Keiji Marushige

Template properties of liver chromatin

Isolated chromatin of rat liver supports DNA-dependent RNA synthesis catalyzed by added RNA polymerase. The product RNA, which is of high but heterogeneous sedimentation coefficient, possesses a base composition different from that of rat liver DNA and is not self-complementary. The template activity of such chromatin, which is five times less than that of deproteinized rat liver DNA, may be raised to that of the latter by selective removal of chromosomally bound histones by methods which leave chromosomal non-histone protein bound to DNA. Kinetic analysis of the interaction of template and RNA polymerase indicates that the polymerase binds equally well to DNA and to chromatin, but that in the latter, polymerase bound to a portion of the polymerase binding sites is prevented, by the presence of histone, from the execution of the transcription act.

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.

Properties of isolated chromatin from sea urchin embryo

Abstract Isolated chromatin of the sea urchin embryo supports RNA synthesis catalyzed by exogenous RNA polymerase. Such chromatin-primed RNA synthesis possesses the general characteristics known for DNA-dependent RNA synthesis. Pluteus chromatin is more active in support of RNA synthesis than is blastula chromatin. The results suggest that more genes are available for transcription in pluteus chromatin. Removal of proteins associated with DNA in chromatin increases template activity and abolishes the difference in template activity between blastula and pluteus chromatin. Blastula chromatin contains slightly more histone than does pluteus chromatin, whereas nonhistone protein content is substantially greater in the latter.