Genotypic determination of Mycobacterium tuberculosis antibiotic resistance using a novel mutation detection method, the branch migration inhibition M. tuberculosis antibiotic resistance test.

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413-424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in M. tuberculosis, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426-5430, 1994). RMP and PZA resistances are associated with multiple mutations within the rpoB and pncA genes, respectively. M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a "blind study." Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of M. tuberculosis clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).

Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.

A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures. Spontaneous DNA branch migration results in dissociation of these structures when the test and reference sequences are identical. Any base substitution, deletion or insertion inhibits branch migration and produces stable cruciform structures. When suitable ligands are attached to the PCR primers, the cruciform structures can be detected by standard immunochemical methods. This approach was tested using several commonly occurring mutations within the human cystic fibrosis gene. New methods for increasing the specificity of PCR amplifications are described that were used for successful mutation analysis.

Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method.

Luminescent oxygen channeling assay (LOCI) is a homogeneous immunoassay method capable of rapid, quantitative determination of a wide range of analytes--including high and very low concentrations of large and small molecules, free (unbound) drugs, DNA, and specific IgM. Assays have been carried out in serum and in lysed blood. Reliable detection of 1.25 microU/L thyrotropin (TSH) and 5 ng/L hepatitis B surface antigen (HBsAg) corresponds to detection limits approximately 3- and approximately 20-fold lower, respectively, than those of the best commercially available assays. An assay of chorionic gonadotropin is capable of quantification over a 10(6)-fold range of concentrations without a biphasic response. Latex particle pairs are formed in the assay through specific binding interactions by sequentially combining the sample and two reagents. One particle contains a photosensitizer, the other a chemiluminescer. Irradiation causes photosensitized formation of singlet oxygen, which migrates to a bound particle and activates the chemiluminescer, thereby initiating a delayed luminescence emission. Assay times range from 1 to 25 min.

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

Rapid, simple, and reliable doctor's office test for antibodies to human immunodeficiency virus 1 in serum.

This "Unit Test Method" assay for detecting anti-human immunodeficiency virus 1 antibody is suitable for nonlaboratory testing and has a sensitivity comparable with that of present enzyme immunoassay methods. The method does not require instrumentation, gives a result in less than 15 min, and incorporates a procedural control. Little technical expertise and hands-on time are required of the user.

Heparan sulfate proteoglycans from mouse mammary epithelial cells: localization on the cell surface with a monoclonal antibody.

Mouse mammary epithelial cells, of the normal murine mammary gland (NMuMG) cell line, bear a heparan sulfate-rich proteoglycan (HSPG) on their surfaces. A hybridoma (281-2) secreting a monoclonal antibody that recognizes this HSPG was produced by fusion of SP-2/0 myeloma cells with spleen cells from rats immunized with NMuMG cells. The 281-2 monoclonal antibody is directed against the core protein of the cell surface HSPG, as demonstrated by (a) recognition of the isolated proteoglycan but not its glycosaminoglycan chains, (b) co-localization of 281-2-specific antigen and radioactive cell surface HSPG on gradient polyacrylamide gel electrophoresis and on isopycnic centrifugation, and (c) abolition of immunofluorescent staining of the NMuMG cell surface by the intact, but not the protease-digested ectodomain of the cell surface HSPG. The antibody is specific for cell surface HSPG and does not recognize the HSPG that accumulates extracellularly beneath the basal cell surface. Therefore, the 281-2 antibody may be used to isolate the cell surface HSPG and to explore its distribution in tissues.

Synthesis and possible role of proteoglycans during Volvox development.

During the embryonic development of Volvox the cells synthesize proteoglycans of relatively small molecular size up to 14 h after inversion. In the following stages there is a gradual transition towards larger forms, due to an increase in sulfated sugar moieties. The presence of sulfate in the medium was obligatory for this transition. None of these molecules could be dissociated in 4.0 M GuHCl, neither could they be digested by glycosidases. Of all proteases examined subtilisin, a bacterial protease, digested the proteoglycans. The possible role of proteoglycans during Volvox morphogenesis has been examined. Somatic proteoglycans caused spheroids to start their swimming earlier, although they inhibited their expansion. Spheroids cultured in sulfate-free medium start their swimming 24 h later than controls, but the addition of somatic proteoglycans to the sulfate-free medium could induce motility almost as well as in the normal forms. Embryonic weight proteoglycans caused aggregation of the spheroids, an effect that also appeared in the sulfate-free medium.

Altered calmodulin activity in fluphenazine-resistant mutant strains. Pleiotropic effect on development and cellular organization in Volvox carteri.

Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild-type and fluphenazine-resistant strains of the multicellular alga Volvox carteri, were tested for the ability to activate Ca2+-ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild-type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+-ATPase. The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular organization and hormone-independent commitment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild-type cells is shown to block the morphogenic process of embryo inversion and to arrest motility. The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous regulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes.

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.

Effect of carbon source and the role of cyclic adenosine 3',5'-monophosphate on the Caulobacter cell cycle.

The expression of cell cycle events in Caulobacter crescentus CB13 has been shown to be associated with regulation of carbohydrate utilization. Growth on lactose and galactose depends on induction of specific enzymes. Prior growth on glucose results in a delay in enzyme expression and cell cycle arrest at the nonmotile, predivisional stage. Dibutyryl cyclic adenosine 3',5'-monophosphate (AMP) was shown to stimulate expression of the inducible enzymes and, thus, the initiation of the cell cycle. beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. Furthermore, carbon source starvation results in accumulation of the cells at the predivisional stage. The cell cycle arrest therefore results from nutritional deprivation and is analogous to the general control system exhibited by yeast (Hartwell, Bacteriol. Rev. 38:164-198, 1974; Wolfner et al., J. Mol. Biol. 96:273-290, 1975), which coordinates cell cycle initiation with metabolic state. Transfer of C. crescentus CB13 from glucose to mannose did not result in a cell cycle arrest, and it was demonstrated that this carbon source is metabolized by constitutive enzymes. Growth on mannose, however, is stimulated by exogenous dibutyryl cyclic AMP without a concomitant increase in the specific activity of the mannose catabolic enzymes. The effect of cyclic AMP on growth on sugars metabolized by inducible enzymes, as well as on sugars metabolized by constitutive enzymes, may represent a regulatory system common to both types of sugar utilization, since they share features that differ from glucose utilization, namely, temperature-sensitive growth and low intracellular concentrations of cyclic guanosine 3',5'-monophosphate.

Effect of 3':5'-cyclic GMP derivatives on the formation of Caulobacter surface structures.

Exogenous derivatives of 3':5'-cyclic GMP, 8-bromo- and N2,O2'-dibutyryl cyclic GMP, coordinately repress surface structure differentiation in Caulobacter crescentus. Growth in the presence of cyclic GMP derivatives resulted in the loss of flagella and pili formation and concomitant resistance to both DNA phage phiCbK and RNA phage phiCb5 infection without affecting growth rate, stalk formation, and equatorial cell division. The effect of cyclic GMP derivatives was shown to be the repression of synthesis of specific structural proteins. This effect could be reversed by exogenous N6,O2'-dibutyryl 3':5'-cyclic AMP, and mutants resistant to repression by cyclic GMP derivatives exhibited a pleiotropic phenotype affecting a cyclic AMP-mediated event.

A pleiotropic mutation affecting expression of polar development events in Caulobacter crescentus.

A developmental mutant of C. crescentus with altered polar surface structures has been isolated. The mutant lacks a flagellum and pili, and may have an abnormal DNA phage receptor site. A revertant regains the normal structures simultaneously. This point mutation allows normal flagellin synthesis, stalk formation, equatorial cell division, and rate of growth. The mutant phenotype indicates that the assembly of the polar surface structures is coordinately regulated and independent of mechanisms regulating cell polarity and division.