RESUMO
Using in silico approaches and RACE we cloned a full length trinucleotide (CAG) repeat-containing cDNA (cag-3). The cDNA is 2478 bp long and the deduced polypeptide consists of 140 amino acids of which 73 are glutamines. The genomic sequence spans approximately 79 kb on mouse chromosome 7 and the gene is composed of four exons. Standard and real-time PCR analyses of several mouse tissues showed that the gene is exclusively expressed in the brain and is not detected in embryonic stages. Within the brain, it is expressed throughout the forebrain region with predominant expression in the hypothalamus and olfactory bulb and very low levels in the mid- and hindbrain.
Assuntos
Encéfalo/metabolismo , Repetições de Trinucleotídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/metabolismo , Expressão Gênica , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Both erythropoietin (EPO) and the short-form thrombopoietin (TPO) were expressed at low levels whereas the long-form TPO was expressed at high levels in transgenic animals. To elucidate the role of carboxy-terminal half of the long-form TPO which is absent in the short-form, we generated recombinant TPO or EPO expression vectors which contain or lack the carboxy-terminal half of TPO and examined their expression in the HC11 and 293 cells. The long-form TPO was expressed higher than the short-form regardless of the cell types, transfection modes, and promoters. When 3'-half of the long-form TPO cDNA was placed downstream of the EPO cDNA to act as a 3'-untranslated region, expression of EPO was moderately increased at the RNA level, however, no remarkable increase was observed at the protein level. These results suggest that the low expression of EPO, as like as the short-form TPO, is due to absence of the 3'-half in the full-length TPO that confers stability both at the RNA and protein levels.
Assuntos
DNA Complementar/metabolismo , Eritropoetina/genética , Glândulas Mamárias Animais/metabolismo , RNA/metabolismo , Trombopoetina/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Animais , Caseínas/genética , Células Cultivadas , Citomegalovirus/genética , Ensaio de Imunoadsorção Enzimática , Eritropoetina/metabolismo , Humanos , Rim/metabolismo , Glândulas Mamárias Animais/citologia , Camundongos , Regiões Promotoras Genéticas/genética , Estabilidade de RNA/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We identified 13 transcript isoforms of a trinucleotide-repeat-containing gene, cag-8, that is expressed almost exclusively in the mouse brain. The polypeptide deduced from the cDNA consists of 137 AAs, of which 74 are glutamines. The 130-kb gene is composed of 16 exons, from which at least 13 isoforms with variable UTRs are formed by alternative splicing. A strong positive cis-acting element was found in the neuroblastomaxglioma hybrid NG108-15 and human embryonic kidney HEK293 cell lines. The cag-8 protein was localized in the cytosol and in granular bodies within the nucleus. These findings indicate that cag-8 is a novel poly(Q)-encoding gene, the expression of which is confined primarily to the brain.