RESUMO
Isoniazid resistant mechanisms in Mycobacterium tuberculosis have been shown to involve at least two genes, kat G and inh A. Alteration in the kat G gene has been found in a great number of resistant isolates. Percentage of resistant isolates harboring alteration in this gene varied among laboratories suggesting that different mutations were presented in different geographic areas. Fourteen isoniazid resistant and five multidrug resistant isolates from the Central Chest Hospital, Thailand, were examined for the kat G gene mutations in the region between base position 17 to 299. No different pattern of mutations were found between these two groups. Among nineteen isolates, there were nine isolates which showed point mutations and five isolates with base insertions of the kat G gene. The remaining five isolates revealed gene deletion. Heteroduplex formation technique also confirmed base alterations in these nine mutants.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Isoniazida/farmacologia , Mycobacterium tuberculosis/genética , Catalase/genética , DNA Bacteriano/análise , Humanos , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Nucleicos Heteroduplexes , Peroxidases/genética , TailândiaRESUMO
The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.
Assuntos
DNA Viral/genética , DNA Viral/metabolismo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genes pX , Humanos , Técnicas In Vitro , Zíper de Leucina/genética , Dados de Sequência Molecular , Transcrição Gênica , Ativação TranscricionalRESUMO
The trans activator protein of Bovine Leukaemia Virus (tax) increases the rate of transcription from the virus promoter through 21 bp sequences located in three tandem copies in the virus LTR. Based on data obtained by three different experimental approaches we concluded that the central CRE-like motif found in each of the BLV 21 bp repeats plays an important and indispensable role in tax mediated trans activation. These include (i) in vivo analysis of the function of mutant 21 bp sequences in transient transfection, (ii) gel mobility shift assay to show that CREB binds to BLV 21 bp repeats in vitro and (iii) the demonstration that the production of antisense CREB mRNA inhibits tax trans activation. Further studies with different deletion mutant CREB proteins suggest that although CREB alpha can interact with factors involved in BLV trans activation, it does not promote transcription initiation; consequently some other member/s of the CREB/ATF family must be involved.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Produtos do Gene tax/metabolismo , Vírus da Leucemia Bovina/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores Ativadores da Transcrição , Sequência de Bases , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , DNA Viral/metabolismo , Células HeLa , Humanos , Vírus da Leucemia Bovina/genética , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
The gene coding for the trans-activating factor (Tax) of the human T-cell leukemia virus, type I (HTLV-I) was mutagenized in vitro using oligonucleotide-directed mutagenesis and recombinant DNA techniques. All except one of the mutagenized tax constructs failed to trans-activate the HTLV-I LTR in a eukaryotic test system. Moreover, negative Tax mutant Arg-39----Gly was found to be trans-dominant. This observation suggests that Tax contains distinct functional domains mediating different interactions of the protein in the process of trans-activation.