Ru-chih C. Huang

The Biology of Isolated Chromatin

The isolated chromatin of higher organisms possesses several properties characteristic of the same chromatin in life. These include the presence of histone bound to DNA, the state of repression of the genetic material, and the ability to serve as template for the readout of the derepressed portion of the genome by RNA polymerase. The important respect in which isolated chromatin differs from the material in vivo, fragmentation of DNA into pieces shorter (5 x 106 to 20 x 106 molecular weight) than the original, does not appear to importantly alter such transcription. The study of isolated chromatin has already revealed the material basis of the restriction of template activity; it is the formation of a complex between histone and DNA. Chromatin isolated by the methods now available, together with the basis provided by our present knowledge of chromatin biochemistry and biophysics, should make possible and indeed assure rapid increase in our knowledge of chromosomal structure and of all aspects of the control of gene activity and hence of developmental processes.

[96] Isolation and characterization of chromosomal nucleoproteins

Publisher Summary Chromosomes are ordinarily obtained from cells during interphase and are, therefore, in the extended form known as chromatin. The advancement in the understanding of chromosomal structure and function has been made possible by the development of new methods for the handling of chromatin and chromosomal constituents. The isolation of chromatin is based upon differential centrifugation followed by sucrose density gradient centrifugation. Chromatin is among the most pelletable components of a tissue homogenate. The tissue is, therefore, ground in a suitable medium, freed of unruptured cells and membrane fragments by filtration, and sedimented at 1000–4000 g, conditions that do not bring down mitochondria. The pellet is then washed by repeated suspension and pelleting, finally layered on sucrose solution, and centrifuged for an appropriate period. By these methods, 60–75% or more of the DNA present in the original tissue is recovered as purified chromatin. The basic steps for the isolation of the highly contracted metaphase chromosomes are (1) accumulation of a large proportion of cells in metaphase by treatment with colchieine or other mitotic poison, (2) homogenization of the cells without damage to the chromosomes, (3) separation of the released chromosomes from cell debris. The separation of chromosomal nucleoprotein into its component entities can now be accomplished by the methods that are relatively mild and nondestructive as compared to those used in the past. An excellent example is the separation of histones from DNA by banding in a cesium chloride density gradient.

Physical and biological properties of soluble nucleohistones

Soluble nucleohistones have been reconstituted by gradient dialysis from individual histone fractions and a common preparation of DNA. The nucleohistones thus obtained sediment at rates approximately that of native nucleohistone and the preparations appear to consist to a substantial degree of individual DNA molecules fully complexed with histone. The complexes reconstituted from different histone fractions differ in their ability to support DNA-dependent RNA synthesis by pea chromosomal or Escherichia coli RNA polymerase, varying from inactive to highly active. The histone fractions differ also in the extent to which they stabilize DNA against thermal denaturation. The histone of lowest arginine-lysine ratio stabilizes DNA most and yields the nucleohistone least active in support of RNA synthesis.

Properties of chromosomal nucleohistone.

The deoxyribonucleohistone component may be physically separated from the other components of pea embryo chromatin. Such nucleohistone differs from deproteinized pea embryo DNA in its higher melting temperature, in its histone/DNA mass ratio (1·3: I) and in its inactivity in the support of DNA-dependent RNA synthesis. Pea embryo chromatin contains DNA not only as nucleohistone but also as DNA not so complexed. This is indicated by the two-step melting profile of chromatin as well as by the ability of chromatin to support DNA-dependent RNA synthesis. Approximately 80% of the DNA of pea embryo chromatin is bound in the form of nucleohistone; the remaining 20% appears to be present as DNA not complexed with histone.

CHROMOSOMALLY DIRECTED PROTEIN SYNTHESIS.

The different kinds of specialized cells of a higher organism differ among themselves in the kinds of enzymes and other proteins which they contain. Nonetheless, each possesses in the chromosomes of its nucleus the complete genomal DNA containing information concerning the manufacture of all of the kinds of enzymes contained in all of the kinds of cells of that creature. It is clear, therefore, that there is in the nucleus some mechanism for the control of genetic activity, a mechanism responsible for that orderly repression and derepression of gene activity which makes possible development. The present work concerns the control of genetic activity.

Enzymatic synthes is of RNA

Abstract This report concerns the synthesis of ribonucleic acid, RNA, by an enzyme system from pea embryos. The enzyme, as prepared, requires as substrate the four riboside triphosphates, ATP, CTP, GTP and UTP, and contains not only protein but a substantial fraction of the cellular DNA.