RESUMO
A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.
Assuntos
Quimera , Marcação de Genes , Camundongos Knockout/genética , Fator de Crescimento Derivado de Plaquetas/genética , Animais , Desenvolvimento Embrionário e Fetal/genética , Vetores Genéticos , Camundongos , Recombinação GenéticaRESUMO
The gene coding for human keratin 18 (K18), a type I intermediate filament protein found in a variety of simple epithelia, is regulated correctly in transgenic mice but is promiscuously expressed after direct transfection into cell culture lines. We have begun an investigation of the mechanisms responsible for the correct regulation of K18 with a comparison of the chromatin state of K18 in permissive and nonpermissive transgenic mouse tissues to identify seven expression-specific, DNase-hypersensitive sites that correlate with known or potential regulatory regions of the gene. Four of these sites are associated with the proximal promoter region and the first intron that has been implicated previously in the transcriptional control of K18. Two hypersensitive sites are associated with a conserved Alu repetitive sequence located immediately upstream of the proximal promoter elements. Transcription of this Alu element in a direction opposite that of K18 was correlated with K18 expression in transgenic tissues. The final hypersensitive site was mapped to exon 6. The potential importance of this region for the expression of K18 was supported by the results of transient expression of the gene and various deleted constructions. In addition, exon 6 and the intron 1 regulatory region were distinguished from the remainder of K18 by differential DNA methylation in expressing and nonexpressing tissues. The CpG-rich proximal promoter and first exon regions remain unmethylated in both permissive and nonpermissive tissues. These results suggest that DNA methylation is not the primary mechanism of control of the gene. An Alu RNA polymerase III transcription unit and exon 6 are implicated in regulation of K18.
Assuntos
Cromatina/metabolismo , Queratinas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Mapeamento Cromossômico , DNA/metabolismo , Metilases de Modificação do DNA , DNA Recombinante , Desoxirribonucleases/metabolismo , Éxons/genética , Humanos , Queratinas/biossíntese , Metilação , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , RNA/análise , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , TransfecçãoRESUMO
Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.
Assuntos
Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , alfa 1-Antitripsina/genética , Animais , Sequência de Bases , Humanos , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Baço/metabolismo , Especificidade por Substrato , alfa 1-Antitripsina/metabolismoRESUMO
Human cDNAs coding for angiogenin were isolated from Li7 hepatoma. Analysis of the nucleotide sequences of isolated cDNA clones and its comparison with recently published sequences of cDNA and human angiogenin gene permitted to suggest that an intron is present in the 5' region of the gene, dividing the coding and 5' untranslating regions. The size of the intron exceeds 1700 b.p. Up to now it has been known that the angiogenin gene is a single copy gene. However our results of blot-hybridization with genomic DNA of some mammals showed that there are 2-3 copies of the gene in their genomes. According to our results the angiogenin gene is conserved in mammalian genomes.
Assuntos
Indutores da Angiogênese/genética , Sequência de Bases , Substâncias de Crescimento/genética , Proteínas/genética , Ribonuclease Pancreático , Homologia de Sequência do Ácido Nucleico , Animais , Clonagem Molecular , DNA/genética , Éxons , Genoma Humano , Humanos , Dados de Sequência Molecular , Hibridização de Ácido NucleicoRESUMO
The 17-mer oligonucleotide probe homologous to the fragment of the gene for human erythrocyte differentiation factor erythropoietin was used to screen the human genomic library for this gene. Restriction analysis and partial sequencing of one of the identified clones have confirmed that the clone does contain the human erythropoietin gene. We are planning to use the cloned human erythropoietin gene for developing a stably transfected mammalian cell line that should secrete erythropoietin.
Assuntos
Clonagem Molecular , Eritropoetina/genética , DNA/genética , Sondas de DNA , Humanos , Hibridização de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
The gene coding for the bovine growth hormone was isolated from a lambda-phage library. The restriction map of the 3'-region (about 10 kb) was determined by restriction analysis and by Southern blot analysis. A comparison of the 5'- and 3'-flanking regions of the bovine growth hormone gene revealed some patches of homology.
Assuntos
Sequência de Bases , DNA/genética , Hormônio do Crescimento/genética , Homologia de Sequência do Ácido Nucleico , Animais , Bovinos , Enzimas de Restrição do DNA , Desoxirribonuclease EcoRI , Hibridização de Ácido Nucleico , Plasmídeos , Recombinação GenéticaRESUMO
Isolation of genomic clones containing the human insulin gene by screening the human genomic library for this gene using the cDNA rat insulin probe is reported. The analysis of promoter-enhancer region and exons sequences has revealed their identity to analogous sequences determined earlier.
Assuntos
Clonagem Molecular , Insulina/genética , HumanosRESUMO
The library of genes was obtained from erythroleukemic AKR cells (C-1), that were maintained as suspension culture. Thirty four clones that had homology with 60-70S RNA of Rauscher Leukemia virus (RLV) were separated from this library. The restriction mapping was carried out with 14 clones, that contained most extensive proviral sequences. One clone (107) contains proviral sequences that are derived from one of the components of the RLV complex. The other 13 clones contain sequences of endogenous xenotropic viruses. The endogenous retroviral sequences obtained differ in restrictive maps from proviruses of ecotropic and xenotropic infectious endogenous MuLV and, apparently, might be attributed as non-inducible infectious xenotropic MuLV of class III. Some of the cloned retroviral sequences had symmetrical structure, that is typical for integrated proviruses, i. e. these sequences were separated from flanking cellular ones by long terminal repeats. All investigated retroviral sequences are deletion mutants of MuLV proviruses. It was shown that the inner regions of proviruses diverged more than the long terminal repeats. The expression of the main inner MuLV polypeptide (p30) was detected in NIH 3T3 cells, transfected with DNA of some clones.
Assuntos
Clonagem Molecular , DNA Viral/genética , Vírus da Leucemia Murina/genética , Leucemia Eritroblástica Aguda/microbiologia , Recombinação Genética , Animais , Linhagem Celular , Mapeamento Cromossômico , DNA/genética , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , Leucemia Eritroblástica Aguda/genética , Camundongos , Camundongos Endogâmicos AKR , Plasmídeos , Vírus Rauscher/genéticaRESUMO
Provirus from a component of Rauscher leukaemia virus (RLV) has been cloned. The provirus (the size of 5000 b. p.) contains two LTR sequences and shares expressed sequence homology with Mo-MuLV. Restriction analysis and determination of the LTR nucleotide sequence and of the site from 3'-end of proviral genome have shown the cloned provirus to be the SFEV component of RLV. LTR from this cloned provirus contains all sites necessary for transcription: CAAT and TATA sequences, "cap" site and polyadenylation signal. The LTR of the cloned provirus from SFEV component of RLV has been shown to function as a promoter in E. coli cells.
Assuntos
Clonagem Molecular , DNA Viral/genética , Leucemia Eritroblástica Aguda/genética , Vírus Rauscher/genética , Animais , Sequência de Bases , Transformação Celular Neoplásica , Transformação Celular Viral , Enzimas de Restrição do DNA , Eritroblastos/microbiologia , Leucemia Eritroblástica Aguda/microbiologia , CamundongosAssuntos
Clonagem Molecular/métodos , Genes Virais , Leucemia Eritroblástica Aguda/microbiologia , Vírus Rauscher/genética , Animais , DNA Recombinante , DNA Viral/genética , Vetores Genéticos , Leucemia Experimental/microbiologia , Camundongos , Camundongos Endogâmicos AKR , Hibridização de Ácido NucleicoRESUMO
The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.
