RESUMO
Two morphological forms of programmed cell death, apoptosis and autophagic cell death, remove unneeded or damaged cells during animal development. Although the mechanisms that regulate apoptosis are well studied, little is known about autophagic cell death. A shotgun proteome analysis of purified dying larval salivary glands in Drosophila was used to identify proteins that are expressed during autophagic programmed cell death. A total of 5661 proteins were identified from stages before and after the onset of cell death. Analyses of these data enabled us to identify proteins from a number of interesting categories including regulators of transcription, the apoptosis, autophagy, lysosomal, and ubiquitin proteasome degradation pathways, and proteins involved in growth control. Several of the identified proteins, including the serine/threonine kinase warts (Wts), were not detected using whole-genome DNA microarrays, providing support for the importance of such high-throughput proteomic technology. Wts regulates cell-cycle arrest and apoptosis, and significantly, mutations in wts prevent destruction of salivary glands.
Assuntos
Autofagia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteoma/metabolismo , Proteômica , Esteroides/metabolismo , Animais , Drosophila melanogaster/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/ultraestrutura , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Glândulas Salivares/citologia , Glândulas Salivares/ultraestruturaRESUMO
To evaluate the role of uridylyl-transferase, the Sinorhizobium meliloti glnD gene was isolated by heterologous complementation in Azotobacter vinelandii. The glnD gene is cotranscribed with a gene homologous to Salmonella mviN. glnD1::Omega or mviN1::Omega mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that glnD and mviN are members of an indispensable operon in S. meliloti.
Assuntos
Proteínas de Bactérias/genética , Nucleotidiltransferases/genética , Óperon , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Conjugação Genética , Genes Bacterianos , Genes Essenciais , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Nucleotidiltransferases/metabolismo , Proteínas PII Reguladoras de Nitrogênio , Plasmídeos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Sinorhizobium meliloti/genéticaRESUMO
To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized. Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was contranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote. glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.
