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PubMed1980

Histone H2B genes of yeast encode two different proteins.

The two genetically unlinked histone H2B genes isolated from Saccharomyces cerevisiae have been sequenced. The genes encode H2B proteins that are 130 amino acids in length and that differ by 4 amino acids. The changes betwen them are Ala leads to Ser, Lys leads to Ala, Thr leads to Val and Ala leads to Val at amino acid positions 2, 3, 27 and 35, respectively. A comparison of yeast H2B histones with those of higher eucaryotes demonstrates a high degree of homology clustered mainly at the carboxyl terminus. There is extensive base substitution between the two H2B genes in nucleotides that do not affect the amino acid sequence. DNA prelude sequences show 64% divergence. The coding regions differ at 49 of the 390 bases (12.6% divergence). 41 of these changes are in silent positions. By using the number of amino acid differences in the proteins we estimate that the two H2B genes are the result of an ancient duplication event.

Amino Acid Sequence↗
PubMed2000

Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides.

The human nuclear autoantigenic sperm protein, NASP, is a testicular histone-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In this study to define the boundaries of antigenic regions and epitope recognition pattern, recombinant deletion mutants spanning the entire protein coding sequence and a human NASP cDNA sublibrary were screened with vasectomy patients' sera. Employing panel sera from 21 vasectomy patients with anti-sperm antibodies, a heterogeneous pattern of autoantibody binding to the recombinant polypeptides was detected in ELISA and immunoblotting. The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Antigenic sites preferentially recognized by the individual patients' sera were located within aa 32-352 and aa 572-787. Using a patient's serum selected for its reactivity to the whole recombinant protein in Western blots, cDNA clones positive for the C-terminal domain of the molecule were identified. The number and location of linear epitopes in this region were determined by synthetic peptide mapping and inhibition studies. The epitope-containing segment was delimited to the sequence aa 619-692 and analysis of a series of 74 concurrent overlapping 9mer synthetic peptides encompassing this region revealed four linear epitopes: amino acid residues IREKIEDAK (aa 648-656), KESQRSGNV (aa 656-664), AELALKATL (aa 665-673) and GFTPGGGGS (aa 680-688). All individual patients' sera reacted with epitopes within the sequence IREellipsis.GGS (aa 648-688). The strongest reactivity was displayed by peptides corresponding to the sequence AELALKATL (aa 665-673). Thus, multiple continuous autoimmune epitopes in NASP involving sequences in the conserved C-terminal domain as well as in the less conserved testis-specific N-terminal region comprising the histone-binding sites, as predicted for an antigen-driven immune response, may be a target of autoantibodies in vasectomized men and may provide a relevant laboratory variable to describe more accurately the spectrum of autoantibody specificities associated with the clinical manifestation of vasectomy.

Amino Acid Sequence↗
PubMed2004

Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes.

The mechanism of an organism's adaptation to high temperatures has been investigated intensively in recent years. It was suggested that the macromolecules of thermophilic microorganisms (especially proteins) have structural features that enhance their thermostability. We compared mRNA sequences of 72 fully sequenced prokaryotic proteomes (14 thermophilic and 58 mesophilic species). Although the differences between the percentage of adenine plus guanine content of whole mRNAs of different prokaryotic species are much lower than those of guanine plus cytosine content, the thermophile purine-pyrimidine (R/Y) ratio within their mRNAs is significantly higher than that of the mesophiles. The first and third codon positions of both thermophiles and mesophiles are purine-biased, with the bias more pronounced by the thermophiles. Thermophile mRNAs that display the highest R/Y ratio (1.43-1.69) are those of the ribosomal proteins, histone-like proteins, DNA-dependent RNA polymerase subunits, and heat-shock proteins. Within mesophilic prokaryotes and five eukaryotic species, the R/Y ratio of the mRNAs of heat-shock proteins is higher than their average over coding part of the genome. Polypurine tracts (R)(n) (with n > or = 5) are much more abundant within the thermophile mRNAs compared with mesophiles. Between two sequential pure-purinic codons of thermophile mRNAs, there is a rather strong tendency for the occurrence of adenine but not guanine tracts. The data suggest that mixed adenine.guanine and polyadenine tracts in mRNAs increase the thermostability beyond the contribution of amino acids encoded by purine tracts, which highlights the importance of ecological stress in the evolution of genome architecture.

Animals↗
PubMed2006

Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants.

Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.

Arabidopsis↗
PubMed1998

Cloning of the murine transcriptional corepressor component SAP18 and differential expression of its mRNA in the hematopoietic hierarchy.

A differentially expressed sequence tag (EMegR4) was isolated from a subtractive hybridization between a single bipotent erythroid/megakaryocytic precursor (E/Meg) and a single bipotent neutrophil/macrophage precursor (N/M). It was used to clone a novel murine cDNA referred to as mSAP18. The full-length mSAP18 cDNA contains 3415 nucleotides and codes for a protein of 153 amino acids. The open reading frame shows 98% identity to a human protein, SAP18, recently identified in a complex with the transcriptional corepressor mSin3 and histone deacetylase. mSAP18 cDNA harbours four polyadenylation sites and a B2 small interspersed repetitive element (SINE) in its 3' untranslated region. We demonstrate that mSAP18 RNA is expressed with stage and lineage specificity in the hematopoietic hierarchy, and that alternative polyadenylation sites are used.

Alternative Splicing↗
PubMed1984

Genes encoding the core proteins of adenovirus type 2.

The nucleotide sequence of the HindIII-D fragment of adenovirus type 2 has been determined. The sequence, which is located between coordinates 41.8 and 51.0, covers most of the L2 cotermination family. It includes three major open translational reading frames encoding the carboxyl-terminal part of the penton base as well as the major core polypeptides V and VII. An additional minor open translational reading frame encoding a highly basic polypeptide was detected in the sequence. The L2 region has a very compact organization with very short distances between the different genes, although no overlapping coding sequences were found. The predicted amino acid sequences of core proteins V and VII reveal that they are highly basic proteins and polypeptide VII resembles the arginine-rich H4 histones in its amino acid composition, but no striking similarities are apparent at the amino acid sequence level. The candidate polypeptide encoded by the newly discovered translational reading frame contains 29% basic residues and includes a hypothetical recognition sequence for the adenovirus-encoded endopeptidase. In conjunction with previously published sequences and those reported in accompanying papers (Akusjärvi, G., Aleström, P., Pettersson, M., Lager, M., Jörnvall, H., and Pettersson, U. (1984) J. Biol. Chem. 259, 13976-13979; Roberts, R. J., O'Neill, K. E., and Yen, C. E. (1984) J. Biol. Chem. 259, 13965-13975) a complete sequence can now be reconstructed for the 35,937-base pairs adenovirus type 2 genome.

Adenoviruses, Human↗
PubMed1998

Raf-1 kinase, a potential regulator of intracellular pH in Xenopus oocytes.

Xenopus laevis oocytes undergo an increase in intracellular pH (pHi) from 7.2 to 7.7 due to the up-regulation of Na+/H+ antiporters in their plasma membrane during oocyte meiotic maturation. Up-regulation of Na+/H+ exchangers (NHE) found in other cell systems appears to be controlled, in some cases, by direct phosphorylation of the exchanger. A number of active protein kinases can be found in maturing Xenopus oocytes. These include, c-mos kinase, Raf-1 kinase, mitogen-activated kinase kinase (MAPKK), MAPK, ribosomal S6 kinase (RSK), and histone H-1 kinase. Our previous study indicated that c-mos kinase, was involved in regulating the increase in oocyte pHi. In the current study, we show that when mRNA coding for a constitutively active form of Raf-1 kinase (delta N-Raf-1) was microinjected into oocytes, the protein product induced an increase in oocyte pHi. On the contrary, the injection of mRNA coding for wild-type Raf-1 (WT-Raf-1) or a kinase-deficient form of Raf-1 (KD-Raf-1) had no effect on the recipient oocyte pHi. 8-Br-cAMP and forskolin blocked the increase in pHi during oocyte meiotic maturation, but had no effect on the Raf-1-induced increase in oocyte pHi. Studies using antisense c-mos oligos demonstrated that Raf-1 was not working via a feedback loop to endogenous c-mos mRNA within the recipient oocytes. Experiments using the selective MAPKK inhibitor, PD 98059, indicated that the Raf-1 effect on oocyte pHi was not mediated by the downstream kinase, MAPKK. Therefore, Raf-1 appears to act independently of c-mos kinase in a pathway, not involving MAPKK, leading to the up-regulation of the Na+/H+ antiporters in Xenopus oocytes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Topoisomerase I is preferentially associated with normal SV40 replicative intermediates, but is associated with both replicating and nonreplicating SV40 DNAs which are deficient in histones.

Based on the use of equilibrium centrifugation in CsCl to separate covalent complexes between topoisomerase I and DNA from protein-free DNA, it was concluded previously that the topoisomerase is preferentially associated with replicating SV40 DNA (Champoux, J. J. 1988. J. Virol. 62:3675-3683). One explanation for the failure to find the enzyme associated with nonreplicating viral DNA is that most of the completed DNA is rapidly sequestered for encapsidation and inaccessible to topoisomerase I. This explanation has been ruled out in the present work by the finding that topoisomerase I in COS-1 cells is also preferentially associated with the replicative form of an SV40 origin-containing plasmid that lacks the genes coding for the virion structural proteins and therefore cannot be encapsidated. Thus it appears that some structural feature of the replicating DNA or the replication complex specifically recruits the topoisomerase to the DNA. SV40 DNA which is produced in the presence of the protein synthesis inhibitor, puromycin, is deficient in histones and as a result lacks normal chromatin structure. Topoisomerase I was found to be associated with SV40 DNA under these conditions whether or not it was replicating. This observation is interpreted as an indication that under normal conditions, chromatin structure limits access of topoisomerase I to the nonreplicating viral DNA.

Animals↗
PubMed2006

Chromatin structure and gene regulation in T cell development and function.

Transcription factors control gene expression programs in the context of the chromatin structure of their target genes. DNA methylation, post-translational histone modifications such as acetylation and methylation, and higher order chromatin organization allow the maintenance of gene expression patterns through mitosis, but how do they accommodate developmentally regulated changes in gene expression programs? Although histone acetylation and deacetylation are in dynamic equilibrium and mechanisms for the removal of methyl groups from histones are emerging, the extent to which there is active demethylation of DNA remains controversial. Looking at chromatin in the three-dimensional space of the nucleus, recent work demonstrates that gene regulation involves contacts between regulatory elements within genes or gene clusters on the same chromosome (in cis) and between different chromosomes (in trans). Finally, non-coding RNAs make a significant contribution to transcriptional and post-transcriptional gene silencing. Together, these advances contribute to an understanding of how gene expression programs are established, maintained and modified during development.

Animals↗
PubMed1986

Characterization of two nonallelic pairs of late histone H2A and H2B genes of the sea urchin: differential regulation in the embryo and tissue-specific expression in the adult.

Two nonallelic pairs of late H2A and H2B genes of the sea urchin Psammechinus miliaris were isolated on two different cosmid clones. The genes of cosmid PmL1 are separated by 11 kilobases of DNA and code for the late H2A-2 and H2B-2 variants. The genes of clone PmL2 are divergently transcribed with 1,060 base pairs of intergenic spacer DNA and code for novel variants of the H2A-2 and H2B-2 type. A comparison of the promoter sequences revealed little homology upstream of the TATA box with the exception of a 24-base-pair-long conserved sequence which is present at the same position in both late H2B promoters and part of which is identical with the "H2B-specific" 5' element. The mRNAs of the H2A and H2B genes of cosmid PmL1 reach their maximal levels early in the mesenchyme blastula embryo, whereas the transcripts of both genes of clone PmL2 accumulate maximally only later in the pluteus larva. In the adult sea urchin all four mRNAs are present in the tube foot but not in the intestine and lantern muscle. This pattern of differential expression in the embryo and tissue-specific expression in the adult suggests cell lineage-specific regulation of the late H2A-2 and H2B-2 genes. Another class of late histone genes represented by the H2A-3 and H2B-1 genes was shown to be expressed in all three adult tissues tested, whereas transcripts of the late H2A-1 genes could not be detected, suggesting that these genes are active exclusively during sea urchin development.

Alleles↗
PubMed2000

Proteome analysis of activated murine B-lymphocytes.

Proteins extracted from murine B-lymphocytes after in vitro stimulation by lipopolysaccharide were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Structural information on the protein entities from 153 spots was obtained. Since many of these spots occur as members of spot families, a smaller number --98 genes-- was found to be coding for the identified spots. The elucidated proteins belong to groups of functional categories; we found 26 enzymes, 36 regulatory proteins, 15 chaperones, 15 structural proteins, 4 immunoglobulins, 1 ribosomal and 1 histone protein. A comparison between expected and observed molecular masses yields a good correlation for the majority of the compared spot entities. This set of proteins now identified in the context of a lymphocyte 2-D gel pattern should advance further studies on lymphocyte functions.

Animals↗
PubMed1985

(AT)n is an interspersed repeat in the Xenopus genome.

We have observed (AT)34 and (AT)23 tracts close to the coding sequences of the Xenopus laevis tadpole alpha T1 and adult beta 1 globin genes, respectively. We show that (AT)n sequences are found as interspersed repeats within the Xenopus globin and histone gene loci. Using (AT)n co-polymer in filter hybridisation experiments we estimate that there are 10(4) (AT)n tracts per haploid Xenopus genome. Hybridisation to genomic blots of DNA from yeast, slime mold, trypanosome, fruit fly, salmon, chicken, rat, human, crab and Xenopus species shows that strictly alternating AT of sufficient length to hybridise appears to be most abundant in Xenopus and crab genomes. We show that the specificity of the co-polymer probe for strictly alternating AT is, however, dependent on the length of the probe. Hybridisation experiments using (TG)n copolymer suggest that this highly conserved repeat is found as clustered repeats in the Xenopus genome in contrast to other eukaryotic genomes so far studied.

Animals↗
PubMed1992

The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli.

CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain. This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein. We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I.

Antigens, Bacterial↗
PubMed2006

Radiation and the regulatory landscape of neo2-Darwinism.

Several recently revealed features of eukaryotic genomes were not predicted by earlier evolutionary paradigms, including the relatively small number of genes, the very large amounts of non-functional code and its quarantine in heterochromatin, the remarkable conservation of many functionally important genes across relatively enormous phylogenetic distances, and the prevalence of extra-genomic information associated with chromatin structure and histone proteins. All of these emphasize a paramount role for regulatory evolution, which is further reinforced by recent perspectives highlighting even higher-order regulation governing epigenetics and development (EVO-DEVO). Modern neo2-Darwinism, with its emphasis on regulatory mechanisms and regulatory evolution provides new vision for understanding radiation biology, particularly because free radicals and redox states are central to many regulatory mechanisms and free radicals generated by radiation mimic and amplify endogenous signalling. This paper explores some of these aspects and their implications for low-dose radiation biology.

Animals↗
PubMed1999

[Clinical sequelae of mutation of the CBP gene].

Gene CBP codes for a transcriptional coactivator, which can interact with many transcriptional factors. It modifies the process of transcription stimulated by these factors by specific binding to RNA polymerase II holoenzyme or by histone acetylation. CBP gene mutation is the molecular cause of autosomal dominant genetic disease called Rubinstein-Taybi syndrome that is manifested by mental and growth retardations, by typical face malformations and broad thumbs and broad big toes. The CBP gene can be affected by the t(8;16)(p11;p13.3) translocation resulting in production of the MOZ/CBP chimeric protein and in induction of acute myeloblastic leukaemia. Therapy using topoisomerase II inhibitors can induce the t(11;16)(q23;13.3) translocation causing acute myeloid or lymphoid leukaemia or myelodysplasia through production of the MLL/CBP protein chimera.

CREB-Binding Protein↗
PubMed2006

In Vitro and in Vivo nucleosome positioning on the ovine beta-lactoglobulin gene are related.

Although positioned nucleosomes are known to play a direct, localised role in regulating access to DNA sequence, they also have the potential, through their long-range distribution, to affect the detailed structure of the higher-order chromatin fibre. To investigate this possibility, we firstly mapped, in vitro, the sequence-dependent positions that the core histone octamer adopts when reconstituted onto DNA containing the ovine beta-lactoglobulin gene. These positioning sites are discussed in terms of their relative affinity for the histone octamer, their locations with respect to the gene sequence and their periodic distribution throughout the gene region. Secondly, we mapped, in vivo, the sites that nucleosomes occupy on the same sequence in liver nuclei, where the gene is transcriptionally inactive. Although the sequence is largely packaged into regularly spaced nucleosomes, reflecting a fibre of uniform higher-order structure, this organisation is disrupted by a number of unusual chromatin structures in a region stretching from the second to the third introns of the gene. A comparison of the in vitro and in vivo nucleosome positioning data shows that they are qualitatively and quantitatively related, suggesting that the structure of the higher-order chromatin fibre containing the beta-lactoglobulin gene is determined, in part, by the long-range organisation of the non-coding sequences within which the gene is embedded.

Animals↗
PubMed2004

Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation.

The histone acetyltransferase Elongator was originally isolated as a component of the elongating form of RNA polymerase II (RNAPII) and a plethora of data has since supported a role for the factor in transcription. However, recent data has suggested that it is predominantly cytoplasmic and does not associate with the DNA of transcribed genes in vivo. Here, we report that Elongator binds to RNA both in vitro and in vivo. Using a modified chromatin immunoprecipitation procedure, RNA immunoprecipitation (RIP), we show that Elongator is indeed present at several actively transcribed genes and that it associates with the nascent RNA emanating from elongating RNAPII along the entire coding region of a gene. These results strongly support a role for Elongator in transcript elongation.

Acetyltransferases↗
PubMed2006

Expression profiles and single-nucleotide polymorphism analysis of human HANP1/H1T2 encoding a histone H1-like protein.

Recently we cloned the Hanp1 cDNA that encodes a histone H1-like haploid germ cell-specific nuclear protein in the mouse. Homozygous Hanp1 mutant male mice were infertile, while females were fertile. Although a substantial number of sperm were recovered from the epididymis, their shape and function were abnormal. Hanp1 protein is essential for nuclear formation in functional spermatozoa, and is specifically involved in the replacement of histones with protamines during spermiogenesis. To investigate the roles of human HANP1 (h-HANP1) and its relation to male infertility, we isolated h-HANP1 cDNA from a human cDNA plasmid library using mouse Hanp1 cDNA as a probe. h-HANP1 is expressed in the testes and its genomic construct also intronless as mouse Hanp1. We found that the h-HANP1 coding region have 5 single-nucleotide polymorphisms in Japanese men.

Amino Acid Sequence↗