J. Logan Irvin

Electrophoresis of histones and histone fractions on polyacrylamide gels

Abstract Zone electrophoresis of histones in polyacrylamide gels provides resolution of components which is superior to that obtained with starch gels, and the reproducibility of the method is excellent. The method has been applied to a comparison of unfractionated calf thymus histones with various fractions obtained by the methods of Ui and Johns and Butler. The very lysine-rich histone fraction yielded 1 mobile band in starch gel and 9 bands in polyacrylamide gel. Crystalline pancreatic RNase yielded 2 major and 6 minor bands on electrophoresis in polyacrylamide gel, and the mobilities were comparable to those of some of the histone components. A method is described for estimation of relative amounts of electrophoretically separated components by determination of bound dye.

The histones of rat testis

Abstract Polyacrylamide gel electrophoresis of the histones of the nuclei of seminiferous epithelial cells of rat testis revealed the five principal histone fractions which are found in liver and other somatic tissues, but, in addition, three unusual bands (desginated X1, X2, and X3) were observed. Fraction X1 had a mobility slightly less than that of F1 and was isolated with F1 in the fractionation procedure of Johns. F1 and X1 were separated by chromatography on carboxymethylcellulose, and they were shown by amino acid analyses to be closely related lysine-rich histones. However, X1 had lower content of lysine and alanine and higher content of arginine, aspartic acid, serine, proline, valine and leucine than F1. Both of these fractions had blocked amino-terminal residues, and both had a lysine residue at the carboxyl terminus. These fractions had similar molecular weights by electrophoresis on sodium dodecyl sulfate gels. Fraction X2 migrated between histone fractions F1 and F3 on electrophoresis while X3 migrated between fractions F2b and F3. Fraction X3 was isolated with F2b during fractionation by the Johns procedure. Fraction X2 has received minimal study, and this fraction may not be unique to the testis inasmuch as a faint band in approximately the position of X2 can be seen in electrophoretic patterns of rat liver histones. The results of the treatment of the histone fractions with alkaline phosphatase indicated that the electrophoretic differences between X1 and F1, or X3 and F2b are not attributable to phosphorylation.

Changes in basic chromosomal proteins during spermatogenesis in the mature rat.

Abstract Nuclei of the seminiferous epithelial cells of rat testis were filtered through glass wool to remove sperm heads, flagellae and late-stage spermatids and then centrifuged through sucrose gradients to yield three fractions. The cellular origins of the predominant nuclei in these fractions were identified through the kinetics of labeling with [3H]thymidine. The relative amounts of the different histone fractions changed during the various stages of spermatogenesis in an interesting and systematic manner. For example, the ratio of the trailing (acetylated) to the leading member of the histone F2a1 doublet was greater in spermatid nuclei than in nuclei of a fraction enriched in primary spermatocytes. Similarly, the ratio X 1 F 1 was greatest in spermatid nuclei. On the other hand, the ratio X 3 F 2b was greater in the nuclei of pachytene-diplotene primary spermatocytes than in the fraction enriched in nuclei of spermatogonia and preloptotene primary spermatocytes. A basic protein fraction with some of the properties of a protamine was extracted from rat sperm heads and from the nuclei of spermatids. This protein fraction has high contents of arginine and cysteine (after reduction), and it appears to be identical with the protamine described by Kistler et al. In addition, a new protamine was isolated from rat sperm heads which has high arginine content but appears to be devoid of lysine and cyst(e)ine. Two other basic protein fractions with high electrophoretic mobilities were extracted with acid from the nuclei of testicular seminiferous epithetial cells without prior reduction. One of these proteins may be identical with the testis-specific protein of Kistler et al.

POSSIBLE ROLE OF HISTONES IN REGULATION OF NUCLEIC ACID SYNTHESIS.

ALTHOUGH this paper will be concerned principally with our studies on the possible role of histones in regulation of nucleic acid synthesis, it seems pertinent to comment first upon the gel electrophoresis of histones. Butler has stated that the most satisfactory way of characterizing histones is the application of starch gel electrophoresis [S, 10, 111. We have found [ 121 that polyacrylamide gels are superior to starch gels in the separation of histones, and Reisfeld et al. [ 131 have reported success with this gel system in the electrophoresis of other cationic proteins. The gels are formed by mixing buffered tetramethy-ethylenediamine catalyst with 60 per cent acrylamide and 0.4 per cent N,N’-methylenebisacrylamide followed by addition of ammonium persulfate. The gels are formed in glass tubes and have a pH of 3 after polymerization. Samples are applied directly upon the gels in a density-stabilized layer, and electrophoresis is conducted in a glycine buffer at pH 4 for 4.5 hr at 15 ma. per tube. The gels are stained in amido black, and excess stain is removed electrically. Patterns obtained by electrophoresis of calf thymus histones are shown diagrammatically in Fig. 1 in comparison with patterns obtained by electrophoresis in starch gels. The bands shown in Fig. 1 C were found consistently by electrophoresis in polyacrylamide gels, and the bands are designated by Roman numerals. Bands II and IV, found in all calf thymus histone preparations examined in this laboratory, stained more heavily than the other bands and provided easily recognized and very reproducible reference points. The inclusion of a sample of unfractionated histone in each electrophoretic run of 6 samples facilitated the assignment of band identities in preparations of histone fractions. Band II was arbitrarily assigned a relative mobility (1~‘)

Effects of age and hypophysectomy upon relative proportions of various histones in rat testis.

Abstract Various histone fractions of seminiferous epithelial cells of the testis of rats change in relative proportions during development and maturation of the testis with increasing age after birth, and these changes in the histones occur in the reverse direction in testis of mature rats during involution of the testis resulting from hypophysectomy. The changes are particularly striking in histone sub-fractions, designated X 1 , X 2 , and X 3 , which are especially characteristic of testis and may be involved in meiosis.

Acetylation of histones of rat testis

Abstract When [1- 14 C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X 1 . In early periods of in vivo incorporation of [ 3 H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage. [ 3 H]Amino acids were incorporated into histone fractions X 1 and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other. Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.

Isolation of histone TH1-xB from rat testis

Summary The lysine-rich histones TH1-xA and TH1-xB, previously detected in rat testis nuclei by two-dimensional gel electrophoresis ( Levinger et al . (9) ), have been isolated and partially characterized. Histone TH1-xB has an unusually high arginine content for a H1 histone. Although TH1-xA is present in small amounts in testes of rats at 5 d after birth, TH1-xB is not detectable at 5 d or 14 d, but is present at 18 d. Both TH1-xA and TH1-xB are present in nuclei of pachytene primary spermatocytes and early spermatids.

Separation and quantitative analysis of rat testis histones by one-dimensional gel electrophoresis☆

Abstract A procedure is presented for direct separation and quantitative analysis of histones of rat testis by use of one-dimensional cylindrical gel electrophoresis in acid/urea/polyacrylamide gels at two concentrations of urea. After separation and staining with amido black the histone fractions are determined by extraction of bound dye and spectrophotometric analysis. This method provides a rapid and accurate procedure for determination of microgram amounts of the histone subfractions which are unique to the testis as well as those which are found in somatic cells, and it is particularly useful for determination of molar proportions of the histones.

Incorporation of glycine into protein fractions of nuclei of liver and hepatoma.

Summary A procedure is described for the separation of the proteins of cell nuclei into several fractions which differ in solubilities and in glycine content. Twenty hours after intraperitoneal injection of glycine-l-C14 into adult rats bearing subcutaneously transplanted hepatomas the specific activities of the protein fractions of the nuclei of liver and tumor were in the descending order: residual lipoprotein > globulins > histone fraction III > histone fraction II. Each of the protein fractions of the nuclei of liver cells had higher specific activities than corresponding protein fractions of the hepatoma with the notable exception of the histones which were in the reverse relationship.

Effect of in vitro histone phosphorylation on template activity of rat liver chromatin

Abstract Conditions for maximal phosphorylation of rat liver chromatin and free histones by a cyclic 3′,5′-AMP-dependent protein kinase preparation from calf liver were determined, and template activities of phosphorylated and non-phosphorylated chromatin for Escherichia coli RNA polymerase were compared. The phosphorylation of chromatin by the protein kinase preparation is stimulated by cyclic AMP, and both histones and non-histone proteins in chromatin are phosphorylated. The 32 P in histones from phosphorylated chromatin is principally in very lysine-rich histones (F1). In contrast, rat liver histones phosphorylated in the free state contain 54 % of the 32 P in slightly lysine-rich histones (F2b) and 14 and 18 % of the 32 P in F1 histones and glycine-arginine-rich histones (F2a1), respectively. The F1 histones bound to native DNA are poor substrates for the protein kinase preparation in comparison with free histones. However, F1 histones bound to denatured DNA are phosphorylated as effectively as free F1 histones. The inhibition of phosphorylation of F1 histones bound to native DNA increases as the ratio of histones to DNA increases, suggesting that F1 histones are bound to the DNA in compact clusters resulting in mutual interference with the phosphorylation sites. Maximally phosphorylated chromatin and the chromatin reconstituted with maximally phosphorylated histones show the same template activity for E. coli RNA polymerase as corresponding non-phosphorylated chromatin in terms of rate of incorporation of labeled nucleotides into RNA. Contrary to expectation, the chromatin reconstituted with histones from regenerating rat liver exhibits a slightly smaller template activity for E. coli RNA polymerase than the chromatin reconstituted with histones from normal rat liver.