II.18 Histones and nuclear proteins of Hiproly barley and an isogenic line.
R. G. McDaniel, Department of Agronomy, University of Arizona, Tucson, Arizona 85721, U.S.A.
Histones and nuclear proteins were isolated from seeds (kernels) and seedlings of Hiproly barley, CI 3947, and an isogenic line, CI 4362, grown adjacently, which were obtained from D. M. Wesenberg. Nuclear proteins were isolated (1) by direct acid extraction of buffered tissue homogenates, and (2) by homogenizing the tissues in sucrose-phosphate-KCl-MgC12-EDTA buffer, filtering through nylon, and centrifuging to pellet chromatin. After washing, the chromatin was suspended in tris buffer. In some experiments, centrifugation through a sucrose gradient was the final purification. Histones were extracted by acidifying the slurry with dilute HCl. Appropriate washing, centrifugation and resusupension procedures were included. Histones were concentrated by lyophilization and resuspended in homogenization buffer. This preparation was used for electrophoresis (Methodology modified from Johns & Butler, Biochem. J. 82:15, 1962; Fambrough & Bonner, Biochem. 5:2563, 1966; Panyim & Chalkley, Biochem. 8:3972, 1969). Electrophoretic techniques were modifications of the methods of Ornstein, (Ann. N. Y. Acad. Sci. 121:321, 1964) for 15%, pH 2.9 gels; and of Reisfeld, et al., (Nature, 195:281, 1962) for 15%, pH 4.3 gels. Urea was included in gels and sample of both systems.
Histones were resolved as seven bands after staining gels with Amido schwarz. When compared with standard calf thymus histones, only quantitative differences were noted between these proteins. Two additional histone fractions were seen in very concentrated barley protein preparations, however, which were not present in the animal preparations. When histone bands of Hiproly and its isogenic line were compared, quantitative differences were observed. Hiproly had a relatively greater amount of histone fraction F1 (very lysine-rich) than did the isogenic line. In addition, Hiproly had less histone fraction F2al (glycine-rich) than its isogenic line. These differences were determined both on a per seed basis and on a total histone basis. Slight quantitative differences in acidic ribonuclear proteins of these lines were also noted. Histone bands of Betzes, CI 6398, and Ubamer, CI 12167, were also quantitatively, and in the case of Ubamer, qualitatively different from histones of Hiproly and CI 4362.
These data demonstrated specific differences between the histones of Hiproly and an isogenic line. As the total protein per seed of Hiproly was greater than that of its isogenic line, the quantitative increase in lysine-rich histone observed in Hiproly may or may not be significant when compared on a total protein or other basis. The possible relation of these data to the problem of lysine levels in barley remains undetermined. Few data, in fact, are extant on the contribution of nuclear proteins to total seed protein constitution. I believe this approach is deserving of further study, however, since a protein whose quantity may be associated with, or responsible for, increased total seed protein would have the potential of being utilized as a breeding tool if its pattern of inheritance were determined (for example, the work of Kirk & Jones, Chromosoma, 31:241, 1970, on histones and B chromosomes in rye and that of Riley and Ewart, Genet. Res. 15:209, 1970, on chromosomes and amino acid content of wheat). Characterization of barley histones by amino acid analysis is presently underway, as is a comparative study of histones and nuclear proteins from a numer of selected barley cultivars. These studies should give us a more comprehensive understanding of the role of these proteins in contributing to the amino acid constitution of barley.