RESUMO
We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.
Assuntos
Apoptose/fisiologia , Citocromos c/genética , Mutação/genética , Transdução de Sinais , Trombocitopenia/etiologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Feminino , Ligação Genética , Humanos , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Oxirredução , Linhagem , Contagem de Plaquetas , Serina/química , Serina/genética , Trombocitopenia/patologiaRESUMO
The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Evolução Molecular , Variação Genética , Plasmídeos/genética , Recombinação Genética , Replicon/genética , Transativadores , Sequência de Bases , Cromossomos Bacterianos/genética , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas/genética , RNA Antissenso/metabolismo , Origem de Replicação/genética , Análise de Sequência de DNARESUMO
Proteins secreted by Mycobacterium tuberculosis may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the M. tuberculosis protein export machinery has not been characterized. A library of M. tuberculosis H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless beta-lactamase gene and an upstream Tac promoter was constructed. Transformation of Escherichia coli with the M. tuberculosis DNA library and selection on plates containing 50-100 micrograms ampicillin ml-1 resulted in the identification of 15 Ampr clones out of a total of 14,000 transformants. Twelve of the beta-lactamase gene fusions conferred high levels of Ampr (up to 1 mg ampicillin ml-1); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for M. tuberculosis antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were > or = 97% identical to hypothetical ORFs in the M. tuberculosis genome, one of which encoded a protein with 35% identity to a low-affinity penicillin-binding protein (PBP) from Streptomyces clavuligerus. Four of the seven hypothetical ORFs encoded putative exported proteins with one or more membrane interaction elements, including lipoprotein attachment sites and type I and II transmembrane (TM) segments. All of the inserts encoded typical signal sequences, with the exception of a possible type II membrane protein. It is concluded that expression of beta-lactamase gene fusions in E. coli provides a useful system for the identification and analysis of M. tuberculosis signal-sequence-encoding genes.
