RESUMO
The insertion sequence IS986 (also known as IS6110) has been widely used for typing Mycobacterium tuberculosis isolates, due to the extensive multiple polymorphism shown using this probe. Although this polymorphism is presumed to be due to the mobility of IS986, transposition of this element has not previously been demonstrated in the laboratory. Using artificial composite transposons constructed in a vector unable to replicate in mycobacteria, we have succeeded in demonstrating the mobility of IS986 in M. smegmatis, apparently through cointegrate formation and transposition. The apparently random nature of IS986 insertions in M. tuberculosis is advantageous for transposon mutagenesis, although its efficient use will require more effective delivery systems.
Assuntos
Cromossomos Bacterianos , Elementos de DNA Transponíveis , Mutagênese Insercional/métodos , Mycobacterium/genética , Transfecção/métodos , Sequência de Bases , Southern Blotting , Análise Mutacional de DNA/métodos , DNA Bacteriano/análise , DNA Recombinante/biossíntese , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
Isolates of Mycobacterium tuberculosis from 88 patients with pulmonary tuberculosis in northern Tanzania were subjected to IS6110 restriction fragment length polymorphism (RFLP) analysis. Of 88 isolates, 73 fell into 11 groups of which 9 contained 2-5 isolates. Of 2 large homology groups one, group H (20 isolates), was isolated only from patients resident in the Kilimanjaro region, whereas 79% of isolates from other groups came from this region. A significant association (P = 0.023) was found between another group, M (24 isolates) and isolation from patients of the Masai tribe. The data from this pilot study support the idea that IS6110 RFLP analysis provides information which may be of value in the control of tuberculosis in Africa.
Assuntos
Mycobacterium tuberculosis/classificação , Polimorfismo de Fragmento de Restrição , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Tanzânia/epidemiologia , Tuberculose Pulmonar/epidemiologiaRESUMO
A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.
Assuntos
Genes Bacterianos , Mycobacterium avium/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis , Biblioteca Genômica , Dados de Sequência Molecular , Polimorfismo GenéticoRESUMO
Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.
Assuntos
Vacina BCG/genética , Sondas de DNA , Elementos de DNA Transponíveis , DNA Bacteriano/química , Mycobacterium bovis/genética , Sequência de Bases , Southern Blotting , Sondas de DNA/química , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.