RESUMO
The inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting of APG1 (albino or pale green mutant 1), an example of a stop-transfer substrate, and atTic40, an example of a post-import substrate. We show that the APG1 transmembrane domain is sufficient to direct stop-transfer insertion. The APG1 transmembrane domain also functions as a topology determinant. We also show that the ability of the post-import signals within atTic40 to target proteins to the IEM is dependent upon their context within the full protein sequence. In the incorrect context, the atTic40 signals can behave as stop-transfer signals or fail to target fusion proteins to the IEM. These data suggest that the post-import pathway signals are complex and have evolved to avoid stop-transfer insertion.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Citoplasma/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cloroplastos/genética , Citoplasma/genética , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologiaRESUMO
BACKGROUND: Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. RESULTS: Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. CONCLUSIONS: uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.
Assuntos
Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gossypium/enzimologia , Regiões Promotoras Genéticas/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Arabidopsis , Sequência de Bases , Códon sem Sentido/genética , Primers do DNA/genética , Flores/metabolismo , Fluorometria , Regulação da Expressão Gênica de Plantas/genética , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Análise de Sequência de DNA , Transgenes/genética , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a human pathogen that causes diverse human diseases including streptococcal toxic shock syndrome (STSS). A GAS outbreak occurred in Brasilia, Brazil, during the second half of the year 2011, causing 26 deaths. Whole genome sequencing was performed using Illumina platform. The sequences were assembled and genes were predicted for comparative analysis with emm type 1 strains: MGAS5005 and M1 GAS. Genomics comparison revealed one of the invasive strains that differ from others isolates and from emm 1 reference genomes. Also, the new invasive strain showed differences in the content of virulence factors compared to other isolated in the same outbreak. The evolution of contemporary GAS strains is strongly associated with horizontal gene transfer. This is the first genomic study of a Streptococcal emm 1 outbreak in Brazil, and revealed the rapid bacterial evolution leading to new clones. The emergence of new invasive strains can be a consequence of the injudicious use of antibiotics in Brazil during the past decades.