The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments.

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that is able to grow at in situ temperatures below 0 degrees C. As abundant members of the microbial community in permanently cold marine sediments, D. psychrophila-like bacteria contribute to the global cycles of carbon and sulfur. Here, we describe the genome sequence of D. psychrophila strain LSv54, which consists of a 3 523 383 bp circular chromosome with 3118 predicted genes and two plasmids of 121 586 bp and 14 663 bp. Analysis of the genome gave insight into the metabolic properties of the organism, e.g. the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system, the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc. D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, nine putative cold shock proteins and nine potentially cold shock-inducible proteins. A comparison of D. psychrophila's genome features with those of the only other published genome from a sulfate reducer, the hyperthermophilic archaeon Archaeoglobus fulgidus, revealed many striking differences, but only a few shared features.

Isolation of segments of homologous genes with only one conserved amino acid region via PCR.

We present a method which allows the isolation of fragments from genes coding for homologous proteins via PCR when only one block of conserved amino acids is available. Sets of degenerated primers are defined by reverse translation of the conserved amino acids such that each set contains not more than 128 different sequences. The second primer binding site is provided by a special cassette that is designed such that it does not allow binding of the second primer prior to being copied by DNA synthesis. The cassette is ligated to partially-digested chromosomal DNA. The second primer is biotinylated to allow elimination of PCR products carrying degenerated primers on both sides via streptavidin binding. Fragments obtained after amplification and enrichment are cloned and sequenced. The feasibility of this method was demonstrated in a model experiment, where degenerated primers were deduced from six conserved amino acids within the family of homologs to the Escherichia coli Vsr protein.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

The msh2 gene of Schizosaccharomyces pombe is involved in mismatch repair, mating-type switching, and meiotic chromosome organization.

We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Delta cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 and mut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2 gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair, S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

Transcription of mutS and mutL-homologous genes in Saccharomyces cerevisiae during the cell cycle.

Transcription of the Saccharomyces cerevisiae DNA mismatch repair genes PMS1, MSH2, and MSH6, a recently discovered homolog of the Escherichia coli mutS gene, was shown to be cell cycle regulated. In contrast, transcription of the MSH1, MSH3 and MLH1 genes was not regulated during the cell cycle. The MSH1 gene, which is thought to be involved in DNA mismatch repair in mitochondria, was also not induced under aerobic growth conditions. Regulation of the PMS1 gene was dependent on intact MluI cell cycle boxes, as demonstrated by analysis of a promoter mutant. Both reduced and increased expression of PMS1 resulted in a mitotic mutator phenotype. Analysis of mRNA levels was performed with a newly developed reverse transcription-PCR (polymerase chain reaction) approach using fluorescently labeled primers and an automated DNA sequencer for detection of PCR products.

Genetic code and phylogenetic origin of oomycetous mitochondria.

We sequenced the 3'-terminal part of the COX3 gene encoding cytochrome c oxidase subunit 3 from mitochondria of Phytophthora parasitica (phylum Oomycota, kingdom Protoctista). Comparison of the sequence with known COX3 genes revealed that UGG is used as a tryptophan codon in contrast to UGA in the mitochondrial codes of most organisms other than green plants. A very high AT mutation pressure operates on the mitochondrial genome of Phytophthora, as revealed by codon usage and by A+T content of noncoding regions, which seems paradoxical because AT pressure causes tryptophan codon reassignment from UGG to UGA in mitochondria of most species. The genetic code and other data suggest that mitochondria of Oomycota share a direct common ancestor with mitochondria of plants and that mitochondria of the ancestor of Planta and Oomycota were acquired in a second endosymbiotic event, which occurred later than the acquisition of mitochondria by other eukaryotes.