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1.
Trends Microbiol ; 9(11): 535-40, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11825713

RESUMO

Most bacterial genomes have very few pseudogenes; notable exceptions include the genomes of the intracellular parasites Rickettsia prowazekii and Mycobacterium leprae. As DNA can be introduced into microbial genomes in many ways, the compact nature of these genomes suggests that the rate of DNA influx is balanced by the rate of DNA deletion. We propose that the influx of dangerous genetic elements such as transposons and bacteriophages selects for the maintenance of relatively high deletion rates in most bacteria; the sheltered lifestyle of intracellular parasites removes this threat, leading to reduced deletion rates and larger pseudogene loads.


Assuntos
Pseudogenes/genética , Bacteriófagos/genética , Cromossomos Bacterianos , Elementos de DNA Transponíveis , Deleção de Genes , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Lisogenia , Modelos Genéticos
2.
FEMS Immunol Med Microbiol ; 22(3): 205-16, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9848681

RESUMO

A lambda gt11 recombinant DNA library of Mycobacterium leprae was screened to isolate recombinant phage clones expressing mycobacterial antigens important for T cell reactivity. The library was plated on a lawn of Escherichia coli Y1090 and recombinant antigens were expressed from isolated phage clones in 96-well plates. Pools of recombinant antigens from 12 wells were tested in T cell proliferation assays with MHC class II restricted human CD4+ Th1 clones secreting interferon-gamma and cytotoxic for antigen pulsed antigen presenting cells. By screening 1750 pools of recombinant antigens with a mixture of eight Th1 clones, we identified two recombinant phage clones that expressed recombinant mycobacterial antigens stimulatory for T cells. MHC restriction analysis and reactivity to a battery of mycobacterial antigens suggested that the two responding Th1 clones recognized mycobacterial antigens/epitopes with different MHC class II (HLA-DR) restriction requirements. Our results suggest that the methodology described in this paper is suited to isolate recombinant phage clones expressing mycobacterial recombinant antigens stimulatory for T cells of protective phenotype. Such antigens may be useful in designing new vaccines and diagnostic reagents against mycobacterial diseases.


Assuntos
Antígenos de Bactérias/imunologia , Micobacteriófagos/genética , Mycobacterium/imunologia , Linfócitos T/imunologia , Células Th1/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Bactérias/análise , Linfócitos T CD4-Positivos/imunologia , Células Clonais , Biblioteca Gênica , Humanos , Ativação Linfocitária , Lisogenia , Micobacteriófagos/metabolismo , Mycobacterium leprae/imunologia , Mycobacterium leprae/virologia , Proteínas Recombinantes/imunologia
3.
Gene ; 183(1-2): 129-36, 1996 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-8996097

RESUMO

Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.


Assuntos
Vetores Genéticos/genética , Micobacteriófagos/genética , Mycobacterium/isolamento & purificação , Clonagem Molecular/métodos , Cosmídeos/genética , Expressão Gênica , Genes Reporter/genética , Cinética , Luciferases/biossíntese , Luciferases/genética , Lisogenia , Testes de Sensibilidade Microbiana/métodos , Micobacteriófagos/crescimento & desenvolvimento , Mycobacterium/virologia , Regiões Promotoras Genéticas/genética , Mapeamento por Restrição , Superinfecção
4.
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
7.
Proc Natl Acad Sci U S A ; 85(18): 6987-91, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2842799

RESUMO

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.


Assuntos
Regulação da Expressão Gênica , Lisogenia , Mycobacterium/genética , Transformação Bacteriana , Clonagem Molecular , Escherichia coli/genética , Canamicina Quinase , Hanseníase/microbiologia , Métodos , Mycobacterium/enzimologia , Fosfotransferases/genética , Plasmídeos , Tuberculose/microbiologia
8.
Tubercle ; 59(3): 203-25, 1978 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-100919

RESUMO

Progress made during the last 15 years in the studies on the relationships between mycobacteria and their bacteriophages is reviewed. The basic biology of the phages and the applications of studies on adaptation and host range are discussed in relation to the development of phage typing systems for epidemiological purposes. The nature of lysogeny, its natural occurrence, its experimental establishment, the effect of the lysogenic state on the host bacterium and the evidence that lysogenic mycobacteria are involved in human disease, especially sarcoidosis, is reviewed.


Assuntos
Micobacteriófagos/genética , Mycobacterium/genética , Adaptação Biológica , Tipagem de Bacteriófagos , Humanos , Lisogenia , Micobacteriófagos/análise , Micobacteriófagos/metabolismo , Mycobacterium/análise , Mycobacterium/metabolismo , Mycobacterium leprae/isolamento & purificação , Mycobacterium tuberculosis/genética , Sarcoidose/etiologia , Transdução Genética
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