RESUMO
Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.
Assuntos
Processamento Alternativo/genética , Sequência Conservada/genética , Fosfoproteínas Fosfatases/genética , Proteínas de Saccharomyces cerevisiae , Transcrição Gênica/genética , Animais , Sequência de Bases , Domínio Catalítico , Morte Celular , Divisão Celular , Linhagem Celular , Clonagem Molecular , Citoplasma/enzimologia , Éxons/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Humanos , Íntrons/genética , Isoenzimas/análise , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/análise , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/imunologia , Biossíntese de Proteínas/genética , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RatosRESUMO
The polymorphism G20210A in the 3' untranslated region of the prothrombin gene is associated with an increased level of factor II activity and confers a twofold to fivefold increase in the risk for venous thromboembolism. Among Caucasian populations, the prevalence of factor II G20210A heterozygotes is 1% to 6%, whereas in non-Caucasian populations it is very rare or absent. The aim of the present study was to discern whether factor II G20210A originated from a single or recurrent mutational events. Allele frequencies of four dimorphisms spanning 16 of 21 kb of the factor II gene were determined in 133 unrelated Caucasian subjects of Jewish, Austrian, and French origins who bore factor II G20210A (10 homozygotes and 123 heterozygotes) and 110 Caucasian controls. Remarkable differences in the allele frequencies for each dimorphism were observed between the study groups (P = .0007 or less), indicating strong linkage disequilibrium and suggesting a founder effect. Indeed, a founder haplotype was present in 68% of 20210A mutant alleles and only in 34% of 20210G normal alleles (P < .0001). These data strongly support a single origin for factor II G20210A that probably occurred after the divergence of Africans from non-Africans and of Caucasoid from Mongoloid subpopulations.