RESUMO
Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochrome b6f complex. One of them, strain abf3, also fails to accumulate holocytochrome c6. We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b6f and cytochrome c6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown alga Odontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25-33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11-12% identity). On the basis of the pleiotropic c-type cytochrome deficiency in the ccs1 mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation.
Assuntos
Cloroplastos/metabolismo , Grupo dos Citocromos c/biossíntese , Proteínas Nucleares/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydomonas reinhardtii , Proteínas de Cloroplastos , Grupo dos Citocromos b/biossíntese , Grupo dos Citocromos b/deficiência , Grupo dos Citocromos b/genética , Complexo Citocromos b6f , Grupo dos Citocromos c/genética , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas de Plantas , Polimorfismo de Fragmento de Restrição , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Alinhamento de SequênciaRESUMO
Nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii has thus far been characterized by integration of the introduced DNA into nonhomologous sites. In this study, the occurrence of homologous recombination events during transformation was investigated with the intent of developing strategies for gene targeting and gene disruption. Homologous recombination was monitored by using nonfunctional 5' and 3' deletion derivatives of the wild-type C. reinhardtii nit1 gene (encodes nitrate reductase) as selectable markers (p5' delta and p3' delta respectively) and the low reverting nit1-305 strain as the transformation recipient. After introduction of the DNA into the cell, intermolecular recombination between p5' delta and p3' delta occurs at a high frequency to restore a functional nit1 gene, indicating the presence of homologous recombination machinery in mitotic cells. Gene-targeting events at the nit1 locus were selected by restoring nit1-305 cells to prototrophy after transformation with only p5' delta and were confirmed by analysis of genomic DNA. By comparing the number of transformants obtained after transformation with p5' delta to the number obtained after transformation with a functional nit1 gene, the frequency of homologous-to-random integration events ranged between 1:1000 after glass bead-mediated transformation and 1:24 after bombardment with DNA-coated tungsten microprojectiles.
Assuntos
Chlamydomonas reinhardtii/genética , Recombinação Genética , Animais , Núcleo Celular/metabolismo , Clonagem Molecular , Troca Genética , Escherichia coli/genética , Genes de Plantas , Vetores Genéticos , Nitrato Redutase , Nitrato Redutases/genética , Plasmídeos , Mapeamento por Restrição , Transformação GenéticaRESUMO
A 9.5-kilobase plasmid of Yersinia pestis, the causative agent of plague, is required for high virulence when mice are inoculated with the bacterium by subcutaneous injection. Inactivation of the plasmid gene pla, which encodes a surface protease, increased the median lethal dose of the bacteria for mice by a millionfold. Moreover, cloned pla was sufficient to restore segregants lacking the entire pla-bearing plasmid to full virulence. Both pla+ strains injected subcutaneously and pla- mutants injected intravenously reached high titers in liver and spleen of infected mice, whereas pla- mutants injected subcutaneously failed to do so even though they establish a sustained local infection at the injection site. More inflammatory cells accumulated in lesions caused by the pla- mutants than in lesions produced by the pla+ parent. The Pla protease was shown to be a plasminogen activator with unusual kinetic properties. It can also cleave complement C3 at a specific site.
Assuntos
Proteínas de Bactérias , Ativadores de Plasminogênio/fisiologia , Yersinia pestis/enzimologia , Yersinia pestis/patogenicidade , Sequência de Aminoácidos , Animais , Contagem de Colônia Microbiana , Escherichia coli/enzimologia , Fibrinolisina/química , Fibrinolisina/metabolismo , Injeções Intravenosas , Cinética , Fígado/microbiologia , Camundongos , Dados de Sequência Molecular , Mutação , Peste/microbiologia , Plasmídeos , Ativadores de Plasminogênio/genética , Proteínas Recombinantes/metabolismo , Baço/microbiologia , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Yersinia pestis/isolamento & purificaçãoRESUMO
We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open reading frame is consistent with our previous conclusion that the two Pla proteins which appear in the outer membrane of pla+ Y. pestis are derived from a common precursor. The deduced amino acid sequence of Pla revealed that it possesses a high degree of homology to the products of gene E of Salmonella typhimurium and ompT of Escherichia coli but does not possess significant homology to other plasminogen activators of known sequence. We also identified a transcription unit that resides on the complimentary strand and overlaps the pla gene.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Genes Bacterianos , Ativadores de Plasminogênio/genética , Salmonella typhimurium/genética , Yersinia pestis/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Genes , Homologia de Genes , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
The 9.5-kilobase plasmid of Yersinia pestis determines plasminogen activator, coagulase, pesticin, and pesticin immunity activities. We have mapped and cloned the loci encoding these activities and demonstrated that both plasminogen activator and coagulase were determined by the same gene, designated pla. The primary translation product of this gene (38 kilodaltons [kDa]) was processed in two sequential steps to produce peptides of 37 and 35 kDa. The first step in this processing occurred rapidly and probably cotranslationally and was blocked when protein export was inhibited. The second step was much slower and resulted in the presence of both the 37- and 35-kDa species in significant quantities. We also showed that the plasmid had a polA-dependent replicon and identified the region that contained its origin of replication and incompatibility functions.
Assuntos
Genes Bacterianos , Plasmídeos , Yersinia pestis/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Bacteriocinas/imunologia , Mapeamento Cromossômico , Clonagem Molecular , Coagulase/genética , Enzimas de Restrição do DNA , Genes , Peptídeo Hidrolases/genética , Ativadores de Plasminogênio/genética , Yersinia pestis/patogenicidadeRESUMO
The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid.