We previously demonstrated (Guo et al., 1987. Nucl. Acids Res. 15, 7081-7090) that purified proheads of bacteriophage phi 29 contain an RNA of 120 bases which is essential for DNA packaging. Here we report that this RNA exists primarily as a polymer of ca. 174 residues in phage-infected cells and that ca. 54 bases are cleaved from its 3'-terminus by adventitious nucleases during the purification of proheads. The long and short forms of the RNA had similar activity in in vitro DNA packaging and phage assembly. We report the sequence of the long form of the RNA and show that similar long and short forms can be isolated from the proheads of the phi 29 relatives phi 21, phi 15 and SF5. The concentration dependence in the reconstitution of RNA-free proheads suggests that one copy of the RNA is sufficient to restore DNA packaging activity to RNA-free proheads. However, quantitative measurements indicate that 5 to 6 copies of the RNA are present on proheads isolated from phage-infected cells.
Methyl parathion hydrolase (MPH) from a methyl parathion-degrading Burkholderia cepacia indigenous to Thailand was purified to apparent homogeneity by three steps of column chromatography using Resource S, Sephadex G100, and Octyl Sepharose 4FF columns. Its molecular mass was determined to be 35 kDa, and the pI to be 8.5. The recombinant plasmid pGT1, containing the MPH-encoding gene, mpdB, cloned into pGEX-4T-2 was over-expressed in Escherichia coli as GST-MPH fusion protein. The recombinant MPH was purified to homogeneity by a single step, using GSTPrep FF affinity column, with the molecular mass identical to that of the native enzyme. The purified enzyme had the specific activity of about 1,600 unit mg(-1) protein and the yield of about 75%, a 39-fold increase in recovery compared to that of the native enzyme. The optimal temperature and pH were 25°C and 9.0, respectively. The MPH was stable, with its activity unchanged for 48 h at 4°C, and reduced to 50% after 5 h and to 45% after 48 h at 25°C. The enzyme activity remained 80-90% after 8-15 h at pH 6-7. Cd(2+), Co(2+), and Zn(2+) ions at the concentration of 1 mM enhanced the activity; while sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and ethylenediaminetetraacetate (EDTA) reduced it. The enzyme also showed cross reactivity with other insecticides within the organophosphate group, and the kinetic parameters for individual substrates were investigated. Since MPH from B. cepacia has wide potential applications in detoxification and detection of organophosphate compounds, this study provides important basis for its future use.
PCR amplification of the 531-bp fragment of the Mycobacterium leprae pra gene in fresh biopsy and slit skin smear samples was evaluated for its usefulness in the detection of leprosy bacilli in patients in Thailand. In multibacillary patients, 87.1% (27 of 31) of biopsy specimens and 41.9% (13 of 31) of slit skin smear specimens were positive by PCR, whereas in paucibacillary patients, 36.4% (8 of 22) of biopsy specimens and 18.2% (4 of 22) of slit skin smear specimens yielded detectable PCR amplification. Compared with other diagnostic procedures, PCR showed a clear advantage over both microscopic examination of slit skin smears and serologic detection of anti-phenolic glycolipid 1 antibody, especially in paucibacillary patients when bacterial indexes were 0 and seropositivity was only 6.25%. PCR was also evaluated for its potential to help monitor bacterial clearance in some of these patients during chemotherapeutic treatment. The PCR results on slit skin smear samples at 1, 3, and 6 months of chemotherapy showed that the number of PCR-positive cases of both multibacillary and paucibacillary types decreased sequentially. The results of this study are encouraging. However, investigation of a larger number of clinical specimens with an improvement in PCR methods, especially on slit skin smears, needs to be done before PCR can be established as a diagnostic procedure for leprosy patients and subclinical cases or as a tool for drug assessment. Leprosy, caused by Mycobacterium leprae, is still considered a major health problem in many developing countries. It is a chronic infectious disease of skin, nasal cavity, and peripheral nerves which eventually leads to disability, disfiguration, and socioeconomic problems (9, 11). There is no useful serologic test for the diagnosis of leprosy (6, 11). Early detection of the causative microorganisms is, therefore, the key element to early identification and treatment of patients and subclinical cases before the disease progresses and neural involvement occurs. These organisms are not cultivable on artificial media, and attempts to identify them by inoculating a susceptible animal such as the armadillo (13) and mouse footpads (3, 10, 14) have proved cumbersome and time-consuming. The routine bacteriologic diagnostic test, the demonstration of acidfast bacilli in skin smears (25), is not sufficiently sensitive or specific. This study was undertaken to evaluate the use of PCR as a means of diagnosis of leprosy and its potential to help assess the bacterial load reduction in patients during the course of chemotherapeutic treatment. MATERIALS AND METHODS Specimen collection. Slit skin smears and punch biopsies were obtained according to standard procedures (25) from 53 untreated leprosy patients at three institutes under the Department of Communicable Disease Control, Thailand: two skin clinics in Bangkok and a leprosy hospital in Samutprakarn province. Specimens were collected between October 1992 and March 1994. These patients were of both the paucibacillary (PB; having a negati...
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