RESUMO
Protein kinase Cdelta (PKCdelta) is a widely expressed calcium-independent PKC isozyme that is induced at mRNA and protein levels upon stimulation of different cellular pathways. We found the rat PKCdelta gene to consist of 19 exons and to span approximately 29 kb. The exon-intron junctions follow the GT/AG rule. The 5' untranslated region is nearly 12 kb in length, and the transcription initiation site is surrounded by CG-rich sequences. The 5' flanking region contains putative binding sites for activator protein 1 (AP-1), nuclear factor kappa B (NFkappaB), stimulatory protein-1 (Sp-1) and nerve growth factor induced-C (NGFI-C) transcription factors. The PKCdelta gene is localized at the rat chromosome 19p14. The cloned gene will help to elucidate the role of PKCdelta in growth, differentiation and death of mammalian cells.
Assuntos
Genes/genética , Isoenzimas/genética , Proteína Quinase C/genética , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Cromossomos/genética , DNA/química , DNA/genética , DNA/isolamento & purificação , Éxons , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Proteína Quinase C-delta , Ratos , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Two repetitive elements of the chicken CR1 family, each located in the 5' flanking region of the avidin-related genes Avr4 and Avr5, have been cloned and sequenced. Both elements are 721 bp in length with 72% identity to a CR1 consensus sequence. They had a 191 bp deletion in a region corresponding to the functional silencer regions previously detected within the CR1 elements upstream of the chicken lysozyme and apoVLDLII genes.
Assuntos
Avidina/genética , Galinhas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avr1-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.
Assuntos
Avidina/genética , Galinhas/genética , Sequência de Aminoácidos , Animais , Avidina/química , Avidina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biotina/metabolismo , Clonagem Molecular/métodos , Sequência Conservada , DNA/genética , DNA/isolamento & purificação , Éxons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Precursores de Proteínas/química , Precursores de Proteínas/genética , Pseudogenes , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Estreptavidina , Transcrição GênicaRESUMO
Immunohistochemical analysis of avidin and ovalbumin expression in the normally developing chick oviduct was compared to those changes induced by exogenous estrogen. Oviduct maturation was found to occur in two consecutive phases: slow proliferation and rapid differentiation. Mitosis was induced in the epithelium by estrogen, whereas it was inhibited by progesterone. Endogenous progesterone may retard the proliferation and prevent the differentiation, an effect that is overridden by increased estrogen concentration at the beginning of differentiation. Short secondary stimulation was shown to closely mimic normal maturation. When chicks treated with diethylstilbestrol (DES) for 1 month were allowed to mature, there were marked alterations in oviduct histology and laying behavior. The tubular glands were found to form from the surface epithelium as budlike invaginations, and these cells also contained avidin and ovalbumin. Ovalbumin production was stable in tubular glands. In contrast, the intensity of avidin staining was variable between gland cells even in the same sections. It was conspicuous that the number of avidin-expressing gland cells diminished markedly when estrogen treatment was prolonged over 1 week. After 2-week stimulation with DES, avidin was expressed predominantly by cells of the basal layer of pseudostratified surface epithelium, and ovalbumin mainly by tubular glands and cells of the luminal layer of surface epithelium. Neither of these proteins was expressed by goblet cells. Expression of progesterone receptor, characterized by two antibodies (polyclonal IgG-RB and monoclonal PR6), did not explain the heterogeneity of expression of avidin and ovalbumin, but probably reflects various differentiation stages of epithelial cells.
Assuntos
Avidina/biossíntese , Ovalbumina/biossíntese , Oviductos/crescimento & desenvolvimento , Progesterona/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Galinhas , Dietilestilbestrol/farmacologia , Células Epiteliais , Feminino , Histocitoquímica , Técnicas Imunoenzimáticas , Mitose/efeitos dos fármacos , Oviductos/citologia , Oviductos/efeitos dos fármacos , Receptores de Progesterona/biossíntese , Maturidade SexualRESUMO
The chicken genomic library was screened using the 32P-labelled 3'-end of avidin cDNA as a hybridization probe. A positive clone, lambda gAV12201, containing a 15-16 kb insert, was detected. The EcoRI subclones, pgAV0.4, pgAV1.8, pgAV3.3 and pgAV3.7 from the genomic clone were subjected to hybridization and restriction enzyme mapping analysis. The preliminary results suggest the existence of three structurally related genes for chicken avidin. Whether the natural gene is within the subclones can only be established when sequencing analyses of the subclones have been completed.
Assuntos
Avidina/genética , Clonagem Molecular , Genes , Animais , Galinhas , DNA/genética , Enzimas de Restrição do DNA , Mapeamento de Nucleotídeos , Homologia de Sequência do Ácido NucleicoRESUMO
A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe.