Histones and their measurement rna's rice embroys

Abstract

The ungerminated embryos contain all the information essential for the development of a whole organism. The process of germination represents a transition from a dormant to a metabolically active state. The nuclei isolated from ungerminated embryos were inactive in transcription, while the nuclei from embryos germinated for 12 and 24 hours showed vigorous synthesis of RNA. The analysis of RNA synthesized in 24-hour nuclei showed the synthesis of heterodisperse type of RNA. The synthesis of RNAs larger than 16S and smaller than 5S was unaffected in the presence of -amanitin, and therefore these may represent rRNAs and 4-5S tRNAs and their precursors, respectively. The synthesis of 7-16S RNA was abolished in the presence of -amanitin, indicating the messenger RNA (mRNA) nature of these RNAs.

Histones were extracted from the isolated nuclei, separated on acid-urea-acrylamide gels, and identified by their electrophoretic mobilities, differential incorporation of lysine and arginine, and the absence of incorporation of tryptophan. The syntheses of histones and DNA were parallel, with a maximum at 18 hours during the early phase of germination. The phosphorylation of histone H1 showed a significant increase during germination and seemed to be coordinated with DNA synthesis.

The ratio of histone/DNA in the nuclei from dry embryos was found to be 2.7 by weight, which decreased during the course of germination and reached near unity by 48 hours. However, the purified chromatin contained histone and DNA in equal amounts, suggesting that the excess histones found in the nuclei of dry embryos may exist in the nucleosol. The analysis of the basic proteins extracted from the nucleosol showed the presence of all the histones. When the fresh synthesis of basic proteins was inhibited by cycloheximide to the extent of 85%, the synthesis of DNA remained unaffected, which may indicate the utilization of the nucleosolic store of histones for complexing with newly synthesized DNA.

The cytoplasmic RNA from ungerminated embryos showed the incorporation of ³H-leucine into proteins in an in vitro translation system derived from rice embryos, while the nuclear RNA was inactive in protein synthesis. This indicated the presence of conserved mRNA in the dry embryos and its localization in the cytoplasm. The RNA from ungerminated embryos was fractionated into poly(A)+ and poly(A) fractions. Proteins synthesized by poly(A) RNA contained all the histones, which suggested the storage of histone mRNAs in dry embryos. The electrophoresis of poly(A) RNA from dry embryos on 6% polyacrylamide gels showed the presence of four discrete bands in the 9-12S region. The RNAs from individual bands were eluted out and translated in vitro. The mRNAs from bands I, II, III, and IV were found to code for histones H1, H2 & H2B, H3, and H4, respectively. The isolation and characterization of individual mRNAs for histones from rice embryos would be useful in studying the organization of histone genes in plants.