Lamin B content in chromatin fractions after purification of nuclear matrix from cells of different types.

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.

[Endogenous DNases as a tool for isolation of nuclear matrix: critical parameters of nucleolysis].

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.

Functional association of immediate early gene c-fos with nuclear matrix.

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.

Dual effects of endogenous DNases on transcriptionally active and inactive chromatin.

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.

[Circadian rhythm of tRNA aminoacylation in hepatocyte culture].

In the monolayer of rat hepatocytes in vitro, the circadian rhythms were revealed of 3H-leucine incorporation in proteins and in aminoacyl-tRNA(Leu) fraction. The oscillations were mainly synphasic, though coordination between aminoacylation and protein synthesis was not stable in the coarse of time. In cell free system with the excess of ATP and aa-tRNA, the rhythm of 3H-leucine incorporation was also clear.