Guest: a 98 by inverted repeat transposable element in Neurospora crassa

The region immediately 3 of histidine-3 has been cloned and sequenced from two laboratory strains of the ascomycete fungus Neurospora crassa; St Lawrence 74A and Lindegren, which have different derivations from wild collections. Amongst the differences distinguishing these sequences are insertions ranging in size from 20 to 101 by present only in St Lawrence. The largest of these is flanked by a 3 by direct repeat, has terminal inverted repeats (TIR) and shares features with several known transposable elements. At 98 bp, it may be the smallest transposable element yet found in eukaryotes. There are multiple copies of the TIR in the Neurospora genome, similar but not identical to the one sequenced. PCR amplification of Neurospora genomic DNA, using 26 by of the TIR as a single primer, gave products of discrete sizes ranging from 100 by to about 1.3 kb, suggesting that the element isolated (Guest) may be a deletion derivative of a family of larger transposable elements. Guest appears to be the first transposable element reported in fungi that is not a retrotransposon.     

Achlya mitochondrial DNA: gene localization and analysis of inverted repeats

Summary Mitochondrial DNA from four strains of the oomycete Achlya has been compared and nine gene loci mapped, including that of the ribosomal protein gene, var1. Examination of the restriction enzyme site maps showed the presence of four insertions relative to a map common to all four strains. All the insertions were found in close proximity to genic regions. The four strains also cotained the inverted repeat first observed in A. ambisexualis (Hudspeth et al. 1983), allowing an examination by analysis of retained restriction sites of the evolutionary stability of repeated DNA sequences relative to single copy sequences. Although the inverted repeat is significantly more stable than single copy sequences, more detailed analysis indicated that this stability is limited to the portion encoding the ribosomal RNA genes. Thus, the apparent evolutionary stability of the repeat does not appear to derive from the inverted repeat structure per se.Abbreviations ATPase 6, 9 genes for ATPase subunits 6 and 9 - COI, II, III genes for cytochrome oxidase subunits 1, 2, and 3 - COB gene for apocytochrome b - L-, S-RNA genes for the mitochondrial large and small ribosomal RNAs - mtDNA mitochondrial DNA - var1 gene for the S. cerevisiae mitochondrially, encoded ribosomal protein - m.u. map units - bp base pairs - kb kilobase pairs     

Chloroplast DNA insertions into the nuclear genome of rice: the genes,sites and ages of insertion involved

Rice (Oryza sativa) is one of three predominant grain crops, and its nuclear and organelle genomes have been sequenced. Following genome analysis revealed many exchanges of DNA sequences between the nuclear and organelle genomes. In this study, a total of 45 chloroplast DNA insertions more than 2 kb in length were detected in rice nuclear genome. A homologous recombination mechanism is expected for those chloroplast insertions with high similarity between their flanking sequences. Only five chloroplast insertions with high sequence similarity between two flanking sequences from an insertion were found in the 45 insertions, suggesting that rice might follow the non-homologous end-joining (NHEJ) repair of double-stranded breaks mechanism, which is suggested to be common to all eukaryotes. Our studies indicate that the most chloroplast insertions occurred at a nuclear region characterized by a sharp change of repetitive sequence density. One potential explanation is that regions such as this might be susceptible target sites or “hotspots” of DNA damage. Our results also suggest that the insertion of retrotransposon elements or non-chloroplast DNA into chloroplast DNA insertions may contribute significantly to their fragmentation process. Moreover, based on chloroplast insertions in nuclear genomes of two subspecies (indica and japonica) of cultivated rice, our results strongly suggest that they diverged during 0.06–0.22 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogenetic characterization of the purple sulfur bacterium Thiocapsa sp. BBS by analysis of the 16S rRNA, cbbL, and nifH genes and its description as Thiocapsa bogorovii sp. nov., a new species

Strain BBS, the purple sulfur bacterium assigned initially to the species Thiocapsa roseopersicina, is the best studied representative of this species. However, no molecular phylogenetic analysis has been performed to confirm its systematic position. Based on the results of analysis of the sequences of 16S rRNA, cbbL, and nifH genes, DNA-DNA hybridization with the T. roseopersicina type strain, and comparative analysis of the phenotypic characteristics of various species belonging to the genus Thiocapsa, we suggest that strain BBS should be assigned to a new species of the genus Thiocapsa, Thiocapsa bogorovii sp. nov. Original Russian Text ? T.P. Tourova, O.I. Keppen, O.L. Kovaleva, N.V. Slobodova, I.A. Berg, R.N. Ivanovsky, 2009, published in Mikrobiologiya, 2009, Vol. 78, No. 3, pp. 381–392.     

The uvp1 gene of plasmid pR cooperates with mucAB genes in the DNA repair process

Summary We show that a DNA fragment that contains the uvp1 gene of the plasmid pR directs the synthesis in Escherichia coli minicells of a protein of apparent molecular weight 20 kDa. Inspection of the nucleotide sequence of the region reveals an open reading frame that has the capacity to encode a protein of 198 amino acids. The uvp1 gene product has been found, in two different systems, to enhance the recombination activity of E. coli cells. We have also observed a striking similarity to resolvase and invertase proteins. The significance of this finding for the function of the uvp1 gene product requires further investigation. We conclude that the uvp1 gene encodes a 20 kDa protein which appears to be responsible for enhancement of both UV survival and recominational activity in E. coli.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Phylogenetic relationships of the Japanese minnows,Pseudorasbora (Cyprinidae), as inferred from mitochondrial 16S rRNA gene sequences

The phylogenetic relationships among threePseudorasbora fishes (Cyprinidae, Sarcocheiichthyinae) occurring in Japan (P. parva, P. pumila pumila andP. pumila subsp. sensu Nakamura [1963]) were inferred from nucleotide sequences of the mitochondrial 16S rRNA gene. The sequences. of 1240 bp, were determined and compared for 22 specimens from 2–8 populations for each taxon, with a singlePungtungia herzi specimen as an outgroup. A total of 171 sites (13.8%) were variable among the specimens, but only 0–2 sites within each population. The phylogenetic relationships estimated by neighbor-joining, maximum-parsimony and maximum-likelihood methods confirmed a sister relationship between the twoP. pumila subspecies, with a high level of confidence. However, their genetic distinction from each other (4.1±0.4SD % sequence difference on average) was at a level similar to that between them andP. parva (5.9±0.5%). The geographic distribution of the twoP. pumila subspecies, which are separated by the Fossa Magna region, suggests that the genetic divergence of the two subspecies originated from a vicariant process separating the freshwater ichthyofaunas of eastern and western Honshu.Pseudorasbora parva populations were divided into two genetic groups (1.8±0.2% sequence difference), one group comprising continental and part of the Japanese populations, and the other the remaining Japanese populations. This suggests that at least two genetically divergent lineages had been originally distributed in Japan, but a strong possibility remains that the present situation has resulted from artificial transplantation.     

Chloroplast DNA phylogeny of subtribe Dendrobiinae (Orchidaceae): Insights from a combined analysis based onrbcL sequences and restriction site variation

Phylogenetic analyses using two chloroplast DNA data sets, derived from variation of the ribulose-bisphosphate carboxylase gene (rbcL) and restriction sites, were performed to examine relationships among 13 taxa in subtribe Dendrobiinae, one of the most taxonomically complicated groups in Orchidaceae, and its putative sister groups. Owing to a limited number of informative substitutions, therbcL data set did not provide conclusive evidence in itself. The data set combiningrbcL and restriction site mutations, however, provided the following insights: (1)Pseuderia belongs with tribe Podochileae rather than tribe Dendrobieae. (2) Subtribe Dendrobiinae is monophyletic ifPseuderia is excluded. (3) ExcludingPseuderia, Dendrobiinae comprises three major clades: Clade 1 (Dendrobium sectionSpatulata, Cadetia, Diplocaulobium, andFlickingeria); Clade 2 (Dendrobium sectionsDendrobium andCallista); and Clade 3 (Epigeneium). (4)Epigeneium diverged early from the lineage including Clades 1 and 2. (5) Relative toCadetia, Diplocaulobium, andFlickingeria, Dendrobium is shown to be para-/polyphyletic. (6)Diplocaulobium andFlickingeria constitute a monophyletic clade, from which cladeDendrobium sectionSpatulata andCadetia form succesive sister groups. Among these results, (1) and (5) are especially stable in view of the congruence between the separate and combined analyses as well as robust internal support.     

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.     

Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge

The bacterial and archaeal communities of three deep-sea hydrothermal vent systems located on the Mid-Atlantic Ridge (MAR; Rainbow, Logatchev and Broken Spur) were investigated using an integrated culture-dependent and independent approach. Comparative molecular phylogenetic analyses, using the 16S rRNA gene and the deduced amino acid sequences of the alpha and beta subunits of the ATP citrate lyase encoding genes were carried out on natural microbial communities, on an enrichment culture obtained from the Broken Spur chimney, and on novel chemolithoautotrophic bacteria and reference strains originally isolated from several different deep-sea vents. Our data showed that the three MAR hydrothermal vent chimneys investigated in this study host very different microbial assemblages. The microbial community of the Rainbow chimney was dominated by thermophilic, autotrophic, hydrogen-oxidizing, sulfur- and nitrate-reducing Epsilonproteobacteria related to the genus Caminibacter. The detection of sequences related to sulfur-reducing bacteria and archaea (Archaeoglobus) indicated that thermophilic sulfate reduction might also be occurring at this site. The Logatchev bacterial community included several sequences related to mesophilic sulfur-oxidizing bacteria, while the archaeal component of this chimney was dominated by sequences related to the ANME-2 lineage, suggesting that anaerobic oxidation of methane may be occurring at this site. Comparative analyses of the ATP citrate lyase encoding genes from natural microbial communities suggested that Epsilonproteobacteria were the dominant primary producers using the reverse TCA cycle (rTCA) at Rainbow, while Aquificales of the genera Desulfurobacterium and Persephonella were prevalent in the Broken Spur chimney.     

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

Chloroplast DNA variation among five species ofRanunculaceae: Structure,sequence divergence,and phylogenetic relationships

A restriction site map of the chloroplast genome ofCaltha palustris L. (Ranunculaceae) has been constructed for 13 restriction endonucleases using filter hybridization with cloned tobacco chloroplast DNA fragments. A size of 153.8 kb has been estimated for theCaltha chloroplast genome. Forty-six chloroplast genes and four open reading frames have been mapped using small tobacco chloroplast gene probes. Chloroplast DNA sequence divergence has been estimated for all pairs of five species ofRanunculaceae, Caltha palustris, Ranunculus bulbosus, R. fascicularis, R. recurvatus, andTrollius ledebourii, and ranges between 0.2% and 9.6% for the total genome. Divergence values are much higher in the small and large single copy regions than in the inverted repeat. Phylogenetic relationships between the five species have been hypothesized using chloroplast DNA restriction site mapping. One hundred and six informative restriction site mutations have been detected using eleven restriction endonucleases. Cladistic analyses of the restriction site mutations have been performed using Wagner and Dollo parsimony algorithms, and confidence intervals have been calculated for the resulting monophyletic groups using bootstrapping. It is demonstrated that restriction site comparisons are applicable to theRanunculaceae on intergeneric level, with the exception of groups having extensive genomic rearrangements. Moreover, sequence divergence is low enough at the interspecific level to allow phylogenetic analyses within genera such asRanunculus.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.