RESUMO
A site of rat DNA (about 1800 b. p.) adjacent to the first ceruloplasmin gene contains, apart from regulatory sequences common for all eukaryotic promotors, cis-elements, which are potential binding sites for soluble nuclear receptors of some hormones. Sequences characteristic of genes expressed in liver cells and mammary gland cells during lactation were detected. Full-length fragment of this locus of ceruloplasmin gene (1800 b. p.) was synthesized by PCR and used in gel shift experiments. It was found that soluble proteins extracted from purified nuclei of mammary gland cells during lactation and from the liver of adult and newborn rats, contain proteins specifically interacting with the PCR product. A fragment of chromosome gene containing exons encoding the central part of rat ceruloplasmin was cloned in pTZ19 bacterial vector. Gel shift assay showed that the cloned fragment contained binding sites for specific transcription factor YY1, whose level in nuclear protein fractions varied during ontogeny (according to immunoblotting data). Monoclonal antibodies detected protein YY1 in the complex of cloned DNA-nuclear proteins. Possible mechanisms of tissue-specific regulation of ceruloplasmin gene varying during ontogeny are discussed.
Assuntos
Ceruloplasmina/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Região 5'-Flanqueadora/genética , Animais , Sítios de Ligação/genética , Clonagem Molecular , Proteínas de Ligação a DNA/análise , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Ligação de DNA Eritroide Específicos , Lactação/genética , Fígado/metabolismo , Mamíferos/genética , Mamíferos/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas Nucleares/imunologia , Ratos , Fator de Transcrição Sp1/análise , Fator de Transcrição Sp1/metabolismo , Distribuição Tecidual , Fatores de Transcrição/análise , Fator de Transcrição YY1RESUMO
An individual clone, presumably carrying a 3 bp fragment of ceruloplasmin receptor cDNA was isolated from the expression library of human placenta cDNA using polyclonal specific antibodies to ceruloplasmin receptors. EcoR1-hydrolysate of isolated DNA was cloned in a pTZ19 bacterial vector and sequenced in the forward and reverse direction. The comparison of the revealed sequence with known sequences of human genome revealed its high similarity to ceruloplasmin cDNA.