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1.
Appl Microbiol Biotechnol ; 75(3): 633-45, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17318539

RESUMO

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.


Assuntos
Fermentação , Nitritos/metabolismo , Saccharomycetales/metabolismo , Tabaco/metabolismo , Northern Blotting , Southern Blotting , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , Nitrosaminas/metabolismo , Filogenia , RNA Ribossômico 18S/genética , Saccharomycetales/classificação , Saccharomycetales/genética , Fatores de Tempo
2.
Int J Food Microbiol ; 103(3): 295-304, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16099314

RESUMO

The subtype of the Saccharomyces cerevisiae yeast species known as S. cerevisiae Hansen CBS 5926 was formerly believed to be a separate species, Saccharomyces boulardii. It is widely considered non-pathogenic and is used as a probiotic agent for treatment and prevention of diarrhea. The biological properties of Saccharomyces spp. show considerable intraspecies variability and the beneficial properties of probiotic yeasts are considered strain-specific. Septicemia and fungemia caused by S. boulardii have recently been described in both immunocompromised and immunocompetent patients receiving biotherapy with this yeast. It cannot be distinguished from other S. cerevisiae strains by phenotypic criteria, so identification of these infections requires molecular typing. To identify the most effective approach for distinguishing S. boulardii, we typed 35 isolates of S. cerevisiae, of which 27 were from various clinical specimens and 8 were isolates of S. boulardii (6 obtained from probiotic preparations and 2 from clinical specimens) using four different molecular methods, two based on PCR-restriction enzyme analysis or sequencing of rDNA spacer regions, the third based on microsatellite polymorphism analysis of the S. cerevisiae genes YKL139w and YLR177w, and the last based on hybridization analysis with retrotransposon Ty917. Several clinical isolates appeared to be identical to one or more other isolates with the first three methods used, whereas with the Ty917 hybridization method all of the isolates tested appeared to be very heterogeneous. The eight S. boulardii isolates were clearly distinguishable from the clinical S. cerevisiae isolates only with Ty917 hybridization and microsatellite DNA analyses. In the latter method, the eight S. boulardii isolates exhibited an allelic variant at one of loci tested that was not shared with any other strain. Our results suggest that microsatellite polymorphism analysis of the YKL139w and YLR177w genes, as well as the analysis by Ty917 hybridization, are the most useful tools for a correct identification of S. boulardii strains.


Assuntos
DNA Fúngico/análise , Probióticos , Saccharomyces/classificação , Saccharomyces/genética , Southern Blotting , Repetições de Microssatélites , Técnicas de Tipagem Micológica , Filogenia , Polimorfismo Genético , Saccharomyces/patogenicidade , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Especificidade da Espécie
3.
J Biol Chem ; 279(43): 44847-57, 2004 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-15308633

RESUMO

The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids. Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse. Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the beta-subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis. We present here our studies on four putative accD genes found in C. glutamicum. Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid. Overexpression of accD1 and accBC, encoding the carboxylase alpha-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA. Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed. These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs. Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and alpha-branch directed by the pks-encoded polyketide synthase. This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.


Assuntos
Carbono-Carbono Ligases/química , Corynebacterium glutamicum/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Policetídeo Sintases/química , Southern Blotting , Proliferação de Células , Escherichia coli/metabolismo , Ácidos Graxos/química , Deleção de Genes , Genoma Bacteriano , Genótipo , Metabolismo dos Lipídeos , Lipídeos/química , Malonil Coenzima A/química , Modelos Biológicos , Modelos Genéticos , Mutação , Peptídeos/química , Fenótipo , Filogenia , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Fatores de Tempo
4.
Fontilles, Rev. leprol ; 24(5): 441-453, mayo 2004. tab, ilus
Artigo em Es | IBECS | ID: ibc-34632

RESUMO

Se ha desarrollado un ensayo colorimétrico de hibridación en microplacas para simplificar la detección de Mycobacterium leprae en muestras clínicas. La técnica detecta los productos amplificados por un ensayo muy sensible RT-PCR con diana sobre una secuencia especie-específica del rRNA 16S bacteriano. El test detectó hasta 10 bacilos aislados de los nódulos linfáticos de ratones desnudos infectados o biopsias cutáneas humanas. La sensibilidad para el diagnóstico en nuestras clínicas se evaluó en 58 biopsias cutáneas de 58 pacientes de lepra sin tratar. El ensayo detectó amplificados RT-PCR de M. leprae en el 100 por ciento de las biopsias de pacientes con lepra multibacilar y el 80 por ciento de biopsias de pacientes paucibacilares, con una sensibilidad total de 91.3 por ciento. El test resultó ser muy específico ya que no se detectaron amplificaciones en las biopsias de pacientes normales o afectados de otras enfermedades distintas a la lepra. La variante colorimétrica es más rápido, sensible y simplifica la detección de los amplificados RT-PCR comparado con el análisis por Southern blot. Puede ser útil para el diagnóstico de los casos difíciles de lepra y como el RNA se degrada rápidamente después de la inactivación celular siendo útil para la evaluación de la respuesta al tratamiento y distinción de recidivas de reacción (AU)


Assuntos
Adulto , Feminino , Masculino , Pessoa de Meia-Idade , Humanos , Colorimetria/métodos , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/patogenicidade , Biópsia/métodos , Sensibilidade e Especificidade , RNA Complementar , Ribonucleases/isolamento & purificação , Ribonucleases , Eletroforese em Gel de Ágar/métodos , Southern Blotting/métodos , Reação em Cadeia da Polimerase/métodos , Colorimetria/classificação , Colorimetria/tendências , Meios de Cultura/isolamento & purificação , Rifampina , Ofloxacino
5.
Yeast ; 21(2): 119-26, 2004 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-14755637

RESUMO

A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.


Assuntos
Genes Fúngicos , Glicerolfosfato Desidrogenase/genética , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Impressões Digitais de DNA , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , DNA Fúngico/metabolismo , Teste de Complementação Genética , Glicerol-3-Fosfato Desidrogenase (NAD+) , Dados de Sequência Molecular , Mutagênese Insercional , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Alinhamento de Sequência
6.
J Bacteriol ; 185(23): 6870-82, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617651

RESUMO

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.


Assuntos
Epotilonas/genética , Complexos Multienzimáticos/genética , Mycobacterium/genética , Sequência de Aminoácidos , Toxinas Bacterianas , Southern Blotting , Epotilonas/análise , Humanos , Macrolídeos , Dados de Sequência Molecular , Complexos Multienzimáticos/análise , Mycobacterium/enzimologia , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Alinhamento de Sequência
7.
Antonie Van Leeuwenhoek ; 84(2): 81-8, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14533711

RESUMO

Pulse field gel electrophoresis karyotypes of 41 strains of the genus Debaryomyces, including 35 strains confirmed as D. hansenii species by D1/D2 ribosomal DNA sequence analysis, were performed. Electrophoretic karyotypes of the 41 strains exhibited 4 to 10 chromosomal bands ranging between 0.7 Mb and 4.2 Mb. Among D. hansenii species, the patterns of strains obtained from the CBS collection and cheese isolates differed strongly from D. hansenii var. hansenii CBS767T. Both D. hansenii var. hansenii and D. hansenii var. fabryii showed chromosome length polymorphism. Electrophoretic karyotypes of the D. hansenii strains were analyzed by Southern hybridization with various species-specific probes isolated from D. hansenii var. hansenii CBS767T. Repeated sequences including the F01pro, M18pro, the Ty1-copia retrotransposon Tdh5 and hypothetical telomeric sequence hybridized to several chromosomal bands, while a D1/D2 probe derived from the large ribosomal sub-unit hybridized only to the largest chromosome. Unique probes such as those hybridizing to actin ACT1, glycerol-3-phosphate dehydrogenase GPD1 and beta-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon.


Assuntos
Cromossomos Fúngicos/genética , Polimorfismo Genético , Saccharomycetales/genética , Sequência de Bases , Southern Blotting , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Cariotipagem , Saccharomycetales/classificação , Saccharomycetales/isolamento & purificação , Especificidade da Espécie
8.
J Bacteriol ; 183(20): 6009-16, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11567001

RESUMO

Analysis of proteins recovered in the S100 precipitate fraction of Streptomyces griseus after ultracentrifugation led to the identification of a 52-kDa protein which is produced during the late growth phase. The gene (eshA) which codes for this protein was cloned from S. griseus, and then its homologue was cloned from Streptomyces coelicolor A3(2). The protein was deduced to be 471 amino acids in length. The protein EshA is characterized by a central region that shows homology to the eukaryotic-type cyclic nucleotide-binding domains. Significant homology was also found to MMPI in Mycobacterium leprae, a major antigenic protein to humans. The eshA gene mapped near the chromosome end and was not essential for viability, as demonstrated by gene disruption experiments, but its disruption resulted in the abolishment of an antibiotic (actinorhodin but not undecylprodigiosin) production. Aerial mycelium was produced as abundantly as by the parent strain. Expression analysis of the EshA protein by Western blotting revealed that EshA is present only in late-growth-phase cells. The eshA gene was transcribed just preceding intracellular accumulation of the EshA protein, as determined by S1 nuclease protection, indicating that EshA expression is regulated at the transcription level. The expression of EshA was unaffected by introduction of the relA mutation, which blocks ppGpp synthesis.


Assuntos
Antraquinonas/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Genes Bacterianos , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Southern Blotting , Clonagem Molecular , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição Genética
9.
Microbiology (Reading) ; 147(Pt 6): 1557-1564, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11390686

RESUMO

Expression of a gene encoding a novel protein antigen of 40 kDa (p40) was detected in IS901(+) strains of Mycobacterium avium, but not in any other species or subspecies of Mycobacterium tested, including IS901(-) M. avium and the other members of the M. avium complex. Although Southern hybridization revealed that the p40 gene is widely distributed within the genus, expression of the antigen could not be detected on Western blots of mycobacterial cell lysates. Nucleotide sequence analysis of the cloned p40 gene, and a database search, revealed high levels of sequence identity with a homologous gene in IS901(-) M. avium, M. avium subsp. paratuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium smegmatis and Mycobacterium tuberculosis. Further analysis of upstream sequences identified a putative promoter region. The p40 gene is the first example of a gene that is widely distributed within the genus Mycobacterium but expressed only in association with the presence of a genomic insertion element, in this case IS901, in strains of M. avium isolated from birds and domestic livestock.


Assuntos
Antígenos de Bactérias/genética , Elementos de DNA Transponíveis , Genes Bacterianos , Mycobacterium avium/genética , Animais , Antígenos de Bactérias/metabolismo , Sequência de Bases , Southern Blotting , Western Blotting , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Epitopos , Regulação Bacteriana da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mycobacterium avium/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
10.
J Clin Microbiol ; 39(7): 2485-93, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11427558

RESUMO

Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.


Assuntos
Antígenos de Bactérias , Antígenos de Bactérias/imunologia , Proteínas de Bactérias , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/diagnóstico , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Sequência de Bases , Southern Blotting , Western Blotting , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes , Análise de Sequência de DNA , Tuberculose Pulmonar/microbiologia
11.
Tuber Lung Dis ; 79(5): 299-308, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10707258

RESUMO

SETTING: Molecular Research Laboratory, Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital. OBJECTIVE: Characterize Mycobacterium tuberculosis homologue of the Streptomyces coelicolor, sporulation specific, whiB regulatory gene. DESIGN: The M. tuberculosis whiB3 gene was isolated by enriched cloning of a 2.8 kb BamHl fragment to which the S. coelicolor whiB gene hybridized. Expression of the gene was analysed by S1 nuclease analysis and promoter studies. RESULTS: An open reading frame within the 2.8 kb BamHl fragment was identified as the M. tuberculosis whiB3 gene, one of four whiB homologues in the M. tuberculosis genome. The deduced amino acid sequence has a 92% identity with a M. leprae protein, and 32% identity with the S. coelicolor WhiB protein. S1 nuclease analysis showed that the M. tuberculosis whiB3 gene is constitutively expressed by the cells in liquid culture. Primer extension analysis revealed three transcriptional start sites. Expression from the three potential promoters is growth phase-dependent. CONCLUSION: The M. tuberculosis whiB3 gene is expressed throughout growth, but expression from the individual promoters is growth phase dependent.


Assuntos
Proteínas de Bactérias/genética , Mycobacterium tuberculosis/genética , Streptomyces/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/química , Southern Blotting , Humanos , Mycobacterium/genética , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estrutura Secundária de Proteína , RNA Bacteriano/genética , RNA Mensageiro/genética , Fatores de Transcrição/química
12.
Immunology ; 86(4): 519-24, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8567015

RESUMO

Although mice acquire only a slight degree of protection against tuberculosis by immunization with Mycobacterium leprae (M. leprae) hsp65 in incomplete Freund's adjuvant, protection is substantial following immunization by injection with J774 macrophage-like tumour cells that express the protein from the mycobacterial gene via a retroviral vector. We here took the same vector, used it to transfect the gene into normal murine bone marrow cells in vitro, and then used the transfected cells to reconstitute haematopoiesis in lethally irradiated mice. Bone marrow-cell clonal expansion and production of the protein in vivo resulted in specific delayed-type hypersensitivity and protection against challenge with Mycobacterium tuberculosis (M. tuberculosis) in about half of recipients. Counts of live bacteria in liver at 3 weeks were fivefold lower in delayed-type hypersensitivity (DTH)-positive than in DTH-negative mice. Other mice acquired neither DTH nor protection despite the presence of the protein in peripheral blood.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias , Transplante de Medula Óssea , Chaperoninas/imunologia , Mycobacterium leprae/imunologia , Tuberculose/prevenção & controle , Animais , Southern Blotting , Chaperonina 60 , Feminino , Genes Bacterianos , Hipersensibilidade Tardia/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/genética , Retroviridae/genética , Transfecção , Irradiação Corporal Total
13.
Scand J Immunol ; 40(2): 165-70, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8047837

RESUMO

The gene for the extracellular alpha antigen of Mycobacterium scrofulaceum (S-alpha) was cloned by using the alpha antigen gene fragments of Mycobacterium bovis BCG as probes. The complete nucleotide sequence was determined. The gene was expressed in Escherichia coli. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein. The deduced amino acid sequences were highly homologous to the alpha antigen of the species of other mycobacteria. Interestingly, the alpha antigens of MAIS complex (Mycobacterium avium-Mycobacterium intracellulare-M. scrofulaceum) were closely homologous even at the C-terminal regions which were variable among those of M. bovis BCG, Mycobacterium leprae and Mycobacterium kansasii.


Assuntos
Antígenos de Bactérias/genética , Genes Bacterianos , Mycobacterium scrofulaceum/genética , Sequência de Aminoácidos , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/química , Sequência de Bases , Southern Blotting , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos
14.
Int J Lepr Other Mycobact Dis ; 62(2): 237-44, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7519225

RESUMO

A genomic library of Mycobacterium leprae in the expression vector lambda gt11 was screened with rabbit polyclonal hyperimmune antiserum elicited with a sonicated extract of M. leprae. Numerous reactive clones were isolated by this immunoscreening, indicating a broad antibody response of the rabbit. None of the recombinant clones was reactive with monoclonal antibodies to the previously well-characterized M. leprae recombinant clones. One of the clones isolated, clone A, encoded a large, 132-143-kDa beta-galactosidase fusion protein expressing an epitope of M. leprae. Monospecific antibodies eluted from this fusion protein reacted on Western blots with a 64-kDa M. leprae protein. The DNA of this clone was shown to be distinct from the gene encoding the well-characterized immunodominant 65-kDa protein by DNA hybridization. We have identified a new lambda gt11 recombinant clone encoding a fusion protein with a 64-kDa protein. This protein is recognized by the humoral immune response of the rabbit in response to a challenge with an M. leprae cell extract.


Assuntos
Antígenos de Bactérias/genética , Mycobacterium leprae/imunologia , Antígenos de Bactérias/análise , Antígenos de Bactérias/biossíntese , Bacteriófagos , Southern Blotting , Western Blotting , Clonagem Molecular , DNA Bacteriano/análise , DNA Bacteriano/química , Eletroforese em Gel de Poliacrilamida , Epitopos/análise , Epitopos/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Humanos , Lisogenia , Mycobacterium leprae/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
15.
Int J Dermatol ; 32(10): 710-3, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7693602

RESUMO

BACKGROUND: Diagnosis of paucibacillary leprosy is often difficult. A method that could confirm the diagnosis is the polymerase chain reaction (PCR) of M. leprae DNA. This reaction was applied to biopsied tissues of leprotic patients to determine the suitability and sensitivity of the reaction. METHODS: Biopsy samples were taken from previously untreated patients with multibacillary (5 patients) and paucibacillary (3 patients) leprosy, fixed in formalin, and embedded in paraffin. DNA was extracted from paraffin blocks and PCR applied. The sensitivity of the PCR method was tested by using the serially diluted DNA sample as the template. RESULTS: All eight patients showed a positive PCR for M. leprae DNA. The sensitivity was such that a single organism of M. leprae, as counted by staining of the acid-fast bacilli was identified by the PCR. CONCLUSIONS: The PCR method is simple, sensitive, specific, and does not require the use of radioisotopes. It can be applied to the unequivocal diagnosis of paucibacillary leprosy which is difficult by other means. The diagnosis can be obtained within 10 hours.


Assuntos
DNA Bacteriano/análise , Formaldeído , Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/patologia , Mycobacterium leprae/genética , Inclusão em Parafina , Reação em Cadeia da Polimerase , Fixação de Tecidos , Southern Blotting , DNA Bacteriano/isolamento & purificação , Amplificação de Genes , Humanos , Hanseníase Virchowiana/microbiologia , Hanseníase Tuberculoide/microbiologia , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Coloração e Rotulagem
16.
J Med Microbiol ; 39(4): 298-304, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8411091

RESUMO

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.


Assuntos
DNA Bacteriano/análise , Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Sequência de Bases , Southern Blotting , Humanos , Hanseníase Dimorfa/diagnóstico , Hanseníase Virchowiana/diagnóstico , Hanseníase Tuberculoide/diagnóstico , Leucócitos Mononucleares/microbiologia , Linfa/microbiologia , Dados de Sequência Molecular , Mycobacterium leprae/genética , Sensibilidade e Especificidade , Pele/microbiologia
17.
Int J Lepr Other Mycobact Dis ; 61(2): 227-35, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8371032

RESUMO

Screening of a lambda gt11 genomic library has been used as an approach for molecular cloning of the Mycobacterium tuberculosis repetitive DNA shown to be present on a previously described 5.6-kb Alu I genomic fragment. The recombinant clone R18.8.2, which strongly hybridized with the radiolabeled 5.7-kb Alu fragment, carried two Eco RI inserts of 2 kb and 1.4 kb in size. Southern hybridization of each of these fragments to restriction endonuclease-cleaved M. tuberculosis DNA clearly demonstrated the 2 kb to contain the repetitive DNA sequence, while the 1.4 kb is represented in a single copy. When replica plaque lifts from the genomic library were probed, the 5.6-kb genomic fragment and the cloned 2-kb repetitive insert hybridized to an identical number of plaques, indicating the similarity and the high copy number of the repeating unit shared by the two fragments. Restriction mapping and Southern hybridization patterns indicated that the 2-kb repetitive and the 1.4-kb single-copy DNA sequences originated from a contiguous piece of genomic DNA. Both fragments were found to be unique to members of the M. tuberculosis complex, except that the 2-kb insert exhibited a weak hybridization with M. kansasii DNA. Finally, a 169-bp region from one end of the single-copy sequence has been amplified by polymerase chain reaction (PCR) in a manner specific to members of the M. tuberculosis complex. The sensitivity of the PCR was such that as few as 9-10 bacilli could be detected.


Assuntos
DNA Bacteriano/análise , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Sequência de Bases , Southern Blotting , Clonagem Molecular , Eletroforese em Gel de Ágar , Genoma Bacteriano , Biblioteca Genômica , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Sondas de Oligonucleotídeos , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e Especificidade
18.
J Appl Bacteriol ; 74(5): 549-56, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-8098028

RESUMO

Chromosomal DNA of nine strains of the Lactobacillus acidophilus (Hansen and Mocquot) group in which heterogeneity had previously been revealed by biochemical characteristics and DNA-DNA hybridization studies were further investigated by restriction analysis, Southern hybridization, conventional and pulsed-field gel electrophoresis (PFGE) analyses. Industrial strains were characterized and identified as Lact. acidophilus. For this species, the number of rRNA gene clusters was estimated to be at least four from analyses of rRNA gene restriction patterns. The chromosome size of type-strains of Lact. acidophilus and Lact. gasseri was estimated from PFGE analysis to be 1.85 and 2.02 Mb respectively and Lact. gasseri strains were found to contain large-sized linear plasmids.


Assuntos
Genoma Bacteriano , Lactobacillus acidophilus/genética , Plasmídeos , Técnicas de Tipagem Bacteriana , Sequência de Bases , Southern Blotting , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Lactobacillus acidophilus/classificação , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição
19.
Curr Genet ; 23(5-6): 443-9, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8391396

RESUMO

Three novel linear plasmids, pDHL1 (8.4 kb), pDHL2 (9.2 kb) and pDHL3 (15.0 kb), were discovered in the halophilic (salt-tolerant) yeast Debaryomyces hansenii. Exonuclease treatment indicated that all three plasmids were blocked at their 5' ends, presumably, by analogy with most other eukaryotic linear plasmids which involved protein attachment. The Debaryomyces plasmids were entirely cured simply by growing cells in normal culture medium, but were stably maintained in culture medium containing salts, sorbitol or glycerol at suitable concentrations. This suggested that the pDHL plasmids required an osmotic pressure for stable replication and maintenance. The Debaryomyces yeast secreted a killer toxin against various yeasts species. Toxin activity was demonstrated only in the presence of salts such as NaCl or KCl, but this killer phenotype was not associated with the pDHL plasmids. Analysis of the plasmid-curing pattern suggested that pDHL3 may play a key role in the replication of the Debaryomyces plasmids. Southern hybridization showed that an extensive homology exists between specific regions of pDHL1 and pDHL2, whereas pDHL3 is unique.


Assuntos
Plasmídeos , Saccharomycetales/genética , Southern Blotting , Replicação do DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel Bidimensional , Endonucleases/metabolismo , Exonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Fatores Matadores de Levedura , Micotoxinas/metabolismo , Pressão Osmótica , Mapeamento por Restrição , Saccharomycetales/metabolismo , Sais
20.
J Clin Microbiol ; 31(3): 665-70, 1993 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8458960

RESUMO

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.


Assuntos
Colorimetria/métodos , DNA Bacteriano/isolamento & purificação , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Southern Blotting , Sondas de DNA , Humanos , Dados de Sequência Molecular , Mycobacterium leprae/genética , Hibridização de Ácido Nucleico/métodos , Sensibilidade e Especificidade
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