Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis.

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells.

Comparative enzymatic degradation of H1 subfractions from Syrian hamster tissues.

An additional hydrolysis site recognized by thrombin on histone H1 molecules was found. Snakes venom proteases from Agkistrodon rhodostoma, Bothrops marajoensis and Bothrops moojeni were further used for the analysis of H1 histones. The presence of the main cleavage site on H1 histone molecules has been established.

Histones and their modifications.

Histones constitute the protein core around which DNA is coiled to form the basic structural unit of the chromosome known as the nucleosome. Because of the large amount of new histone needed during chromosome replication, the synthesis of histone and DNA is regulated in a complex manner. During RNA transcription and DNA replication, the basic nucleosomal structure as well as interactions between nucleosomes must be greatly altered to allow access to the appropriate enzymes and factors.

Distribution of the H1 histone subfractions in Syrian hamster chromatin fractions.

Chromatin from two Syrian hamster tissues: the Kirkman-Robbins hepatoma and the liver, has been separated into soluble (S) and insoluble (P) fractions. Both fractions contain the complete set of five main histones but differ in respect of H1 subfractions. The hepatoma chromatin is known to contain an unusual H1 subfraction, H1 slow [12, 13], probably identical with a similar subfraction present in hamster testes.

Occurrence of the low-mobility H1 histones subfraction in embryonic, differentiated, and neoplastic tissues of the Syrian hamster.

Electrophoretically slow H1 histone subfractions with mobilities identical to that of the subfraction found in the Kirkman-Robbins hamster hepatoma chromatin have been shown to be present in 12-day hamster embryos and in a sarcoma-type hamster tumor induced by SV40. No subfractions of such mobility were found in hamster liver, regenerating liver, thymus, spleen, and a fast-growing transplantable amelanotic hamster melanoma. A suggestion is made that some defective mechanisms of differentiation may affect the regulation of expression of the genes coding for the H1 histone subfractions.

Chemical synthesis of nucleoside-gamma-[32P]triphosphates of high specific activity.

A simple chemical procedure for the preparation of four common ribonucleoside 5-gamma-[32P]triphosphates of high specific activity (up to 10 Ci/mmole) based on the condensation of orthophosphoric acid with the corresponding nucleoside 5-diphosphate in the presence of ethyl chloroformate as well as the methods of purification and identification of the products are described.

Effect on transcription of low-molecular-weight RNA from calf thymus chromatin.

1. Two low-molecular-weight nuclear RNA fractions were isolated from the loosely-bound non-histone proteins of calf thymus chromatin: RNA I (of 4 - 5.5S, heterogeneous on polyacrylamide-gel electrohoresis), and RNA IV (of about 3S).

Molecular cloning of the restriction fragments derived from double EcoRI/PstI digestion of the calf satellite I DNA and their restriction analysis.

Calf satellite I DNA digested with EcoRI and PstI gives three fragments 643, 621 and 83 bp long. Two of them, the 643 and 621 bp were cloned using pBR 322 vector and analyzed by means of the MspI and HaeIII restriction enzymes.

The 1360 bp long basic repeat unit of calf satellite I DNA contains GC rich nucleus of about 140 bp.

Fine melting profiles of calf satellite I DNA and its fragments obtained after digestion with endoR.EcoRI and endoR.AluI nucleases were investigated.

The fractionation of calf thymus DNA by multiple formation of sedimentable complexes with homologous lysine-rich histone KAP. Derivative melting curves and ultracentrifugation in CsCl concentration gradients.

The process of fractionation of total calf thymus DNA using a step precipitation of DNA by means of increasing concentrations of the homologous histone KAP was investigated. In addition to the known fractions three so far undescribed ones/in thymus/,characterized by buoyant densities in CsCl equal 1.692, 1.

The fractionation of calf thymus DNA on histone KAP-Sepharose 4B columns.

It was found that fractionation of calf thymus DNA on homologous histtone KAP covalently bound to CNBr activated Sepharose 4B depends on the molecular weight of DNA. The maximum of elution of high molecular DNA (m. wt.