Preliminary Study On The Core Histone Gene Cluster And The Antibacterial Activity Of Histone H2A From Chlamys Farreri

Zhikong scallop is the major specie in aquaculture and contributes enormously to the economic development of coastal provinces in China. Since the summer of 1997, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, resulting in the production decreasing drastically. The durative outbreak of diseases has accelerated intensive efforts for the development of better health management strategies and characterization of original immune efforts for disease control. Although it is still not very clear about the pathogens for the scallop mortality, the immune response of scallop to the pathogen infection plays a key role in controlling outbreak of disease. Understanding the immune defense mechanisms of scallop may contribute to develop strategies for management of the disease and for long-term sustainability of scallop or mollusk farming.The identification and characterization of genes involved in scallop immune responses are now considered to be essential for the elucidation of immune defense mechanisms and disease control because of their potential use as therapeutic agents and genetic improvement biomarkers on disease-resistant strain selection. Antimicrobial effectors constitute the first line of innate immunity for scallop exposed to various potential pathogens in the aquatic environment by exerting broad-spectrum microbicidal activity.The nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri was of 5671 bp, which contained a copy of the four core histone genes H4, H2B, H2A and H3 amplified and identified by the techniques of homology cloning and genomic DNA walking. In the 3'flanking region of all the histone genes in the cluster had the structures in their 3'flanking region which related to the evolution of histone gene expression patterns throughout the cell cycle, including two different termination signals, the hairpin structure and at least one AATAAA polyadenylation signal. In their 5'region, the transcription initiation sites with a conserved sequence of 5'-PyCATTCPu-3'known as the CAP site were present in all genes except to H2B, generally 37–45 bp upstream of the start code. Canonical TATA and CAAT boxes were identified only in certain histone genes. In the case of the promoters of H2B and H2A genes, there was a 5'-GATCC-3'element, which had been found to be essential to start transcription at the appropriate site. After this element, in the promoter of H2B, there was another sequence, 5'-GGATCGAAACGTTC-3', which was similar to the consensus sequence of 5'-GGAATAAACGTATTC-3'corresponding to the H2B-specific promoter element. The presence of enhancer sequences was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes (5'-TGATATATG-3'). There were several slightly more complex repetitive DNA in the intergene regions. The presence of the series of conserved sequences and reiterated sequences was consistent with the view that mollusc histone gene cluster arose by duplicating of an ancestral precursor histone gene, the birth-and-death evolution model with strong purifying selection enabled the histone cluster less variation and more conserved function. Meanwhile, the H2A and the H2B were demonstrated to be potential good marks for phylogenetic analysis. All the results will be contributed to the characterization of repeating histone gene families in mollusc.Histone H2A is reported to participate in host-defense response through producing novel AMPs from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not been reported in mollusca to our knowledge. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate if the similar mechanism existed in mollusca. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not up-regulated after bacterial stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His+ Mut+) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G+) and Gram-negative (G-) bacteria. The antibacterial activity toward G+ bacteria was two and a half times more than that against G- bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. But if H2A was really involved in scallop immune response mechanisms needed to be further investigated.