Most Mycobacterium tuberculosis strains contain multiple copies of insertion sequence(IS) 6110. Since the integration of IS 6110 occurs randomly, IS 6110 sequences are found in the chromosomes with varying numbers and locations. The restriction
fragment
length polymorphism (RFLP) analysis of Korean M. tuberculosis isolates were performed using a part of IS 6110 as a probe. Seventeen clinical isolates showed highly diverse RFLP patterns. The DNA fragment of 1.6kb, 2.4kb, and 3.5kb were found very
frequently among which the 3.5kb fragment was the most frequent one.
To understand the mechanisms of this phenomenon, the 3.5kb RFLP fragment was cloned into pUC18 and the nucleotide sequence of the 3' -flanking region of the IS 6110 DNA was determined. The DNA fragment attached to IS 6110 at this site had 3
cleavage
sites for NaeI. The sizes of NaeI fragment were 320bp, 430bp, and 5.2kb. Either 320bp or 430bp NaeI fragments hybridized with only 3.5kb fragment, as expected, but also 2.4kb, 4.7kb. and 11.7kb fragments in M. tuberculosis isolates which showed
3.5kb
RFLP band in the RFLP analysis.
The nucleotide sequence of 320bp NaeI fragment showed characteristic GGC and GGT repeats, inverted repeat, and characteristic chi octomer sequence which was know to be used by Rec BCD proteins involved in the recombination events. These strongly
suggested that the multiple presence of hybridizing bands with either 320bp or 430bp NaeI DNA was caused by the recombination events of the sequences.
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