RESUMO
Blastoderm cells from chicken embryos of a donor breed (Green-legged Partridgelike; GP) were transferred to embryos of a recipient breed (White Leghorn; WL) to form chimeric progeny that, after inter se mating, permitted successful reconstitution of the donor breed. Among 23 chimeric chicks hatched from WL embryos injected with GP cells, 20 (87%) were raised until maturity, and progeny were tested by mating with GP birds to determine the ability of blastodermal cells to form germline chimeras. Six of the tested birds (30%) produced recipient-derived and donor-derived offspring, indicating that they were germline chimeras. The mean percentages of donor-derived germ cells in these birds were 21.1 (17.6 to 50.0%) and 16.9 (5.3 to 23.1%) in males and females, respectively. Among 477 chicks, resulting from mating the germline chimeric male with four germline chimeric females, 10 chicks (2.1%) exhibited a GP phenotype, indicating that the original donor stock had been reconstituted. Only one germline chimeric hen produced GP offspring, but the expected and calculated percentages of GP offspring were similar (2.99 and 2.08, respectively). Two methods of DNA analyses (RFLP and PCR amplification of polymorphic microsatellite loci) of chimeras and their offspring indicated that through mating of a relatively small number of chimeras it is possible to reconstitute a highly diverse population.
Assuntos
Galinhas/genética , Quimera/genética , Mutação em Linhagem Germinativa , Animais , Blastoderma/transplante , Embrião de Galinha/fisiologia , Cruzamentos Genéticos , Feminino , MasculinoRESUMO
Elements controlling high expression of the 17-1A antigen gene in pancreatic carcinoma cells (Capan 2) reside within the two regions: proximal (-193 to +3) and distal (-877 to -518). We demonstrate here that some factors present in nuclear extracts from nonexpressing cells bind specifically to the control elements, important for gene expression. Our results suggest that nonexpressing cells may either lack at least one of the factors necessary for activation or may contain their modified forms. A major difference between expressing and nonexpressing cells was found in the region containing core enhancer sequence. Moreover, nonexpressing cells display a complex pattern of DNA-protein interactions in this region, suggesting that these cells contain factors acting negatively mainly on the enhancer sequence. Our results however, indicate that the mechanism of repression is much more complicated than expected.
Assuntos
Antígenos de Neoplasias/genética , Carcinoma de Células Escamosas/genética , Carcinoma/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Antígenos de Neoplasias/biossíntese , Sequência de Bases , Carcinoma/patologia , Carcinoma de Células Escamosas/patologia , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Transcrição Gênica , Células Tumorais Cultivadas/metabolismoRESUMO
The interaction of low mobility group proteins (LMG), isolated from chromatin of pancreatic carcinoma cells (CAPAN-2), with fragments of 5'-flanking region of the antigen 17-1A gene was studied by gel retardation assay. The LMG proteins, which formed complexes with DNA were extracted from the gels and identified by polyacrylamide gel electrophoresis under denaturing conditions. The proteins of Mw about 100, 60, 55 and 48 kDa, which formed specific complexes with fragments of 5'-flanking region of the antigen 17-1A gene, were identified.
Assuntos
DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Animais , Antígenos de Neoplasias/genética , Sequência de Bases , Cromatina/química , DNA de Neoplasias/análise , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/análise , Oligonucleotídeos , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Ligação Proteica , Ratos , Células Tumorais CultivadasRESUMO
To determine the location of sites important for the function of the 17-1A antigen gene promoter and to characterize the protein factors binding to these sites, fragments of the promoter region were analysed by gel retardation assay with nuclear extracts from Capan 2 cell line. At least two separate regions, which specifically bind nuclear proteins, were identified within the 5'flanking region of the 17-1A antigen gene. These regions have been located between nucleotides -877 to -518 (distal region) and -193 to +3 (proximal region) and presumably participate in regulation of expression of the 17-1A antigen gene.
Assuntos
Antígenos de Neoplasias/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Regiões Promotoras Genéticas , TATA Box , Adenocarcinoma , Sequência de Bases , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/isolamento & purificação , Oligodesoxirribonucleotídeos/síntese química , Neoplasias Pancreáticas , Ligação Proteica , Mapeamento por Restrição , Especificidade por SubstratoRESUMO
The protein of molecular weight about 160 kD (designated LMG160) was isolated from purified low mobility group chromatin proteins. Polyclonal antibody directed against the LMG160 protein in mouse was raised. The specificity of the antibody was determined with the use of ELISA. Using chemical cross-linking procedure followed by immunoprecipitation with the antiLMG160 antibody complex formation with chromatin proteins was demonstrated. Among the proteins that form complexes with LMG160, histones H3, H2A, and H4 were identified (Western blotting technique).
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reagentes de Ligações Cruzadas , Histonas/metabolismo , Fígado/metabolismo , Succinimidas , Animais , Anticorpos , Western Blotting , Cromatografia de Afinidade , Proteínas Cromossômicas não Histona/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Peso Molecular , RatosRESUMO
Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fígado/metabolismo , Fatores Etários , Animais , Embrião de Galinha , Cromatina/isolamento & purificação , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , EndodesoxirribonucleasesRESUMO
Electrophoretic profiles of [3H] tryptophan labelled non-histone chromatin protein fractions of liver were studied during the embryonic development of chicken. In embryonic chicken liver a characteristic of non-histone chromatin protein fraction K (molecular weight 18 000) was synthesized. The synthesis of this fraction changed quantitatively during various days of chicken development. The greatest changes of non-histone chromatin proteins were observed on the 21st day of development (day of hatch), when besides the synthesis of a group of low molecular weight proteins (fractions J,K,L,M) the synthesis of fractions of non-histone proteins of high molecular weight was also higher than on other days. Significantly, a high molecular weight fraction, not analyzable in 7.5% PAGE, was present only in the early days (7th-12th) of our investigations.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Fígado/embriologia , Fatores Etários , Animais , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Triptofano/metabolismoRESUMO
In fetal and adult rat liver synthesis of [3H] tryptophan labelled non-histone chromatin proteins was low. At birth, synthesis of non-histone chromatin proteins dramatically increased, especially fractions of high molecular weight. The role of these fractions appearing only at the first day of life (after parturition) is not clear.