RESUMO
The tropomyosin gene tmy-1/lev-11 of Caenorhabditis elegans spans 14.5 kb and encodes three isoforms by alternative splicing. To identify, characterize and compare the genome and tissue expression of a fourth isoform, the technique of rapid amplification of cDNA ends and microinjection with lacZ and gfp fusion plasmids were employed. We elucidated CeTMIV, a fourth isoform of tmy-1, which encoded a 256 residue polypeptide. CeTMIV isoform had a similar promoter region to CeTMIII isoform, but was alternatively spliced to generate a cDNA that differed in two exons. The tmy-1::lacZ and tmy-1::gfp fusion genes, with 3.2 kb promoter sequence and 1.1 kb of CeTMIV isoform specific exons, were expressed in the pharyngeal and intestinal cells. Further unidirectional deletion of the sequence located the primary promoter region 853 bp upstream from the initial codon. We show within the upstream region, the presence of B and C subelement-like sequences of myo-2, which may be used to stimulate pharyngeal expression. Despite the presence of a ges-1 like sequence, we were unable to locate the two GATA sites required for intestinal expression. Reassessing tissue expression for CeTMIII isoform with newly constructed fusion plasmids, we showed further expression in germ-line tissue and intestinal cells in addition to pharyngeal expression. Finally, to demonstrate that tropomyosin is essential for development, we inactivated the body wall and pharynx-specific isoforms by RNA-mediated interference. In addition to 50-75 % embryonic lethality in both cases, the worms that survived body wall interference had abnormal body morphology and uncoordinated movements, and those that survived pharynx interference had deformed pharynges and gut regions. These results show the function of tropomyosin in normal muscle filament assembly and embryonic development, and illustrate the different expression patterns characteristic of tropomyosin isoforms in C. elegans.
Assuntos
Processamento Alternativo/genética , Padronização Corporal , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Mucosa Intestinal/metabolismo , Faringe/metabolismo , Tropomiosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Expressão Gênica , Regulação da Expressão Gênica , Genes de Helmintos/genética , Genes Reporter/genética , Células Germinativas/metabolismo , Intestinos/citologia , Intestinos/embriologia , Íntrons/genética , Dados de Sequência Molecular , Músculo Liso/metabolismo , Faringe/embriologia , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tropomiosina/análise , Tropomiosina/química , Tropomiosina/genéticaRESUMO
Human rotavirus (HRV) infection and its seasonal distribution was studied over a 12-month period in Ghana. A total of 561 stool samples, 447 diarrhoea stools and 114 non-diarrhoea stools (controls), were obtained from children attending three polyclinics in Accra. Rotavirus was detected during 10 of the 12 months and showed a seasonal trend. It was high during the relatively cool dry months and low during the wet season. Peaks of infection were in February (26.2%) and September (24.5%). HRV was detected in 67 of 447 of the diarrhoea stools (15.0%) and in eight of 114 controls (7.0%). The HRV isolation rate was highest (20.2%) in the under-18-months age group. The RNA electropherotype of the HRV isolates was predominantly (83.6%) of the long type. Non-group A HRV was detected in 14.9% of the HRV-positive samples.