A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis.
Taking advantage of the known sequence conservation of portions of bacterial sigma factor proteins, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction (PCR) to amplify DNA sequences from the chlamydial genome. The PCR products were used as a probe to recover the genomic fragments from a library of cloned murine Chlamydia trachomatis DNA. Sequence analysis of one of these clones revealed striking homology to the sigma-70 protein of Escherichia coli and the sigma-43 protein of Bacillus subtilis, strongly implying that this locus (sigA) encodes the major vegetative sigma factor of murine C. trachomatis. This PCR-based approach will be broadly applicable to the cloning of major sigma factors from other eubacteria.
Engel
J N
JN
Department of Microbiology and Immunology, University of California, San Francisco 94143.
Ganem
D
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D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005051:Eubacterium / Q000235:genetics*; D015723:Gene Library; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014157:Transcription Factors / Q000235:genetics*
D015345:Oligonucleotide Probes; D012808:Sigma Factor; D014157:Transcription Factors