Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.     

Analysis of group B streptococcal gene scaAB encoding the protein involved in adherence and aggregation

The portion of group B streptococcal (GBS) sequence encoding the ABC transport system was cloned and sequenced. The genetic region under study included scaAB gene, which was capable of encoding for putative adherence and aggregation factor, and scaR, which was encoding for putative represser. After the insertional inactivation of scaAB gene on the GBS chromosome streptococci increased the tendency for aggregation. The level of GBS adherence to vaginal epithelium was also different after scaAB inactivation. GBS sea operon was compared to similar ABC encoding bacterial operons.     

Isolation of a cDNA clone encoding the RNase-superfamily-related gene highly expressed in chicken bone marrow cells.

The RNase gene superfamily combines functionally divergent proteins which share statistically significant sequence similarity. Known members assigned to this family include secretory and nonsecretory RNases; angiogenin; eosinophil cationic protein; eosinophil-derived neurotoxin; sialic-acid binding lectin and anti-tumor protein P-30. We report the cDNA cloning of the chicken RNase Super Family Related (RSFR) gene that is specifically overexpressed in normal bone marrow cells and bone marrow-derived AMV transformed monoblasts. It codes for a 139 amino acid protein with a putative signal peptide and remarkable conservation of active-site residues, other residues known to be important for substrate binding and catalytic activity and half-cystine residues common for all RNase family members. Phylogenetic tree analysis shows that RSFR defines a new group of genes within the family. We also conclude that an amino acid sequence block CKXXNTF(X) 11C is a "shortest RNase superfamily signature" which is both necessary and sufficient to identify all previously recognized family members as well as chicken RSFR.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV.

The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.     

Effect of insertional mutation in the cspA gene encoding the major cold-shock protein on radiation resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA+). The radioprotective effect of this plasmid is abolished or drastically reduced in mutants recA13 and rpoH15 defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to gamma-irradiation of E. coli mutants with an intermediate level of radioresistance (Gamr445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gamr445 mutant. In the chromosome of E. coli with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of gamma-rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. coli cells to the lethal effect of gamma-rays and UV light.     

Mutation of the gene encoding protein kinase C 1 stimulates mitotic recombination in Saccharomyces cerevisiae.

We have isolated a recessive allele of the yeast protein kinase C gene (PKC1) which promotes an elevated rate of mitotic recombination and confers a temperature-sensitive growth defect. The rate of recombination was increased between genes in direct repeat and at a series of heteroalleles and was dependent upon the RAD52 gene product. The mutant pkc1 allele was sequenced and found to encode a single amino acid change within the catalytic domain. Osmotic stabilizing agents rescued the temperature-sensitive growth defect but not the hyperrecombination phenotype, indicating that the two traits are separable. This separability suggests that the PKC1 gene product (Pkc1p) regulates DNA metabolism by an alternate pathway to that used in the regulation of cell lysis. The regulation of recombination is a previously unidentified role for Pkc1p.