Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by u

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Cloning of rainbow trout (Oncorhynchus mykiss) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

Rainbow trout historic H3 (RH3) promoter was cloned via high fidelity PCR. The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1, resulting in an expression vector pRH3FGFP-1. The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope. Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage. The results indicate that RH3 promoter, as a piscine promoter, could serve in producing transgenic Cyprinoid such as rare minnow. Promoter activity of RH3, CMV and common carp beta-actin (CA) were compared in rare minnow by the expression of respective recombinant EGFP vectors. The expression of pCMVEGFP occurred earlier than the following one, pRH3EGFP-1, and then pCAEGFP during the embryogenesis of the transgenics. Their expression activities demonstrated that the CMV promoter is the strongest one, followed by the CA and then the RH3.

Genomic organization, nucleotide sequence analysis of the core histone genes cluster in Chlamys farreri and molecular evolution assessment of the H2A and H2B

This work represents the nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri. The tandemly repeated unit of 5671 bp containing a copy of the four core histone genes H4, H2B, H2A and H3 was amplified and identified by the techniques of homology cloning and genomic DNA walking. 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'-PyATTCPu-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 (5'-TGATATATG-3') was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes. 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 molluscs.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. 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 whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. 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 upregulated after 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 2.5 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. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex. (C) 2010 Elsevier Inc. All rights reserved.

Identification and molecular evolution of cow CENP-A gene family

The centromere protein A (CENP-A), a histone H3-like protein, provides an essential role for chromosomal segregation during mitosis and meiosis. In this study we identified ten new CENP-A-like genes (excluding the original CENP-A gene) in cow by searching

The nuclear matrix of Euglena gracilis (Euglenophyta): A stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracillis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Regulation of zygotic gene activation in the preimplantation mouse embryo: Global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A tan inhibitor of histone deacetylases) or aphidicolin tan inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

Hir/Hira (histone regulation) genes were first identified in yeast as negative regulators of histone gene expression. It has been confirmed that HIRA is a conserved family of proteins present in various animals and plants. In this paper, the cDNAs of the Hira homolog named CagHira and CaHira were isolated from gynogenetic gibel carp (gyno-carp) and gonochoristic color crucian carp (gono-carp) respectively. The full-length CagHira is 3,860 bp in length with an open reading frame (ORF) of 3,033 bp that encodes 1,011 amino acids, while the full-length CaHira is 3,748 bp in length and also has an ORF of 3,033 bp. The deduced amino acid sequences of both Hira homologs contain seven WD domains and show high identity with other HIRA family members. RT-PCR analyses revealed strong expression of Hira in the ovaries, whereas no expression was detected in the testes of either of the fishes. Hira transcription was not detected in the liver of gyno-carp, but a high level of Hira mRNA was observed in gono-carp. The temporal expression pattern showed that the Hira mRNA is consistently expressed during all embryonic development stages in gyno-carp. However, the abundance of CaHira mRNA significantly decreased (P < 0.05) shortly after fertilization and then increased again and remained stable from gastrula till hatching. The varying spatiotemporal expression patterns of Hira genes in gyno-carp and gono-carp may be associated with the differing reproductive modes used by these two closely related fishes. Our results suggest that Hira may play a role not only in the decondensation of sperm nucleus and the formation of pronucleus during fertilization, but also in gastrulation and the subsequent development of embryos.

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone, full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis. (C) 2001 Elsevier Science B.V. All rights reserved.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone I-Il as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.