Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Temperature-induced changes in viability, diphenoloxidase and permeability of Mycobacterium leprae

Among mycobacteria secretion of the enzyme diphenoloxidase has been established as a property of Mycobacterium leprae. The antileprosy drug dapsone (DDS), which completely inhibits the enzyme from plant and mammalian sources, does not readily penetrate intact M. leprae. When the drug is complexed with polylysine, it easily permeates the bacteria and produces 100% inhibition of its diphenoloxidase, suggesting a permeability barrier of the cytoplasmic membrane of M. leprae to dapsone. In this study: (1) when the organisms, purified from fresh tissues of experimentally infected armadillos, were treated with dilute alkali or exposed to warmer temperatures, DDS penetrated the bacteria and inhibited the diphenoloxidase. Washing with trypsin had no effect. Dapsone easily permeated the bacilli, purified from tissues stored at 0 degrees C or at -80 degrees C. (2) Diphenoloxidase of freshly-prepared M. leprae was stimulated when the bacteria were exposed to 50 degrees C for 10 min; at 60 degrees C the activity decreased, and at 100 degrees C the enzyme was completely inactivated. When the enzyme was assayed at temperatures below 37 degrees C, the activity was considerably lower, indicating that M. leprae may not be a psychrophilic organism in this respect. (3) The bacteria exposed to 50 degrees C failed to multiply in mouse footpads. M. leprae remained viable in tissues stored at 0 degrees C or -80 degrees C; but when the bacteria purified from these tissues were frozen, they lost their viability. On the other hand, the organisms separated from fresh tissues remained viable when frozen at -80 degrees C. The inhibition of diphenoloxidase of M. leprae by dapsone could serve as an indirect method to assess the integrity of the bacterial cell membrane and to predict whether the bacteria would retain their viability on freezing.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions

Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40-45 degrees C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4 degrees C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9 degrees C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1 x SSC, 45 degrees C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4-11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of ATP generation and decay in Mycobacterium leprae in vitro

The intracellular ATP content of Mycobacterium leprae isolated from armadillo tissue was approximately 1.5 X 10(-16) g per bacillus. During in vitro incubation of bacilli at 4 degrees C, 33 degrees C or 37 degrees C there was an exponential decrease in ATP content, the rate depending on the medium and the temperature. M. leprae incorporated phosphate into ATP and into other nucleotide materials during in vitro incubation.     

Adenylate kinase activity in Mycobacterium leprae

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent Km values of M. leprae adenylate kinase for ADP and Mg2+ were 1 X 10(-4) M, respectively. The enzyme was heat-labile: loss of activity by 80% at 45 degrees C and over 90% at 60 degrees C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity.     

Effect of lipid physical state on the rate of peroxidation of liposomes.

The effect of cholesterol on the rate of peroxidation of arachidonic acid and 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) in dimyristoylphosphatidylcholine (DMPC) liposomes was examined above and below the phase transition temperature (Tm) of the lipid. The rate of peroxidation of arachidonic acid was more rapid below the phase transition temperature of the host lipid. At a temperature below the Tm (4 degrees C), increasing concentrations of cholesterol reduced the rate of peroxidation of arachidonic acid as judged by the production of thiobarbituric acid reactive substances. Above Tm (37 degrees C), cholesterol increased the rate of peroxidation of the fatty acid. Similarly, PAPC was peroxidized more rapidly at 4 degrees C than at 37 degrees C. However, cholesterol had little effect on the rate of peroxidation of PAPC at 4 degrees C. The rate of peroxidation of arachidonic acid was related to the lipid bilayer fluidity as judged by fluorescence anisotropy measurements of diphenylhexatriene. The rate of peroxidation increased slowly with increasing rigidity of the probe environment when the bilayer was relatively fluid and more rapidly as the environment became more rigid. The increase in the rate of peroxidation of arachidonic acid in the less fluid host lipid was unrelated to differences in iron binding or to transfer of arachidonic acid to the aqueous phase. Decreasing the concentration of arachidonic acid in DMPC to less than 2 mol% dramatically decreased the rate of peroxidation at 4 degrees C, suggesting that formation of clusters of fatty acids at 4 degrees C is required for rapid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.     

Temperature optimum of Mycobacterium leprae in mice

Shepard, Charles C. (Communicable Disease Center, Atlanta, Ga.). Temperature optimum of Mycobacterium leprae in mice. J. Bacteriol. 90:1271-1275. 1965.-Mycobacterium leprae multiplied most rapidly in foot pads of mice kept at an air temperature of 20 C. At air temperatures of 15 and 25 C, bacillary multiplication was slightly slower; at 10 and 30 C, distinctly slower; and at 4 and 35 C, no bacillary multiplication was detected. The temperature of the foot pad tissues of mice kept at an air temperature of 20 C averaged 27 to 30 C and that of mice kept at 10 and 30 C averaged about 25 and 36 C, respectively. These measurements indicate that the optimal temperature for the growth of M. leprae in mice is in the range several degrees above and below 30 C. The comparative effect of different air temperatures on the growth of M. leprae in foot pads was very similar to that found earlier for M. marinum in this site, thus indicating that the potential growth of M. leprae in vitro might have a similar optimum to M. marinum in vitro, i.e., 25 to 35 C. The optimal temperature for the growth of M. leprae appears to be the same in mice as in humans. It is pointed out that the temperature optimum of M. leprae may be a reflection of the fact that most of the bacilli being excreted into the environment, where they may reach new hosts, have multiplied in the nasal mucosa, a cool tissue.     

Characterization and taxonomic implications of the rRNA genes of Mycobacterium leprae.

The number of rRNA genes of Mycobacterium leprae was determined by restriction analysis of M. leprae total chromosomal DNA. A single set of rRNA genes was found. This set was subcloned from a cosmid library of M. leprae DNA into pUC13 and was characterized by restriction analysis and hybridization with Escherichia coli rRNA genes. The 16S, 23S, and 5S genes of M. leprae were clustered on a 5.3-kilobase DNA fragment. On one hand, restriction analysis of the set of rRNA genes showed the uniqueness of M. leprae among mycobacteria, but on the other hand, it suggested that M. leprae strains of several origins are very much alike. Quantitative hybridization studies between M. leprae rDNA and total DNA of various bacteria demonstrated a close relatedness between M. leprae and corynebacteria, nocardia, and mycobacteria, especially Mycobacterium tuberculosis.     

Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24

Abstract An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers. Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain. A 4.4 kb Bam HI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24. Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp. The highest similarity (∼78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E. coli promoter the S. lividans recA gene could partially complement an Escherichia coli recA mutant.     

The use of oligodeoxynucleotide probes in chaotrope-based hybridization solutions.

Hybridization solutions containing chaotropes may be used to modulate the thermal stability (Tm or Td) of oligodeoxynucleotide (ODN) duplexes or hybrids over a 90 degrees C range. Modulation of Td allows formulation of hybridization solutions that permit ambient temperature hybridization using most combinations of probe length, probe composition, target type, and facilitates development of convenient and rapid assay formats. The conditions required to achieve ODN duplex fidelity, and optimal yields of hybridized product, are described for trichloroacetate, thiocyanate, guanidinium salts and other chaotropic salts. The effects of different solid supports on Td are described. Also, a method is presented that uses chaotropic compounds to reduce background arising from signal ODN probes in a sandwich assay hybridization format.     

Mutation detection by stacking hybridization on genosensor arrays

A new strategy for analysis of point mutations using oligonucleotide array (genosensor) hybridization was investigated. In the new approach, a single-stranded target strand is preannealed with a labeled "stacking oligonucleotide," and then the partially duplex labeled target molecule is hybridized to an array of glass-tethered oligonucleotide probes, targeted to the region on the target immediately adjacent to the stacking oligomer. In this configuration, the base-stacking interactions between the "capture probe" and the contiguously stacking oligomer stabilize the binding of the target molecule to its complementary probe on the genosensor array. The temperature of hybridization can be adjusted so that the target molecule will bind to the glass-tethered probe only in the presence of the stacking oligomer, and a single mismatch at or near the terminal position ol the capture probe disrupts the stacking interactions and thereby eliminates or greatly reduces the hybridization. This stacking hybridization approach was investigated using a collection of synthetic targets, probes, and stacking oligonucleotides, which permitted identification of conditions for optimal base mismatch discrimination. The oligonucleotide probes were tethered to the glass using a simple, improved attachment chemistry in which a 3'-aminopropanol function introduced into the probe during chemical synthesis binds covalently to silanol groups on clean, underivatized glass. "Operating parameters" examined in the stacking hybridization system included length of capture probe, position, type and number of mismatches between the probe and the target, temperature of hybridization and length of washing, and the presence of terminal phosphate group in the probe, at its junction with the stacking oligomer. The results suggest that in the stacking hybridization configuration: 1. Optimal mismatch discrimination with 9-mer probes occurs at 45 degrees C, after which little or no improvement in mispair rejection occurred on lengthy continued washing at 45 degrees C. 2. At 25 degrees C optimal mismatch discrimination occurred with 7- or 8-mer probes, or with 9-mer probes containing an additional internal mismatch. 3. The presence of a phosphate group on the 5'-end of the glass-tethered probe had no general effect on mismatch discrimination, but influenced the relative stability of different mismatches in the sequence context studied. These results provide a motivation for continued development of the stacking hybridization technique for nucleic acid sequence analysis. This approach offers several advantages over the traditional allele-specific oligonucleotide hybridization technique, and is distinct from the contiguous stacking hybridization sitrategy that the Mirzabekov laboratory has introduced (Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov et al. (1996) Nucleic Acids Res. 24, 2998-3004).     

Determination of Herpes simplex virus DNA with the use of solid-phase methods for the detection of PCR products

To determine DNA of herpes simplex virus (HSV), types 1 and 2, the polymerase chain reaction (PCR) method was developed with the subsequent detection of amplification products by means of electrophoresis or the molecular hybridization of nucleic acids (MHNA). Two variants of MHNA have been compared: hybridization in the solution of a biotinylated probe with digoxigenin-labeled PCR with the subsequent sorption of hybridization complexes onto streptavidin-covered plates and solid-phase hybridization of digoxigenin-labeled PCR with a biotinylated probe. Effective hybridization was observed after the denaturation of targets at 95 degrees C in the solution of 50 mM NaOH, but not in neutral solutions. To increase the level of sensitivity of hybridization in solution, the exact selection of the amount of the probe was shown to be necessary, for both its excess and deficiency essentially decreased the method sensitivity. A decrease in the ionic power of hybridization solutions from 6 h SSC to 1 h SSC led to greater specificity of hybridization without a decrease in the method sensitivity. A rise in the temperature of hybridization and subsequent washing to 45 degrees C decreased the sensitivity of the method. The limit of the sensitivity PCR with electrophoretic detection was 30 HSV genome equivalents, and 10 genome equivalents in the presence MHNA in the solution and on the solid phase.     

Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite

The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization.