Nucleotide sequences of two corn histone H3 genes. Genomic organization of the corn histone H3 and H4 genes

Summary Two histone H3 genes have been cloned from a gtWES.B corn genomic library. The nucleotide sequences show 96% homology and both encode the same protein, which differs from its counterpart in wheat and pea by one amino acid substitution. The 5-flanking regions of the two corn H3 genes contain the classical histone-gene-specific consensus sequences and possess several regions of extensive nucleotide homology. A conserved octanucleotide 5-CGCGGATC-3 occurs at approximately 200 nucleotides upstream from the initiation ATG codon. This octanucleotide was found to exist in all of the 7 plant histone genes sequenced so far. Codon usage is characterized by a very high frequency of C (67%) and G (28%) at the third position of the codons, those ending by A (1%) and T (4%) being practically excluded.Comparison of Southern blots of EcoRI, EcoRV and BamHI digested genomic DNA suggests that the corn H3 and H4 genes are not closely associated. The H3 genes exist as 60 to 80 copies and the H4 genes as 100 to 120 copies per diploid genome. re]19851002 rv]19851212 ac]19851216     

Conserved organization of an avian histone gene cluster with inverted duplications of H3 and H4 genes

Summary The organization of histone gene clusters of the duckCairina moschata was studied in the DNA inserts of two recombinant phage that overlap and feature identical histone gene arrangements but differ in sequence details and in the extent of repetition of an AT-rich motif in one of the nontranscribed spacer regions. These few but substantial differences between otherwise nearly identical histone gene groups suggest that we have independently isolated alleles of the same site of the duck genome or that this gene arrangement occurs (with slight variations) more than once per haploid genome. Within the histone gene cluster described, H3 and H4 genes are duplicated (with inverted orientation), whereas one H1 gene is flanked by single H2A and H2B genes. The arrangement of duck histone genes described here is identical to a subsection of the chicken genome but differs from any other published histone gene cluster.     

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.     

The heparin-binding lectin from ovine placenta: Purification and identification as histone H4

The heparin-binding lectin complex from ovine placental cotyledons was purified by affinity chromatography on heparin-agarose column. It showed three protein bands, which had molecular weights of 13 000, 15 000 and 17 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of DNA by agarose gel electrophoresis. The protein components of the complex were separated by reverse-phase HPLC. The minimum inhibitory concentrations of glycosaminoglycans were significantly different for the lectin complex and the separated proteins, suggesting affinity changes upon DNA binding. The haemagglutinating activity specificity allowed the characterization of the fraction with a molecular weight of 13 000 as the heparin-binding lectin. This protein was identified as histone H4 by internal sequencing, thus showing that this is the histone responsible for the heparin-binding property of the complex. The accompanying proteins were tentatively identified as histones H2A and H2B. This revised version was published online in November 2006 with corrections to the Cover Date.     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.     

Nuclease sensitivity and functional analysis of a maize histone H3 gene promoter

A 1 kb region of a maize H3 histone gene promoter has been analysed at a structural and functional level. Micrococcal nuclease digestion of isolated nuclei showed that the promoter region is organized into nucleosomes but a zone extending over approximately one nucleosome (20 to 230 bp upstream of the TATA box) displays remarkable accessibility to digestion. Three DNase I-hypersensitive sites were found within this zone at the vicinity of consensus sequences, some of which are already known to act ascis elements. This promoter region is able to direct faithful expression of the GUS reporter gene in meristematic tissues of transgenic tobacco plants.     

Dramatic diversity of ciliate histone H4 genes revealed by comparisons of patterns of substitutions and paralog divergences among eukaryotes

The accumulation of divergent histone H4 amino acid sequences within and between ciliate lineages challenges traditional views of the evolution of this essential eukaryotic protein. We analyzed histone H4 sequences from 13 species of ciliates and compared these data with sequences from well-sampled eukaryotic clades. Ciliate histone H4s differ from one another at as many as 46% of their amino acids, in contrast with the highly conserved character of this protein in most other eukaryotes. Equally striking, we find paralogs of histone H4 within ciliate genomes that differ by up to 25% of their amino acids, whereas paralogs in other eukaryotes share identical or nearly identical amino acid sequences. Moreover, the most divergent H4 proteins within ciliates are found in the lineages with highly processed macronuclear genomes. Our analyses demonstrate that the dual nature of ciliate genomes-the presence of a "germline" micronucleus and a "somatic" macronucleus within each cell-allowed the dramatic variation in ciliate histone genes by altering functional constraints or enabling adaptive evolution of the histone H4 protein, or both.     

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen

Summary A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacteriumMethanobacterium thermoautotrophicum H has been cloned and sequenced. This DNA complements mutations in both the purE₁ and purE₂ loci ofEscherichia coli. The sequence of theM. thermoautotrophicum DNA predicts that complementation inE. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized inE. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogenMethanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome ofM. thermoautotrophicum has a molar G+C content of 49.7%, whereas the genome ofM. smithii is 30.6% G+C. Conservation of encoded amino acids while accommodating the very different G+C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the clonedM. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene fromM. smithii maintains a codon usage that conforms to the RNY rule.     

Regulation of histone H3K4 methylation in brain development and disease

The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.     

The haemochromatosis candidate gene HFE (HLA-H) of man and mouse is located in syntenic regions within the histone gene cluster

The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3′ portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene. J. Cell. Biochem. 69:117–126, 1998. © 1998 Wiley-Liss, Inc.     

Structure and expression of the human oocyte-specific histone H1 gene elucidated by direct RT-nested PCR of a single oocyte

Oocyte-specific histone H1 is expressed during oogenesis and early embryogenesis. It has been described in mice and some nonmammalian species, but not in humans. Here, we identified the cDNA in unfertilized human oocytes using direct RT-nested PCR of a single cell. Sequencing of this cDNA indicated an open reading frame encoding a 347-amino acid protein. Expression was oocyte-specific. Homology was closest with the corresponding gene of mouse (H1oo; 42.3%), and, to lesser extent, with that of Xenopus laevis (B4; 25.0%). The gene, named osH1, included five exons as predicted by the NCBI annotation project of the human genome, although the actual splicing site at the 3(') end of exon 3 was different by 48 nucleotides from the prediction. The presence of polyadenylation signals and successful amplification of cDNA by RT-PCR using an oligo(dT) primer suggested that the osH1 mRNA is polyadenylated unlike somatic H1 mRNA. Our technique and findings should facilitate investigation of human fertilization and embryogenesis.     

Purification and characterization of histone H1 variants from human placenta

Three histone H1 variants were extracted from human placental tissue in a single process using a high-salt buffer solution, and purified by ion exchange, hydroxyapatite, and reversed-phase chromatography. In the first chromatographic step, a cation exchanger resin, SP-Sepharose FF, was used to remove impurities having molecular weights higher than those of histones. In the second chromatographic step, hydroxyapatite resin was used to remove impurities with relatively low molecular weights. A second round of cation exchange chromatography using high-grade HS POROS resin resulted in two main fractions, each of which appeared as a single band following SDS-PAGE. The first fraction showed a single peak in RP-HPLC, while the second fraction showed two main peaks. These three peaks were further separated and polished by semi-preparative RP-HPLC, and their molecular masses and sequences were determined using MALDI-TOF-MS and N-terminal amino acid sequencing, respectively. The sequences and masses of these three variants corresponded with those of histones H1.2, H1.4, and H1.5. Moreover, all three purified histone subtypes demonstrated cytotoxicity in an MTT assay.     

Evolution of prokaryotic DNA: intragenic and extragenic divergences observed with orthologs from three related species

This study compared orthologous gene pairs from Escherichia coli K12, E. coli O157:H7 EDL933, Salmonella typhimurium LT2, and Yersinia pestis CO92 using only homologs of equal length, and differing nucleotides were counted and mapped. The data showed very clearly how the rates of divergence change with intragenic and extragenic position. The rate of synonymous mutation is lowest near start codons and near stop codons, and, a little surprisingly, the opposite seemed to be true for nonsynonymous substitutions. Analysis outside genes reveals that nucleotide divergences occur less frequently upstream of start codons than downstream of stop codons, and a remarkable drop in divergences is seen for two of the data sets around N = 9 nucleotides upstream of start codons; that is, the Shine-Dalgarno region changes at a lower rate. The explanation is likely to be the link between expressivity and sequence complementarity to the 3' end of 16S ribosomal rRNA. The latter is highly conserved across many bacterial and archaebacterial species.