Prevalence of pSmeSM11a-like plasmids in indigenous Sinorhizobium meliloti strains isolated in the course of a field release experiment with genetically modified S. meliloti strains

Plasmid pSmeSM11a, residing in the indigenous Sinorhizobium meliloti strain SM11 originating from a field in Strassmoos (Bavaria, Germany), was analysed previously at the genomic level. Thirty-seven indigenous S. meliloti strains, originating from two different locations in Germany, were screened for genes identified previously on pSmeSM11a. Seven of these strains harbour accessory plasmids that are very similar to pSmeSM11a. The identified pSmeSM11a-like plasmids are c. 130-150 kb in size and possess nearly identical restriction profiles. Up to 30 genes identified previously on pSmeSM11a could be detected on these plasmids by hybridisation experiments, e.g., the nodulation genes nodP and nodQ, the ethylene level modulation gene acdS and the taurine metabolism gene tauD. A few pSmeSM11a genes were also detected on other plasmids. The reference plasmid pSmeSM11a contains a region that is similar to a segment of S. meliloti strain Rm1021 pSymA. Regions with similarity to pSymA were also detected on the aforementioned seven pSmeSM11a-like plasmids. The specifications of these regions are nearly identical to the one on pSmeSM11a and differ from Rm1021 pSymA as determined by nucleotide sequence analysis. Two further plasmids similar to pSmeSM11a completely lack the pSymA-region. Those strains carrying accessory plasmids that contain the acdS gene encoding 1-aminocyclopropane-1-carboxylate deaminase are able to grow on 1-aminocyclopropane-1-carboxylate as the sole source of nitrogen, demonstrating functionality of the acdS gene product. About 36% of the analysed plasmids, including three pSmeSM11a-like plasmids, could be transferred to another S. meliloti recipient strain, allowing for their dissemination in S. meliloti populations.     

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.     

Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae

The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone [Entian et al., Mol. Gen. Genet. 198 (1984) 50-54]. The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer. Several initiation regions and termination points were located using nuclease S1 mapping. The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts. Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes. Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.     

Distinct origins of tRNA(m1G37) methyltransferase

The enzyme tRNA(m1G37) methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the N1 position of G37 in the anticodon loop of a subset of tRNA. The modified guanosine is 3' to the anticodon and is important for maintenance of reading frame during decoding of genetic information. While the methyltransferase is well conserved in bacteria and is easily identified (encoded by the trmD gene), the identity of the enzyme in eukarya and archaea is less clear. Here, we report that the enzyme encoded by Mj0883 of Methanocaldococcus jannaschii is the archaeal counterpart of the bacterial TrmD. However, despite catalyzing the same reaction and displaying similar enzymatic properties, MJ0883 and bacterial TrmD are completely unrelated in sequence. The catalytic domain of MJ0883, when aligned with the five known structural folds (I-V) that have been described to bind AdoMet, is of the class I fold, similar to the ancient Rossmann fold that binds nucleotides. In contrast, the catalytic domain of the bacterial TrmD has the unusual class IV fold of a trefoil knot structure. Thus, both the sequence and structural arrangements of tRNA(m1G37) methyltransferase have distinct evolutionary origins among primary kingdoms, revealing an unexpected but remarkable non-orthologous gene displacement to achieve an important tRNA modification.     

Nucleotide sequence and evolution of the rat mitochondrial cytochrome b gene containing the ochre termination codon

The nucleotide sequences of the genes for cytochrome b and three potential transfer RNAs (tRNAPro, tRNAThr and tRNAGlu) in cloned rat mitochondrial DNA were determined. The derived amino acid sequence of the cytochrome b protein from the light strand indicated that the C-terminal amino acid is asparagine and the ochre termination codon is encoded in the DNA, in contrast to the the lack of termination codon in the reading frame of human [Anderson et al., Nature 290 (1981) 457] or mouse [Bibb et al., Cell 26 (1981) 167] mitochondrial DNA. The first ATG codon of the cytochrome b gene was spaced five nucleotides from the 5'-end of the tRNAGlu gene on the heavy strand. There was a single nucleotide spacing between the termination codon of the cytochrome b gene and the 5' end of the tRNAThr gene in the light strand. There was also a single nucleotide spacing between the 3'-end of the tRNAThr gene and the 3'-end of the tRNAPro gene on the heavy strand. The amino acid and nucleotide sequences of the cytochrome b genes of mammals and yeast [Nobrega and Tzagoloff, J. Biol. Chem. 255 (1980) 9828] were compared to reveal structural differences in two very different species. At the same time, amino acid substitutions in particular regions of the mammalian gene corresponding to the exon-intron boundaries in the yeast gene were noted. These genetic features are discussed in relation to the extreme compression of genetic information in the mammalian mitochondrial genome as related to the evolution of the gene organization and its sequence.     

A horizontally transferred tRNA(Cys) gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is aminoacylated

Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.