RESUMO
We analyzed the content of lamin B, one of the main proteins of the nuclear membrane, in different chromatin fractions obtained during purification of the nuclear matrix from different cell types. Depending on cell type and nuclear matrix preparation technique, lamin B was found in different not associated with matrix chromatin compartments. This effect was observed after chromatin extraction with ammonium chloride after nucleolysis and after chromatin extraction with sodium chloride before nuclease treatment. These findings suggest that the structure of the nuclear matrix is destabilized in certain extraction procedures and that studies of subcompartmentalization of nuclear macromolecules require additional control of nuclear matrix integrity.
Assuntos
Cromatina/metabolismo , Lamina Tipo B/metabolismo , Matriz Nuclear/metabolismo , Cloreto de Amônio/química , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa , Humanos , Cloreto de Sódio/químicaRESUMO
Experiments on resting hepatocytes with inactive c-fos gene and active albumin gene. We revealed that DNA of the transcribed gene is less susceptible to the influence of endogenous Ca2+/Mg(2+)-dependent DNases in matrix-associated and highly soluble chromatin fractions. In the fraction of low soluble chromatin active gene was more accessible for DNases. Our results indicate that activity of endogenous DNases can change in the transcribed gene locus.
Assuntos
Cromatina/metabolismo , Desoxirribonucleases/metabolismo , Hepatócitos/metabolismo , Transcrição Gênica , Albuminas/genética , Animais , Regulação da Expressão Gênica , Genes fos , Masculino , Ratos , Ratos WistarRESUMO
Accumulation of c-fos gene locus DNA in the nuclear matrix of hepatocyte nuclei was observed during induction of c-fos with cycloheximide. No enhanced association with the nuclear matrix was detected for inactive immunoglobulin gene locus. The use of endogenous DNAses allows isolation of nuclear matrix preparations enriched with transcribing chromatin.
Assuntos
Cromatina/metabolismo , Genes fos , Matriz Nuclear/metabolismo , Animais , Cromatina/genética , Cicloeximida/metabolismo , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/fisiologia , Imunoglobulina G/genética , Masculino , Inibidores da Síntese de Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
Degree of nucleolysis has critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences as demonstrated at the example of inactive (c-fos, c-myc, and Ck) and active (p53, albumin, and 28S rRNA) genes in resting hepatocytes. Optimal degree of nucleolysis features degradation of loop domains of chromatin with preserved relatively uniform molecular weight distribution of DNA. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix.