RESUMO
Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia.
Assuntos
Mesocestoides/crescimento & desenvolvimento , Mesocestoides/genética , Tropomiosina/biossíntese , Animais , Sequência Conservada , DNA de Helmintos/química , DNA de Helmintos/genética , Echinococcus granulosus/genética , Echinococcus multilocularis/genética , Evolução Molecular , Duplicação Gênica , Camundongos , Dados de Sequência Molecular , Músculos/química , Filogenia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Schistosoma mansoni/genética , Análise de Sequência de DNA , Homologia de Sequência , Tropomiosina/genética , Turbelários/genéticaRESUMO
Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition.
Assuntos
Meios de Cultura/farmacologia , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae , Tropomiosina/biossíntese , Actinas/metabolismo , Actomiosina/metabolismo , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Meios de Cultura/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica/efeitos dos fármacos , Músculos/metabolismo , Concentração Osmolar , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/genética , Tropomiosina/genética , Tropomiosina/isolamento & purificação , Tropomiosina/farmacologia , Troponina/farmacologia , ViscosidadeAssuntos
Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Proteínas de Helminto/biossíntese , Tropomiosina/biossíntese , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Echinococcus/genética , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Larva/genética , Larva/crescimento & desenvolvimento , Tropomiosina/genéticaRESUMO
Although numerous studies have reported the production of skeletal muscle alpha-tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg(2+)-ATPase in the absence of troponin. On the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha-tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha-tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg(2+)-ATPase.
Assuntos
Músculo Esquelético/metabolismo , Pichia/genética , Tropomiosina/genética , Actinas/metabolismo , Actomiosina/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Galinhas , Clonagem Molecular/métodos , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Tropomiosina/biossíntese , Tropomiosina/isolamento & purificaçãoRESUMO
A regulação dependente de `Ca²+ï da atividade ATPásica da acto-miosina em concentrações fisiológicas de actina, tropomiosina e troponina ocorre exclusivamente na presença de troponina T (TnT). Nosso grupo demonstrou que um polipeptídeo correspondente aos primeiros 191 aminoácidos da TnT ativa a atividade ATPásica da acto-miosina na presença de tropomiosina e na ausência das outras duas subunidades do complexo troponina (TnI/TnC). Com o objetivo de mapear e caracterizar esse domínio ativatório da TnT, construímos fragmentos de TnT correspondentes às regiões compreendidas entre os resíduos de aminoácidos: 1-157 (TnT1-157), 1-76 (TnT1-76), 77-157 (TnT77-57), 77-191 (TnT77-191) e 158-191 (TnT158-191)...
Assuntos
Actinas/biossíntese , Sequência de Aminoácidos , Músculo Esquelético , Tropomiosina/biossíntese , Troponina T , Dicroísmo Circular , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.
Assuntos
Ambystoma/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Tropomiosina/biossíntese , Tropomiosina/genética , Ambystoma/genética , Ambystoma/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Complementar/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The cDNA for alpha-tropomyosin (TM) was cloned by the polymerase chain reaction (PCR) from a lambda gt11 library constructed with mRNA from juvenile axolotl heart tissues. Subsequently, the nucleotide sequence of the cDNA was determined. This is the first reported cDNA for axolotl alpha-tropomyosin. Comparative analyses of the deduced amino acid sequence of this cDNA with Xenopus skeletal muscle alpha-tropomyosin sequences indicate that the axolotl heart cDNA has 93% and 96% homology in the regions of amino acids 39-80 (exon 2b) and 258-284 (exon 9a), respectively. However, there is about 86.55% homology at the nucleic acid level (coding region) and 97.5% homology at the amino acid level with that of Xenopus skeletal muscle alpha-tropomyosin cDNA. Northern blot analyses with polyA+ RNA from juvenile heart suggest the presence of at least two different transcripts for alpha-tropomyosin in axolotl heart. The results of 3'-RACE concur with those of northern blot analyses.