RESUMO
Tissue-specific regulation of the expression of ceruloplasmin (CP) gene, which encodes major copper-containing extracellular glycoprotein was investigated. A decrease of the CP concentration associated with copper amounts in milk during the first 3 days of lactation was used as phenotypic index for evaluating the CP enzyme activity in the mammary gland. Computer analysis of mammalian CP gene promoter region has revealed conserved sequences of cis-elements, which potentially were capable of regulating the enzyme activity. It has been shown that changes in the nucleotide sequence of specific transcriptional factor binding sites located at 5'-end of CP gene were associated with disturbance of the regular downregulation of CP gene activity during lactation.
Assuntos
Ceruloplasmina/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Lactação/fisiologia , Glândulas Mamárias Humanas/enzimologia , Leite Humano/enzimologia , Elementos de Resposta/fisiologia , Adolescente , Adulto , Ceruloplasmina/genética , Cobre/metabolismo , Feminino , Humanos , Análise de Sequência de DNARESUMO
Alternative expression of ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, is a neuron survival factor, was studied in the current work. Computer analysis showed that Cp-mRNA isoform, coding for 109 kDa polypeptide, can be formed as a result of the transcription from the alternative promoter in 3'-region of intron 2 of rat Cp gene. Alternative Cp form starts with 25 amino acid residues sequence, coded with intron 2 region. It is followed by amino acid sequence of the main Cp isoform starting from Gly 113. In silico data were experimentally confirmed using RT-PCR. It was demonstrated that the predicted mRNA was generally localized in liver and brain cells of adult rats. Direct sequencing of the obtained PCR-product showed the entire coincidence of the real and predicted mRNAs. It was in vitro showed that approximately 110 kDa Cp-like protein was completed and accumulated in the absence of mitochondria. This protein is transferred into the isolated mitochondria in the reconstructed system. Transport is energy-dependent, it is not accompanied with the shortening of Cp polypeptide length and needs the presence of cytosolic factors. Probably import is determined by the inner protein mitochondria import signal with amino acid sequence KVVYREFTDSTFRE, located in 756-769 region of mature Cp. Possible role of Cp in iron metabolism in mitochondria is under discussion.
Assuntos
Ceruloplasmina/genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Ceruloplasmina/metabolismo , Humanos , Ferro/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
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
By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.
