DNA fingerprinting of Mycobacterium tuberculosis by IS6110 restriction fragment length polymorphism analysis requires substantial high-quality DNA. We demonstrated that, despite extraction treatments that might be expected to inactivate this organism, M. tuberculosis remained viable during this process. These data suggest that the extraction of M. tuberculosis DNA should be performed within containment until complete.The standard method employed for DNA fingerprinting of Mycobacterium tuberculosis is IS6110 restriction fragment length polymorphism. This modality requires a large quantity (Ն2 g) of high-molecular-weight DNA (5-7). The DNA extraction protocols currently used for this purpose are based on chemical and enzymatic lysis of the bacterial cells followed by a chloroform-isoamyl alcohol-based DNA extraction, a somewhat lengthy process that raises methodological and biosafety issues. The cultivation of M. tuberculosis requires a biosafety level 3 containment facility and up to 4 weeks of culture time. The extraction necessitates a 2-to 3-day protocol harsh enough to lyse the bacteria yet sufficiently gentle to prevent DNA shearing.Due to the cumbersome nature of extraction protocols, there has been interest in determining at which point M. tuberculosis preparations can be considered inactivated and thus be safely removed from containment. Heating of the culture is widely used, and at least one report has deemed heating at 80°C as sufficient for inactivation (3). However, other reports have raised concerns as to the efficacy of heating (8) and even of further treatment with a combination of lysozyme and proteinase K (2). As such, the question of where and when these preparations may be safely manipulated remains unanswered. It is vital to address this issue, as improper inactivation and premature transfer of extraction mixtures from containment could result in the unnecessary exposure of laboratory personnel to M. tuberculosis.Here, the DNA from clinical M. tuberculosis isolates was extracted using standard protocols (all reagents were from Sigma-Aldrich unless noted). In brief, isolates were inoculated onto Lowenstein-Jensen (L-J) slants, and when luxuriant growth was apparent, all visible colonies were collected into 500 l Tris-EDTA buffer, pH 8.0, and heated for 20 min at 80°C. Lysozyme was then added to each tube (final concentration, 1 mg/ml), followed by incubation at 37°C for 2 h. Ten percent sodium dodecyl sulfate (final concentration, 1.1%) and proteinase K (final concentration, 0.2 mg/ml; Promega Inc.) were then added, and the tubes were vortexed gently and incubated for 20 min at 65°C. A mixture of N-acetyl-N,N,Ntrimethyl ammonium bromide (CTAB; final concentration, 40 mM) and NaCl (final concentration, 0.1 M) was added, followed immediately by the addition of NaCl alone (final concentration, 0.6 M). The tubes were then vortexed until the suspension turned milky and were incubated for 10 min at 65°C. Seven hundred fifty microliters of chloroform-isoamyl alcohol (24:1) was added to each tube, and ...

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Regulators of G-protein signaling (RGSs) are negative regulators of G-protein coupled receptor (GPCR)-mediated signaling that function to limit the lifetime of receptor-activated G(alpha)-proteins. Here we show that four mammalian RGSs differentially inhibit the activation of a FUS1--LacZ reporter gene by the STE2 encoded GPCR in yeast. In order to examine the role of the GPCR in modulating RGS function, we functionally expressed the human somatostatin receptor 5 (SST(5)) in yeast. In the absence of RGSs, FUS1--LacZ activation in response to somatostatin increased in a dose-dependent manner in cells expressing SST(5). In contrast to the results obtained with Ste2p, all RGSs completely inhibited SST(5)-mediated signaling even at concentrations of agonist as high as 10(minus sign5) M. The ability of RGSs to inhibit SST(5) signaling was further assessed in cells expressing modified Gpa1 proteins. Even though SST(5)-mediated FUS1--LacZ activation was 5-fold more efficient with a Gpa1p/G(i3alpha) chimera, response to somatostatin was completely abolished by all four RGSs. Furthermore, we demonstrate that RGS1, RGS2 and RGS5 have reduced ability to inhibit SST(5)-mediated activation of the RGS-resistant Gpa1p(Gly302Ser) mutant suggesting that the ability to interact with the G(alpha)-protein is required for the inhibition of signaling. Taken together, our results indicate that RGSs serve as better GAPs for Gpa1p when activated by SST(5) than when this G-protein is activated by Ste2p.

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