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Complete Genome Sequence of Rickettsia typhi and Comparison with Sequences of Other Rickettsiae. Michael P. McLeod, 2004.Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts . Here we present the complete genome sequence of R . typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R . prowazekii and R . conorii . We identified 877 genes in R . typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts . In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system . The three rickettsial genomes share 775 genes: 23 are found only in R . prowazekii and R . typhi, 15 are found only in R . conorii and R . typhi, and 24 are unique to R . typhi . Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R . typhi, compared to R . prowazekii and R . conorii . In addition, we found a 124-kb R . typhi-specific inversion, starting 19 kb from the origin of replication, compared to R . prowazekii and R . conorii . Inversions in this region are also seen in the unpublished genome sequences of R . sibirica and R . rickettsii, indicating that this region is a hot spot for rearrangements . Genome comparisons also revealed a 12-kb insertion in the R . prowazekii genome, relative to R . typhi and R . conorii, which appears to have occurred after the typhus (R . prowazekii and R . typhi) and spotted fever (R . conorii) groups diverged . The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae . An Alternative Strategy for Adaptation in Bacterial Behavior. Barry L. Taylor, 2004. Convenient Biological Assay for Polyethylene Glycol-Interferons in Patients with Hepatitis C. Anne Boulestin, 2004.The vesicular stomatitis virus cytopathic effect reduction assay is suitable to quantify polyethylene glycol-alpha interferon 2a (PEG-IFN-  2a) and PEG-IFN-2b . Human serum and ribavirin did not interfere with the assay . This bioassay was successfully used for assaying PEG-IFN-2a and PEG-IFN-2b in serum samples from patients undergoing combination therapy .
Treatment of Salmonella enterica Serovar Enteritidis with a Sublethal Concentration of Trisodium Phosphate or Alkaline pH Induces Thermotolerance. Balamurugan Sampathkumar, 2004.The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined . Pretreatment of S . enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H2O2 . Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis . Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively . More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment . Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins . Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation . Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis . In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed . Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells . These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment . Nuclease Activity of Saccharomyces cerevisiae Mre11 Functions in Targeted Nucleotide Alteration. Li Liu, 2003.Oligonucleotides can be used to direct site-specific changes in genomic DNA through a process in which mismatched base pairs in the oligonucleotide and the target DNA are created . The mechanism by which these complexes are developed and resolved is being studied by using Saccharomyces cerevisiae as a model system . Genetic analyses have revealed that in all likelihood the reaction occurs in two phases: DNA pairing and DNA repair . While the former phase involves strand assimilation, the latter phase likely involves an endonucleolytic processing step that leads to joint resolution . In this study, we established the importance of a functioning MRE11 gene in the overall reaction, as yeast strains deficient in MRE11 exhibited severely reduced activity . The activity could be rescued by complementation with wild-type MRE11 genes but not with MRE11 alleles lacking the nuclease function . Taken together, the data suggest that Mre11 provides nuclease activity for targeted nucleotide exchange, a process that could be used to reengineer yeast genes .
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