RESUMO
Trichomonas vaginalis viruses (TVV), which may regulate P270 gene expression in the protozoan pathogen T. vaginalis, are a group of divergent double-stranded (ds) RNA viruses. In the present study, the complete 4674-bp cDNA sequence of a 4.6-kb ds RNA from a newly identified TVV2-1 isolate was determined. The sequence of the plus-strand mRNA contains four open reading frames, which encode overlapping cap and pol genes in the reading frame 2 and reading frame 1, respectively, and two putative serine-threonine-rich basic proteins VP3 and VP4 in the third reading frame. An 85-kDa capsid protein and a 160-kDa CAP-POL fusion protein were identified in crude viruses by Western blotting experiments using antisera raised against gene-specific oligopeptides. In conjunction with the presence of a potential ribosomal slippery heptanucleotide G GGC CCC within the overlap of the cap and pol genes, these observations suggest that the pol gene of TVV2-1 is translated via a -1 ribosomal frameshifting event during translation of the cap gene. Our results also provide insight into the conservation among divergent dsRNA species from TVV and suggest that the genome of TVV2-1 may encode two extra genes in addition to the cap and pol genes.
Assuntos
DNA Complementar/genética , Vírus de RNA/genética , Trichomonas vaginalis/virologia , Sequência de Aminoácidos , Animais , Capsídeo/genética , Clonagem Molecular , DNA Complementar/química , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Vírus de RNA/isolamento & purificação , Vírus de RNA/ultraestrutura , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We have previously used differential mRNA display to identify and clone a range of cDNAs derived from genes expressed in only one developmental stage of Angiostrongylus cantonensis. In this paper we report the characterisation of a gene designated Ac-fmp-1 and its product, expressed only in the adult stage. Full length cDNA of 1.5 kb terminates at the 5' end with the conserved nematode spliced leader (SL) sequence and contains one open reading frame coding for a putative protein of 417 amino-acids. The recombinant protein expressed from this open reading frame is antigenic in the infected host and polyclonal antibodies raised against the recombinant protein recognize a 66 kDa protein present only in adult female worms. This protein localises to the muscle cell membranes adjacent to the pseudocoelom. Nine kb of genomic DNA has been amplified by polymerase chain reaction and 7 kb has been cloned and sequenced. This sequence includes 1.5 kb of 5' flanking region and 5.5 kb of the Ac-fmp-1 gene sequence. In this sequence, corresponding to approximately 0.8 kb of the cDNA, the exon/intron pattern has been determined.
Assuntos
Angiostrongylus cantonensis/genética , Genes de Helmintos , Sequência de Aminoácidos , Angiostrongylus cantonensis/crescimento & desenvolvimento , Angiostrongylus cantonensis/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , DNA Complementar/genética , DNA de Helmintos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genéticaRESUMO
A procedure based on polymerase chain reaction was developed for the study of rotaviruses. A full-length cDNA copy of the gene coding for major neutralizing glycoprotein VP7 of a human rotavirus isolate 1407 with the "long" electrophoretype (classified as the first electrophoretype) was cloned and sequenced. The primary structure of glycoprotein VP7 of isolate 1407 in A and C antigenic regions was found to be similar to that of serotype 1 virus.
Assuntos
Antígenos Virais/genética , Genes Virais/genética , Código Genético/genética , Rotavirus/genética , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase/métodos , Rotavirus/isolamento & purificaçãoRESUMO
A procedure based on polymerase chain reaction use for the detection of rotavirus has been developed. Full length cDNA copy of the VP7 gene coding for the major neutralization glycoprotein of the human rotavirus RK9 with an unusual "wide" electrophoretype is cloned and sequenced. Glycoprotein VP7 of RK9 has a unique amino acid composition in A and C antigenic regions. It shows that strain RK9 represents a new (12) rotavirus serotype.
