Amplification and cloning of the Mycobacterium tuberculosis dnaA gene.
To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.
Rajagopalan
M
M
Department of Microbiology, University of Texas Health Center at Tyler 75710, USA.
Qin
M H
MH
Steingrube
V A
VA
Nash
D R
DR
Wallace
R J
RJ
Madiraju
M V
MV
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D003360:Cosmids; D017931:DNA Primers; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005798:Genes, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria