RESUMO
Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.
Assuntos
Chaperonina 10/genética , Íntrons , Peptídeos/genética , Proteínas da Gravidez/genética , Fatores Supressores Imunológicos , Sequência de Aminoácidos , Animais , Sequência de Bases , Chaperonina 10/metabolismo , Feminino , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Especificidade de Órgãos , Ovário/metabolismo , Peptídeos/metabolismo , Gravidez , Proteínas da Gravidez/metabolismo , RNA Mensageiro/análise , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.
Assuntos
Chaperonina 10/química , Chaperonina 10/genética , Mapeamento Cromossômico , Peptídeos/química , Peptídeos/genética , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Fatores Supressores Imunológicos , Animais , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 9 , Clonagem Molecular , Cricetinae , DNA/genética , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Família Multigênica , Gravidez , RatosRESUMO
Various types of alcoholics have been described and heredity has been shown to be involved in some of these types. An important role of the mesolimbic dopamine system has been suggested in the reinforcing effects of alcohol and recent molecular genetic studies are implicating the gene for the D2 dopamine receptor (DRD2) in alcoholism. In a double-blind study, bromocriptine, a DRD2 agonist, or placebo was administered to alcoholics with either the A1 (A1/A1 and A1/A2 genotypes) or only the A2 (A2/A2 genotype) allele of the DRD2 gene. The greatest improvement in craving and anxiety occurred in the bromocriptine-treated A1 alcoholics and attrition was highest in the placebo-treated A1 alcoholics. The feasibility of a pharmacogenetic approach in treating certain types of alcoholics is suggested.
