Nucleotide sequence and secondary structures of precursor 16S rRNA of slow-growing mycobacteria

Abstract

Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 165, 235 and 55 rRNA being present in that order. The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli. PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-I region separating the 16s rRNA and 235 rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu ' , Mycobacterium simiae and Mycobacterium marinum. The amplified DNA was sequenced. The sequence data, together with those obtained previously for Mycobacterium leprae and M. tuberculosis, were used to identify putative antitermination signals and RNase 111 processing sites within the leader region. Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized. A secondary structure for mycobacterial precursor-I 65 rRNA was devised, based on sequence homologies and homologous nucleotide substitutions. The 18 nucleotides a t the 5'-end of spacer-I have the capacity of binding sequences close to the 5'-and 3'-ends of mature 16s rRNA, suggesting that secondary structure is important to the maturation process. All the slowgrowers, including M. leprae, conform to the same scheme of secondary structure. The scheme proposed for M. tuberculosis is a variant of the main theme. The leader and spacer sequences may prove a useful supplement to 165 rRNA sequences in establishing phylogenetic relationships between very closely related species. 'M. lufu'appears t o be a close relative of M. intracellulare.

Paratuberculose em ruminantes no Brasil

“…François et al (1997), através desta técnica, observaram semelhança dos isolados de Map provenientes de bovinos com a doença de Johne e de pacientes com a doença de Crohn. Adicionalmente, alguns autores utilizaram genes do rRNA, 16S, 23S e 5S, para o estudo de Map (Ji et al 1994, Zakham et al 2012. Outros pesquisadores utilizaram o "multilocus enzyme electrophoresis" (MEE), cujo teste apresenta maior sensibilidade e rapidez em relação ao RFLP e PFGE, além de requerer menor quantidade de DNA para o teste (Lee et al 1994).…”

The ribosomal RNA (rrn) operons of fast-growing mycobacteria: primary and secondary structures and their relation to rrn operons of pathogenic slow-growers

“…3a). A product of identical size was obtained previously when E. coli was transformed with plasmid pJY104 (Ji et al, 1994a). The start of transcription of the r m operon of M .…”

“…tuberculosis, which contains nearly 650 bp of 16s rRNA gene coding region and 550 bp of its 5'-end flanking region, was introduced into the multiple cloning site of the vector pE J 106, which contains a chloramphenicol acetyltransferase (cat) gene lacking its own promoter. The pJY104 construct contains the promoter in the correct orientation for the expression of the cat gene (Ji et al, 1994a). The construct was used to transform both E. coli DH5aF' and M .…”

“…Plasmid DNA was isolated by standard methods exactly as described previously (Ji e t al., 1994a). Genomic DNA was isolated from mycobacteria by the method of Katoch & , avoiding the use of the French pressure cell (see Ji et al, 1994a). RNA was hydrolysed by incubation with 20000 units of RNase T1 (Life Technologies) overnight at 37 "C in TE buffer.…”

“…This procedure reduces the possible errors caused by PCR amplification (Saiki e t al., 1988). DNA sequences were determined by the dideoxy chain-termination procedure using ["SIdATP-as, as described by Ji et al (1994a).…”