression of these genes was examined. The ND4L gene (interrupted by one intervening sequence) potentially encodes an 89 residue long hydrophobic protein that shares about 26% homology (or 41% homology if conservative amino acid substitutions are allowed) with the analogous human mitochondrial protein. The ND5 gene (which contains two introns) encodes a 715 residue polypeptide that shares 23% homology with the human analogue; a 300 amino acid long region is highly conserved (50% homology) in the two ND5 proteins. The stop codon of the ND4L gene overlaps the initiation codon of the downstream ND5 gene, and the two genes are contranscribed and probably cotranslated. A presumed mature dicistronic (ND4L plus ND5) RNA was detected. The postulated mRNA (about 3.2 kb) contains 5 and 3 non-coding regions of about 86 and 730 nucleotides, respectively; this species is generated from very large precursor RNAs by a complex processing pathway. The ND4L and ND5 introns are all stable after their excision from the precursor species.Abbreviations bp base pairs - rRNA ribosomal RNA - ND NADH dehydrogenase - URF unidentified reading frame - kDal kilodaltons; a.a., amino acid     

Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells

Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mt^a and mt) were purified and analysed. The constitutive agglutinin from mt^a cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mt^a cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt cells only doubled the apparent agglutinin activity.Abbreviations mt mating type - DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - YPG yeast-peptone-glucose - PAS periodic-acid-Schiff reagent     

Deletion mapping of mitochondrial transfer RNA genes in Saccharomyces cerevisiae by means of cytoplasmic petite mutants

Summary Mitochondrial transfer RNA genes have been ordered relative to the position of five mitochondrial drug resistance markers, namely, chloramphenicol (C), erythromycin (E), oligomycin I and II (O_I, O_II), and paromomycin (P). Forty-six petite yeast clones that were genetically characterized with respect to these markers were used for a study of these relationships. Different regions of the mitochondrial genome are deleted in these individual mutants, resulting in variable loss of genetic markers. Mitochondrial DNA was isolated from each mutant strain and hybridized with eleven individual mitochondrial transfer RNAs. The following results were obtained: i) Of the seven petite clones that retained C, E, and P resistance markers (but not O_I or O_II), four carried all eleven transfer RNA genes examined; the other three clones lost several transfer RNA genes, probably by secondary internal deletion; ii) Prolyl and valyl transfer RNA genes were located close to the P marker, whereas the histidyl transfer RNA gene was close to the C marker; iii) Except for a glutamyl transfer RNA gene that was loosely associated with the O_I region, no other transfer RNA genes were found in petite clones retaining only the O_I and/or the O_II markers; and iv) Two distinct mitochondrial genes were found for glutamyl transfer RNA, they were not homologous in DNA sequence and were located at two separate loci.The data indicate that the petite mitochondrial genome is the result of a primary deletion followed by successive additional deletions. Thus an unequivocal gene arrangement cannot be readily established by deletion mapping with petite mutants alone. Nevertheless, we have derived a tentative circular map of the yeast mitochondrial genome from the data; the map indicates that all but one of the transfer RNA genes are found between the C and P markers without forming a tight cluster. The following arrangement is suggested:-P-pro-val-ile-(phe, ala, tyr, asp)-glu₂-(lys-leu)-his-C-E-O_I-glu₁-O_II-P-.Supported in part by Cancer Center CCRC 111B-3. Present address: Laboratoire de Biologie Generale, Universite Paris-Sud Orsay, 91405, FranceThe Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract E(11-1)69     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

云斑车蝗线粒体基因组全序列测定与分析

采用长距 PCR 扩增及保守引物步移法并结合克隆测序测定并注释了云斑车蝗 Gastrimargus marmoratus （Thunberg）的线粒体基因组全序列。结果表明：云斑车蝗线粒体基因组全序列为15 904 bp（GenBank登录号为EU527334）,A+T含量略高于非洲飞蝗Locusta migratoria,为76.04%,包括13个蛋白质编码基因,22个tRNA 基因,2个rRNA基因和一段1 057 bp的A+T富集区。蛋白质基因的起始密码子中,除COⅠ和ND5为TTG以外,均为昆虫典型的起始密码子ATN。ND5基因使用了不完全终止密码子T,其余基因均为典型的TAA或TAG。预测了22个tRNA基因的二级结构,发现tRNA^Ser（AGN）缺少DHU臂, tRNA^Ser（UGY）的反密码子环上有9个碱基。预测了云斑车蝗12S和16S rRNA二级结构,分别包括3个结构域30个茎环和6个结构域44个茎环。A+T富集区含有3个串联重复序列。     

Arrangement of the rRNA sequences in the rDNA of Lytechinus variegatus

The arrangement of the 26S RNA and 18S RNA sequences of the ribosomal DNA (rDNA) from the sea urchin Lytechinus variegatus was investigated by an electron microscopic analysis of R-loops formed between the ribosomal RNA genes and the mature ribosomal RNAs. Ninety-eight percent of observed molecules contained R-loops clearly seen as a three-stranded complex. The size of DNA complementary to mature cytoplasmic 18S and 26S ribosomal RNA (rRNA) was calculated by measuring the double-strand (ds) and single-strand (ss) part of the R-loops separately. The values for the 18S R-loop are 1.75±0.24 kb¹ (ss) and 1.56±0.23 kb (ds). The 26S R-loop is 3.34±0.39 kb (ss) and 3.33±0.33 kb (ds). These measurements agree fairly well with the rRNA sizes measured on denaturing sucrose density gradients: 3.23±0.22 kb for the 26S and 1.93±0.10 kb for 18S. The short spacer between the 18S and 26S R-loops is 1.03±0.24 kb and the longer spacer is 5.36±0.53 kb. In long molecules a repeating pattern was observed. The average length of an rDNA repeat unit is 11.33±0.64 kb when computed using double-strand R-loop measurements and 11.50±0.72 when computed using R-loop single-strand lengths.Abbreviations kb kilobases, 1000 bases of RNA or single-strand DNA, and kilobase pairs, 1000 base pairs of duplex DNA or DNA/RNA hybrid - EDTA ethylenediaminetetraacetate - SSC 0.15 M NaCl, 0.015 M sodium citrate - PIPES piperazine-N,N-bis (2-ethanesulfonic acid)-Na_1.4     

Molecular cloning and characterization of the alkB gene of Escherichia coli

Summary Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation. Since the cloned DNA fragment was mapped at 47 min on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we concluded that the cloned DNA fragment contains the alkB gene itself but not other genes that suppress alkB mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkB codes for a polypeptide with a molecular weight of about 27,000. Introduction of a small deletion into the alkB region of the bacterial chromosome resulted in inactivation of both the alkB and ada genes, thereby suggesting that the two genes are adjacent on the E. coli chromosome.Abbreviations Ap ampicillin - Cm chloramphenicol - HPLC high performance liquid chromatography - kb kilobases - kd kilodaltons - MMS methylmethane sulfonate - MNU methylnitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - Tc tetracycline - SDS sodium dodecyl sulfate     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

The linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase

Summary Plasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced. The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs. The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases. The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages. ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively. No homology to any published protein sequence was found for the smaller ORFs. The origin of the terminal protein attached to the 5 ends of pClK1 remains open; several alternatives for its origin are discussed. The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties. Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.