H2A, H2B and H4 histone genes of rice embroys

Abstract

A knowledge of the organization of histone genes in plants is a prerequisite for a greater insight into the regulation of their gene expression during germination. With the idea of obtaining much-needed information on the organization of histone genes in plants, rice embryo histone genes were studied. DNA was isolated from 48-hour germinated rice embryos, digested with BamHI, and cloned in pBR322 at the BamHI site. Recombinant clones were selected by ampicillin resistance and tetracycline sensitivity.

They were further probed by colony hybridization using ³²P-labelled homologous poly(A) and poly(A) 25S, 18S, 9–12S, and 4S RNAs isolated from 18-hour germinated rice embryos. Four clones, two each hybridizing with 9–12S and 4S poly(A) RNA fractions, which are enriched in histone messengers and tRNAs respectively, were selected.

One of the clones hybridizing to the 9–12S poly(A) RNA fraction was found to contain an insert of 6.64 kb in size. Total RNA from 18-hour germinated rice embryos was hybridized to recombinant DNA from both the clones, eluted, and translated in a cell-free translation system derived from dry rice embryos using ³S-methionine or ¹C-amino acids as the labelled precursor. The protein products were analyzed on a Panyim and Chalkley acid–urea polyacrylamide gel along with standard rice histone markers, and the radioactive products were detected by fluorography. The protein bands that were visualized corresponded to histones H2A, H2B, and H4. All the radioactivity incorporated into the proteins was recovered in the three bands, showing that the insert does not hybridize with any other translatable RNA. This demonstrates that the insert codes for only three histone proteins.

The insert from one of the clones, purified on agarose gel and DE-52, was labelled at the 5 ends using -³²P-ATP and T4 phage polynucleotide kinase, cleaved with the single-cut enzyme EcoRI, and the two fragments of sizes 5.35 kb and 1.29 kb were isolated. A restriction map of the insert was constructed using eight restriction endonucleases according to the method of Smith and Birnstiel. The map showed that the sites for GC-specific enzymes tend to cluster at the two ends of the insert, indicating that the histone genes, which are generally GC-rich, may be located at the ends of the insert.

The insert was digested with PstI, EcoRI, and HpaII to give overlapping fragments. The fragments were separated on an agarose gel, transferred onto a nitrocellulose filter, and hybridized to RNAs isolated from 18-hour germinated rice embryos. The hybrid-captured RNAs were eluted, translated in vitro using ³S-methionine, and the products were identified. The results showed that the H2A gene was located at one end of the insert, followed by the H2B gene, and the H4 gene was located at the other end.

RNAs that hybridized to the individual strands separated on an agarose gel following alkali treatment were eluted, translated in vitro, and the products identified. It was found that H2A and H2B were encoded by one strand and H4 by the other, implying bidirectional transcription of histone genes in rice embryos.

The large EcoRI fragment carrying H2A and H2B genes was sequenced from the 5 end using the chemical method of Maxam and Gilbert. A sequence of about 320 nucleotides, including part of the coding region for H2A, was obtained. The 5 region showed typical eukaryotic promoter elements such as the “CAAT” box and the “TATA” box (Goldberg–Hogness box), as well as a histone-specific sequence motif 5 GATC 3.

There are minor differences in the location of these consensus sequences in rice embryo histone genes compared to histone genes from other sources. However, by and large, the organization of histone genes and the promoter elements involved in their expression in rice embryos are similar to those in other systems.