RESUMO
Transmission of foreign mtDNA along the paternal lineage founded by male mice (F0), and distribution of that mtDNA in their progeny at early stages of prenatal development were studied. Transmitochondrial males of F0 obtained after injection of human mitochondria into mouse zygotes has been shown to transmit foreign mtDNA to subsequent generations. Individual peculiarities among the males studied, concerning transmission of foreign mtDNA to the progeny, are likely to exist. Besides, the distribution of human mtDNA among blastomeres of transmitochondrial embryos under study differed from that observed in previous investogation of its inheritance along the maternal lineage.
Assuntos
DNA Mitocondrial/genética , Embrião de Mamíferos/metabolismo , Padrões de Herança , Mitocôndrias/genética , Zigoto/metabolismo , Animais , Quimera , Feminino , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mitocôndrias/química , Fatores Sexuais , Zigoto/crescimento & desenvolvimentoRESUMO
One of the crucial problems of developmental biology is the study of mechanisms of regulation of gene expression in early embryogenesis. Here we studied dynamics of mosaic appearance of a marker fluorescent protein in in vitro developing mice embryo derived from zygotes after microinjections to male pronuclei of cloned DNA fragment carrying EGFP under control of different promoters. Main attention was paid to initial stages of development, when structural rearrangements and reprogramming of both parental genomes, activation of zygotic genes, and control of development by embryo genome take place.
Assuntos
Blastocisto/metabolismo , Proteínas de Fluorescência Verde/genética , Mosaicismo/embriologia , Transgenes , Animais , Blastocisto/citologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microinjeções , Zigoto/citologia , Zigoto/metabolismoRESUMO
Human apolipoprotein A-I gene (apoA-I) plasmid expression vectors were transferred into mice by hydrodynamic injections into tail vein. Two types of expression vectors were used. First one -pCMVcapoAI contains cDNA of apo A-I driven by human cytomegalovirus early gene promoter (CMV). Second one--pAlg contains genomic locus of intron-containing apo A-I under control of own extended 5'-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to appearance of human apo A-I mRNA in the liver and human Apo A-I protein in the serum of injected mice. Dynamics of human Apo A-I content in the serum of mice injected by pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, maximal concentration of human Apo A-I protein in the mouse serum was detected one day after injection with following decline to zero level during next two weeks. Under the same conditions injections of pAlg led to maximal level of human Apo A-I concentration in the mouse serum (up to 20 mkg/ml in some animals) on the 5th-7th day of experiment with following graduate decline during several months (human Apo A-I concentration in the serum of oldest analyzed mouse (6 months after injection) was about 25% of its maximal level in the same animal). Levels of human Apo A-I concentration in the mouse serum were compatible after injections of both expression vectors, in spite of much more strong activity of CMV promoter in comparison with APOAI in cultured human hepatoma cells HepG2. We ascribe the revealed difference in dynamics of human Apo A-I expression to delay of apo A-I transcription from pAlg vector, that was confirmed by nested RT-PCR. Significant level and long-term persistence of human Apo A-I in the serum of mice injected by pAlg could be explained by properties of APOAI or (and) exon-intron structure of genomic apo A-I gene. To test the role of APOAI in long-term expression of human Apo A-I in the mice we performed hydrodynamic injections of plasmid vectors containing cDNA of reporter gene encoding luciferase driven by variants of APOAI. No long-term expression of luciferase was found in the livers of injected mice. Therefore, our data suggest the role of exon-intron structure in maintaining of efficient and long-term expression of transferred human apo A-I.
Assuntos
Apolipoproteína A-I/genética , Expressão Gênica , Técnicas de Transferência de Genes , Fígado/metabolismo , Animais , Sequência de Bases , Primers do DNA , Humanos , Camundongos , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.
Assuntos
DNA/administração & dosagem , Galactose/metabolismo , Polilisina/administração & dosagem , Células 3T3 , Animais , Apolipoproteína A-I/genética , Arteriosclerose/genética , Arteriosclerose/terapia , Cloranfenicol O-Acetiltransferase/genética , Genes Reporter , Terapia Genética , Humanos , Técnicas In Vitro , Óperon Lac , Fígado/metabolismo , Camundongos , Polilisina/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
A new method was developed to study the mechanism of initiation of the retrotransposition cycle: retrotransposons of Drosophila melanogaster, gypsy, copia, and 17.6 were expressed in yeast under the control of potent yeast promoters. Expression of retrotransposons induced formation of viruslike particles (VLPs) associated with full-length Ty1 RNA and DNA sequences. This phenomenon was termed heterologous induction. When the gene for reverse transcriptase of human immunodeficiency virus (HIV) was expressed in yeast, the same results were obtained. These data allowed us to assume the excess of active reverse transcriptase to play the central role in induction of transposition. Possible mechanisms of induction of Ty1 transposition by homologous and heterologous elements are discussed.
Assuntos
DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos , Animais , Clonagem Molecular , Drosophila melanogaster/genética , Vetores Genéticos , Transcriptase Reversa do HIV , HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/genética , Saccharomyces cerevisiae/genéticaAssuntos
Elementos de DNA Transponíveis/efeitos dos fármacos , HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/genética , HIV-1/genética , Poli A/genética , RNA/genética , RNA Mensageiro , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Saccharomyces cerevisiae/genética , Transformação Genética/efeitos dos fármacos , Transformação Genética/genéticaRESUMO
The recombinant plasmid DNA YEp secl-v-sis was constructed. This plasmid was able to code for the synthesis and secretion into the cultural medium of the protein-product of oncogene v-sis. Transcription, copy number and stability of the plasmid DNA were studied under the conditions of galactose induction. The v-sis protein was determined by gel electrophoresis and immunoblotting methods and assayed for cell-proliferation activity in the mammalian cell culture.
Assuntos
Oncogenes , Proteínas Oncogênicas de Retroviridae/biossíntese , Saccharomyces cerevisiae/genética , Fatores de Crescimento Transformadores/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas Oncogênicas v-sis , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Fatores de Crescimento Transformadores/genética , Fatores de Crescimento Transformadores/metabolismoRESUMO
We have made a large scale analysis of prototrophic products of spontaneous and induced mitotic recombination within LYS2 gene of the yeast Saccharomyces. The mutant alleles staying in heterozygote with the wild type allele were uncovered and analysed.Among thirteen lys2 mutations used in the study three had reduced frequencies of mitotic gene conversion. These rarely converting mutations gave a remarkably high proportion of reciprocal events (up to 38%) in pairwise combinations, never seen for any other pair of alleles studied. Two of these mutations are the deletions of large parts of LYS2 gene.The results suggest that mispairing in the region of deletion blocks the hybrid DNA migration and leads to the reduced conversion ability of deletions. Comparison of uncovered alleles ratio in all allele combinations tested lead us to another hypothesis about bidirectional migration of hybrid DNA.