Cloning And Functional Analysis Of Histone H3and H4in Euplotes Octocarinatus

Histones are basic components of nucleosome and essential to chromatin structure and function. Histone posttranslational modifications and sequence variants jointly participate in modification of chromatin and regulation of gene expression. Histones are highly conserved proteins in different organism. However, a high degree of variation was found in ciliate.Euplotes octocarinatus, a free-living unicellular ciliated protozoan, in which programmed genome rearrangement occurs during the conjugation stage. Histones are involved in the degree of fragmentation and amplification of chromosomes during the development of macronuclei. In this study, different variants of histone genes:H3P and H3V; H4A and H4B were systematically investigated. The mian results obtained are as follows:1. Cloning and sequence analysis of H3P and H3V gene from E. octocarinatus. In the study, H3P and H3V were cloned from E. octocarinatus by PCR. Sequences analysis showed that the macronuclear DNA carrying H3P is672bp. The opening reading frame of the H3P gene was432bp, which encoded a143amino acid polypeptide with a predicted molecular mass of16.4ku and isoelectric point of11.38.The macronucleus DNA carrying H3V is1623bp. The opening reading frame of the H3V gene was411bp, which encoded a136amino acid polypeptide with a predicted molecular mass of15.5ku and isoelectric point of11.35. There is no intron in the macronucleus DNA of H3P and H3V. H3P gene shared78%-87%identity with reported other H3gene, while H3V shared85%—88%identity with H3gene from other eukaryotes. H3P shared78.7%identity with H3V. Real-time PCR showed that H3V transcript was higher than H3P. The results indicated that the H3gene have different variant in E. octocarinatus.2. Localization of histone H3P and H3V. To investigate the functions of Histone H3in E. octocarinatus, H3P and H3V were cloned into a macronuclear artificial chromosome of E. octocarinatus which harboring codon-optimized EGFP gene. pEGFP-EoH3P and pEGFP-EoH3V were constructed and transformed into E. octocarinatus. Immunofluorescence localization of pEGFP-EoH3P and pEGFP-EoH3V showed H3P was located in the macronucleus and the H3V was dispersedly distributed in the E. octocarinatus.3. Cloning and sequence analysis of H4A and H4B gene from E. octocarinatus. In the study, H4A and H4B were cloned from E. octocarinatus by PCR. Sequences analysis showed that the opening reading frame of the H4A gene was324bp, which encoded a107amino acid polypeptide with a predicted molecular mass of11.6ku and isoelectric point of10.99. The macronucleus DNA carrying H4B is611bp. The opening reading frame of the H4B gene was384bp, which encoded a127amino acid polypeptide with a predicted molecular mass of14.4ku and isoelectric point of9.93. There is no intron in the macronucleus DNA of H4A and H4B. H4A gene shared a high identity of81%-94%with reported H4gene, while H4B shared an identity of36%-70%with H4gene from other eukaryotes. H4A shared44.7%identity with H4B. Real-time PCR showed that H4A transcript was higher than H4B. The results indicated that the H4gene have different variant in E. octocarinatus.4. Expression and purification of histone H4A and H4B. The H4A and H4B gene were constructed into prokaryotic expression vector pET-28a by recombinant DNA techniques, and the recombinant plasmid pET-28a-H4A and pET-28a-H4B were transformed into E.coli BL21. The fusion protein His6-H4B was expressed with0.5mmol/L IPTG induction6hour in E.coli BL21. Howerve, His~6-H4B was expressed as inclusion body at lower level amounting to10%of the total bacterial protein, which made purification by Ni-NTA sepharose affinity chromatography more difficult.In conclusion, the histone H3and H4from E. octocarinatus have been cloned and analysed in this study. The expression level of histone H3and H4were studied by real-time quantitative PCR. The results indicated that the H3and H4gene have different variant in E. octocarinatus. Furthermore, the localization of histone H3and the expression of histone114in E. octocarinatus were studied for the first time. H3P localized to the macronucleus ofE. octocarinatus, suggesting that H3P may play a role in the DNA rearrangement processes of macronuclear development. These studies provide new data for further exploring the evolution and function of the histone H3and H4.