Douglas M. Fambrough

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.

Limited molecular heterogeneity of plant histones

Abstract 1. 1. The histones of pea-bud chromatin have been fractionated and purified by ion-exchange and gel-filtration chromatography and by preparative disc electrophoresis. 2. 2. Four of the purified histone components are single molecular species as judged by the criteria of chromatographic behavior, disc electrophoresis, N- and C-terminal amino acids and number of tryptic peptides. 3. 3. Two further histone fractions contained pairs of electrophoretically separable components. Each of these fractions appears by the above criteria to represent only two molecular species. 4. 4. Thus pea-bud histone contains only eight molecular species. 5. 5. There is a one-to-one correspondence between the major types of pea-bud and mammalian histones.

[27] Biosynthesis and intracellular transport of acetylcholine receptors

Publisher Summary The acetylcholine receptors of vertebrate skeletal muscles and the electric organs of certain fish are multisubunit, integral membrane proteins that become cation-selective ion channels in response to the binding of acetylcholine. This chapter focuses on techniques and strategies to elucidate events in the biosynthesis and turnover of acetylcholine receptors. Quantitative analyses of acetylcholine receptor number and distribution generally involve the use of very high affinity ligands whose binding sites on the receptor molecule overlap the binding sites for acetylcholine, and the most widely used of these ligands is a-bungarotoxin. Studies on acetylcholine receptor biosynthesis are usually carried out on early postfusion myotubes that possess several hundred receptor sites per square micrometer of cell surface. Among the most common strategies for determining the kinetics of biosynthesis and turnover of molecules in living systems, the metabolic labeling strategy involves supplying a tagged precursor to the system either continuously or for a defined period and measuring the amount of precursor incorporated into the molecule of interest as a function of time.