Phylogenetic study of Staphylococcus and Macrococcus species based on partial hsp60 gene sequences.

A 600 bp partial hsp60 gene sequence has been described previously as a novel genetic marker for species identification and phylogenetic studies within the genus Staphylococcus. In the present study, the 600 bp partial hsp60 gene sequences of 40 validly described Staphylococcus species and subspecies and four Macrococcus species were PCR-amplified and sequenced. Phylogenetic analysis revealed excellent concordance between the unrooted dendrograms based on partial hsp60 and 16S rRNA gene sequences. The genus Macrococcus is clearly separated from the genus Staphylococcus, but is closely related to the 'sciuri group', the only staphylococci that are cytochrome c oxidase-positive. The remaining Staphylococcus species clustered into five broad-based subdivisions, which corresponded to the 'aureus group', the 'epidermidis group', the 'haemolyticus group', the 'saprophyticus group' and the 'intermedius group'. These results agreed remarkably well with the current taxonomy of this diverse family, which is based on classical phenotypic and biochemical testing. Furthermore, pairwise sequence comparisons indicated that the hsp60 gene is more divergent and more discriminatory than the 16S rRNA gene for species differentiation among strains of the genera Staphylococcus and Macrococcus. It is concluded that the hsp60 gene may be an efficient alternative target for taxonomic and phylogenetic studies on members of these genera.

Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences.

The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.

Truncation mutations in ABCA1 suppress normal upregulation of full-length ABCA1 by 9-cis-retinoic acid and 22-R-hydroxycholesterol.

Mutations in ABCA1 uniformly decrease plasma HDL-cholesterol (HDL-C) and reduce cholesterol efflux, yet different mutations in ABCA1 result in different phenotypic effects in heterozygotes. For example, truncation mutations result in significantly lower HDL-C and apoliprotein A-I (apoA-I) levels in heterozygotes compared with nontruncation mutations, suggesting that truncation mutations may negatively affect the wild-type allele. To specifically test this hypothesis, we examined ABCA1 protein expression in response to 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-R-OH-Chol) in a collection of human fibroblasts representing eight different mutations and observed that truncation mutations blunted the response to oxysterol stimulation and dominantly suppressed induction of the remaining full-length allele to 5-10% of wild-type levels. mRNA levels between truncation and nontruncation mutations were comparable, suggesting that ABCA1 expression was suppressed at the protein level. Dominant negative activity of truncated ABCA1 was recapitulated in an in vitro model using transfected Cos-7 cells. Our results suggest that the severe reduction of HDL-C in patients with truncation mutations may be at least partly explained by dominant negative suppression of expression and activity of the remaining full-length ABCA1 allele. These data suggest that ABCA1 requires a physical association with itself or other molecules for normal function and has important pharmacogenetic implications for individuals with truncation mutations.

ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation.

Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.