Kemba N Lee

Centers for Disease Control and Prevention, Atlanta, Michigan, United States

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Publications (2)4.07 Total impact

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    ABSTRACT: DNA sequencing of the region directly downstream of the Anaplasma phagocytophilum (strain MRK) 16S rRNA gene identified homologues of sdhC and sdhD; however, further sequencing by gene walking failed to identify additional sdh gene homologues. The sequence downstream of sdhD identified a partial gene, pep1, predicted to encode a protein >35.3 kDa with 26.3% identity to a hypothetical Ehrlichia canis protein with no known function. The recently completed sequence of the A. phagocytophilum genome confirmed our findings and indicated that the sdhA and sdhB genes are duplicated in a tandem orientation, and located distant from the sdhC and sdhD genes. The expression of the A. phagocytophilum 16S rRNA, sdhC, and sdhD genes was examined by reverse transcriptase PCR which showed that these three genes are expressed as an operon. The pep1 gene was expressed independent of the 16S-sdhCD operon from a promoter between sdhD and pep1. Further analysis of the sdhA and sdhB genes suggested the tandem duplication of the genes in conserved and may be unique to the species A. phagocytophilum. While the conservation of the A. phagocytophilum Sdh proteins, including the residues required for heme- and quinone-binding by SdhC and SdhD, suggests these subunits form an active enzymatic complex, the unusual genomic arrangement and expression pattern of these genes support previous studies (rRNA, ftsZ) indicating that gene rearrangement and operon fragmentation are common in the genomes of Anaplasma and other obligate intracellular bacteria. OMB DISCLAIMER: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC or the Department of Health and Human Services.
    Gene 06/2008; 414(1-2):41-8. · 2.08 Impact Factor
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    ABSTRACT: Degenerate primers corresponding to highly conserved regions of previously characterized ftsZ genes were used to PCR amplify a portion of the ftsZ gene from the genomic DNA of Ehrlichia chaffeensis (ftsZ(Ech)), Anaplasma phagocytophilum (ftsZ(Ap)), and Rickettsia rickettsii (ftsZ(Rr)). Genome walking was then used to amplify the 5' and 3' termini of the genes. The DNA sequences of the resulting amplification products yielded open reading frames coding for proteins with molecular masses of 42.0, 45.7, and 48.3 kDa for A. phagocytophilum, E. chaffeensis, and R. rickettsii, respectively. These homologs are 20 to 70 amino acids longer than the FtsZ proteins characterized in bacteria such as Escherichia coli and Bacillus subtilis, but do not possess the large extended carboxyl-termini found in the FtsZ proteins of Bartonella, Rhizobium, and Agrobacterium species. The functional domains important for FtsZ activity are conserved within the ehrlichial and rickettsial FtsZ protein sequences. The R. rickettsii FtsZ sequence is highly homologous to the FtsZ protein previously described for Rickettsia prowazekii (89% identity), and identical to the FtsZ protein of Rickettsia conorii. The percent identity observed between the A. phagocytophilum and E. chaffeensis FtsZ proteins is only 79% and is particularly low in the carboxyl-terminal region (15.8% identity). Primers were designed to PCR amplify a portion of the variable carboxyl-terminal region of the ftsZ gene, and used to differentiate each agent based on the size of the amplicons: A. phagocytophilum, 278 bp; E. chaffeensis, 341 bp; and Rickettsia spp., 425 bp.
    DNA and Cell Biology 04/2003; 22(3):179-86. · 1.99 Impact Factor