Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.

OBJECTIVE: To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex. DESIGN: One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles). RESULTS: DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature. CONCLUSION: Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.

Effect of metabolic inhibitors on nuclear pore formation during the HeLa S3 cell cycle.

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S(3) cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G(1) phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3-4 h after mitosis, i.e., about 1-2 h before S phase begins.