[Exon-intron structure and organization of the 5'-terminal region of the human p53 gene]. / Ekzon-intronnaia struktura i organizatsiia 5'-kontsevoi oblasti gena p53 cheloveka.

S1 mapping and nucleotide sequencing were used for localization of exon-intron junctions of the human p53 gene. The 3'-end of the gene was localized 12 nucleotides downstream AATAAA sequence, though an mRNA, 94 nucleotides shorter, was observed in one tumor. No typical promoter sequences were found at the 5'-end of the gene. In cell-free HeLa extracts transcription of RNA initiates within the sequence of possible hairpin-loop structure. Organization of the promoter region of the human p53 gene is discussed.

Identification of sequence-specific DNA-binding factors by label transfer: application to the adenovirus-2 major late promoter.

A method of affinity labelling proteins specifically associated with DNA target sequences is proposed. The method utilizes covalent UV-crosslinking of proteins to highly labelled DNA (e.g. in crude cell or nuclear extracts) followed by degradation of the DNA to short oligonucleotides. Proteins selectively labelled by attached residual oligonucleotides are readily amenable to molecular mass determination. Using this approach, we have characterized a HeLa polypeptide specifically bound to a short segment of the adenovirus-2 major late promoter (Ad2 MLP). A molecular mass value (approximately 51 kD) and precise location of the crosslinking site(s) of the protein within the MLP (-55 with respect to the cap site) were determined.

Histone crosslinking patterns indicate dynamic binding of histone H1 in chromatin.

Crosslinking of histone H1 molecules to each other and to the core histones with bifunctional reagents in mouse liver nuclei and chromatin was compared with that under the conditions of random 'contacts' between these molecules. The patterns of crosslinking of the H1 subfractions (H1A, H1B, and H10) to each other in nuclei, chromatin and in solution at different ionic strengths due to random collisions were essentially the same. Moreover, the contacts between the H1 molecules were qualitatively the same in nuclei, chromatin and in solution also at the level of the chymotryptic halves of the H1 molecules. The contacts between the H1 molecules and the core histones in nuclei were similar to those obtained in chromatin at 70 mM NaCl, when H1 molecules readily migrate, and at 0.6 M NaCl, when H1 molecules are dissociated from chromatin. We conclude that spatial arrangement of H1 subfractions and mutual orientation of H1 molecules in isolated nuclei are random-like at least in terms of cross-linking. The static and dynamic models of histone H1 binding to chromatin compatible with the known data are considered. Although unequivocal verification of the models is not possible at present, the dynamic models do correspond better to recent data on the location of the histone H1 in nuclei and chromatin.

[Problems of cellular activity regulation by secondary messengers]. / Problemy reguliatsii kletochnoi aktivnosti posredstvom vtorichnykh messendzherov.

The paper surveys the data obtained during the last years in the Laboratory of Regulation of Cellular Activity of the Institute of Molecular Biology. The data mostly concern the enzymes taking part in metabolism and in biological action of the secondary messengers, as well as the mechanisms of regulation of some metabolic pathways in the cultured cells. The results pertinent to the mechanisms of chromatin condensation and based on this genetic control processes are also discussed.

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes.

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.

[Localization of histone H1 in chromatin. Cross-linking of the N- and C-terminal halves of the molecule with bifunctional reagents]. / Raspolozhenie gistona H1 v khromatine. Sshivanie bifunktsional'nym reagentom N- i C-kontsevykh polovin molekuly.

Mutual arrangement of histone H1 molecules was studied in calf thymus nuclei, extended chromatin and chromatin, isolated and kept in 8 M urea. Histone H1 dimers crosslinked with methyl 4-mercaptobutyrimidate were digested with chymotrypsin and crosslinked fragments obtained were analysed by diagonal gel electrophoresis. In all chromatins tested the N- and C-terminal parts of the H1 molecules were crosslinked in all possible combinations, i.e. C-C, C-N and N-N. These and related data obtained earlier indicate, that the proximity of histone H1 molecules in chromatin is determined by the structure of nucleosomal chain itself and not by chromatin superstructure. The results also suggest that the H1A and H1B subfractions of histone H1 are interspersed in extended nucleosomal chains.

[Localization of histone H1 in chromatin. Cross-linking of central globular regions of H1 molecules with a bifunctional reagent]. / Raspolozhenie gistona H1 v khromatine. Poperechnoe sshivanie tsentral'nykh globuliarnykh chastei molekul H1 bifunktsional'nym reagentom.

Mutual arrangement of histone H1 molecules and central globular parts of H1 was studied by crosslinking with a reversible bifunctional reagent. The yields of histone H1 dimers and dimers of it's globular fragment in nuclei and isolated chromatin were similar. In the presence of 8 M urea the yield of the H1 dimers was approximately threefold decreased, dimers of globular fragment being practically absent. The data suggest that the proximity of H1 molecules in nuclei is stipulated by a structure of a nucleosomal chain itself and not by chromatin superstructure. The results are in accord with the "head" to "head" histone H1 orientation within the nucleosomal chain and do not support participation of the central globular region of H1 molecule in chromatin condensation. A model of H1 arrangement in extended nucleosomal chain is proposed.

Mutual arrangement of histone H1 molecules in extended chromatin. Chymotryptic digestion of cross-linked H1 histone dimers.

Mutual arrangement of histone H1 molecules in chromatin extended in low salt-EDTA buffer and additionally in the presence of urea was studied by means of reversible cross-linking combined with chymotryptic digestion. In the chromatins tested, the chymotryptic halves of H1 were cross-linked in all possible combinations; i.e., C-C, C-N and N-N. The results imply that the mutual arrangement of H1 histones is determined by the structure of extended nucleosomal chain, rather than chromatin superstructure.

[Structure, chemical modification and interaction of histone H1 with chromatin components]. / Struktura, khimicheskaia modifikatsiia i vzaimodeistvie gistona H1 s komponentami khromatina.

Structure, chemical modification, and interaction of histone H1 and its individual fragments with DNA and structural elements of chromatin are considered. Special attention is paid to phosphorylation of histone H1 molecules. Recent data concerning localization and mobility of histone H1 in chromatin as well as mechanisms of nucleosomal chain condensation are reviewed.

[Residual chromatin of mouse spleen nuclei after intensive nuclease treatment. Isolation and properties]. / Ostatochnyi khromatin iader selezenki myshi posle intensivnoi obrabotki nukleazoi. Poluchenie i svoistva.

Two-step treatment of mouse spleen nuclei with staphylococcal nuclease was used to isolate residual nuclear structures lacking a considerable part of chromation. Partial disruption of the nuclear envelope after the first step of digestion was shown to be essential for obtaining residual nuclear structures. Isolated residual nuclear structures contained condensed chromatin (residual chromatin) which was not solubilized upon additional staphylococcal nuclease treatment and amounted to approximately 20% of total nuclear chromatin. Residual chromatin was almost deprived of nonhistone chromosomal proteins. It contained a full complement of histones and consisted of nucleosomal chains having different lengths--from one to 50-60 nucleosomes. Some of the condensed chromatin chains were anchored to the nuclear matrix.

[Properties of mouse spleen residual condensed chromatin associated with the nuclear matrix]. / Svoistva ostatochnogo kondensirovannogo khromatina selezenki myshi, assotsiirovannogo s iadernym matriksom.

Properties of condensed residual chromatin of mouse spleen, a component of residual nuclear structures, were studied. Extraction of the structures with buffers of different NaCl concentrations showed that the condensed chromatin consists of condensed nucleosomal chains. On increasing the ionic strength the complexes gradually fell apart into separate nucleosomal chains. DNA of condensed chromatin was accessible to staphylococcal nuclease and DNAase I, but digestion of this DNA was not accompanied by solubilization of the residual chromatin. Besides the essentially decreased total content of nonhistone chromosomal proteins the condensed chromatin practically did not contain HMG proteins. The nucleosome repeat length of this chromatin was shorter than that of chromatin solubilized by staphylococcal nuclease.

Chemical crosslinking of histone H1o to histone neighbours in nuclei and chromatin.

Crosslinking of histones in mouse liver nuclei and extended chromatin with a bifunctional reagent leads to the formation of H1-H1o heterodimers as well as H1o-H1o homodimers. H1o can be also crosslinked to the core histones. Thus, the location of histone H1o within the basic repeating chromatin structure seems to be analogous to that of H1 histone.

Protection of expressed immunoglobulin genes against nuclease cleavage.

Fragmentation of the actively transcribed kappa immunoglobulin gene in mouse myeloma nuclei with micrococcal nuclease and the restriction nuclease Bsp RI reveals a chromatin structure without the regularity of repeating nucleosomes found in bulk chromatin. Such regularity is restored about 2.2 kb 3' of the coding region. An only moderately increased micrococcal nuclease sensitivity and a 65% average protection of the Bsp RI sites indicates a DNA-protein interaction in the transcribed region which is not very different from that of an inactive gene. As determined by indirect endlabeling the frequency of Bsp RI cleavage both, after very mild and exhaustive digestion, varied moderately from site to site along the gene. In addition, it was not in each case the same at analogous sites on both alleles which are both transcribed. Thus, the experiments demonstrate differences between the chromatin structures of the genes which may be related to regulatory phenomena and thereby corroborate earlier findings made with DNAase I.

Nucleosome distribution on the Jk and Ck immunoglobulin gene segments of mouse liver chromatin.

The chromatin structure of the transcriptionally inactive kappa immunoglobulin gene in mouse liver was investigated by mainly employing indirect endlabeling on Bsp RI restriction nuclease digestions of intact nuclei. The disclosed strong (about 85%) but not uniform protection of the Bsp RI sites by nucleosomes is inconsistent with both a uniquely sequence-oriented localization and a completely random distribution of nucleosomes in this region of the genome. Several possibly applicable models are discussed. A model with multiple phases and non-uniform linker lengths cannot be excluded; however a largely random localization with a weak superimposed organization in two confined areas was tested and found sufficient for explaining the data.

Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei.

In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites. In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele. Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences. Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence. The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions.

Conditions for sliding of nucleosomes along DNA: SV 40 minichromosomes.

'Sliding' of nucleosomes along DNA under nearly physiological conditions was studied using treatment of SV 40 minichromosomes with the single-cut restriction endonucleases EcoRI and BamHI. Each enzyme can convert no more than 20-25% of the circular DNA molecules of minichromosomes into the linear form irrespective of the presence of histone H1. This suggests absence of the nucleosomes lateral migration (sliding) along DNa at least in the vicinity of the restriction endonucleases cleavage sites during several hours of incubation. The sites available for EcoRI and BamHI in minichromosomes seem to be located predominantly in the spacer DNA regions of nucleosomes. Introduction of only one double-strand (but not single-strand) break into the DNA of minichromosomes stripped of histone H1 is sufficient to induce redistribution of the nucleosome core particles due to their sliding along DNA. Thus, sliding of the nucleosome core particles can be induced under physiological conditions by rather low energy expenditures.