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PubMed1988

Cloning and characterization of a core histone gene tandem repeat in Urechis caupo.

A Urechis caupo histone gene tandem repeat has been isolated from a 5.0-kilobase EcoRI genomic library in lambda gtWES.lambda B. Genomic reconstruction experiments indicate that the cloned sequence is repeated approximately 100 times per haploid genome. Unique restriction fragments from the cloned sequence hybridize with individual core histone genes from a histone gene tandem repeat of the sea urchin, Strongylocentrotus purpuratus. No hybridization is detected when restriction digests are probed with a sea urchin H1 histone gene. Hybrid selection and in vitro translation of embryo mRNAs demonstrate that the clone contains sequences complementary to all four core histones; however, no H1 histone is detected among the translation products. Based on a restriction site map of the clone and the subcloned sequences which hybridize to the histone mRNAs, the order of the core histone genes in the clone is shown to be H3 H2A H2B H4. S1 nuclease hybrid protection mapping is used to locate the coding regions and to determine the transcript lengths of the core histone mRNAs. The transcript lengths of H2A, H2B, H3, and H4 mRNAs are approximately 464, 438, 494, and 397 bases, respectively. The S1 nuclease mapping also demonstrates that H2A and H4 are transcribed from one DNA strand while H2B and H3 are transcribed from the other strand. In the tandem repeat, the genes are organized so that transcription of the H2A-H2B and H3-H4 gene pairs is divergent.

Animals↗
PubMed1982

The two yeast histone H2A genes encode similar protein subtypes.

The sequences of the two histones H2A genes in the yeast Saccharomyces cerevisiae have been determined. These genes encode two histone H2A subtypes which are 131 amino acids in length but differ at 2 amino acid positions: an Ala leads to Thr and a Thr leads to Ala change at positions 124 and 125. Thus, the two histone H2A subtypes have identical amino acid compositions. The coding regions of the two H2A genes are homologous at 369 of 393 bases (94%), with all but 2 of the 24 changes being silent. There is only 30% homology in the 5' flanking sequences of the two H2A genes. Like other eukaryotic histone genes, the yeast H2A genes are not interrupted by intervening sequences. When the yeast H2A histones are compared to those from other eukaryotes, there is at least 80% homology in amino acid sequence.

Amino Acid Sequence↗
PubMed1983

Evolution of alpha q- and beta-tubulin genes as inferred by the nucleotide sequences of sea urchin cDNA clones.

Evolutionary studies on the tubulin multigene families were initiated by nucleotide sequence analysis of cDNA clones complementary to sea urchin (Lytechinus pictus) testis alpha- and beta-tubulin cDNA clones (p beta 1, p beta 2, p beta e) demonstrated the existence of tubulin mRNA heterogeneity. p beta 2 and p beta 3 contain identical tubulin-coding regions and extremely similar 3' untranslated sequences, including a polyadenylation signal (AAUAAA). However, p beta 2 contains an additional region of 3' untranslated sequence which includes a second polyadenylation signal. These two sequences may be allelic, representing products of alternative transcription termination or processing pathways. p beta 1 and p beta 2 (or p beta 3) cDNAs almost certainly correspond to transcripts of distinct but evolutionarily related genes. Examination of the available coding portions showed that they differ only by a few silent nucleotide substitutions and the deletion/insertion of one codon; most of the differences are clustered within the last 15 3'-end codons. In contrast, their 3' untranslated sequences are considerably divergent. Nucleotide alignment in this region was feasible by considering specific point and segmental mutations, mainly T in equilibrium or formed from C transitions and small deletions/insertions associated with small direct repeats. The sea urchin alpha- and beta-tubulin cDNA and corresponding protein sequences were compared with previously described tubulin cDNA and protein sequences from other organisms. Both alpha and beta tubulins are very conserved proteins, evolving with a rate comparable to that of histones. Analysis of the nucleotide divergence of the coding cDNA regions showed that replacement sites have changed with a rate 20-175 times lower than that of the silent sites. Among the 177 codons compared between the sea urchin testis and chick brain beta-tubulin cDNAs, there are 7 conservative amino acid replacements and the deletion/insertion of two codons. Most of these changes are clustered near the C-terminus. The 161-amino acid portion of chick brain, rat and porcine alpha-tubulin sequences differs by 3 conservative amino acid replacements from the corresponding sea urchin testis alpha-tubulin sequence. The compared interspecies 3' untranslated sequences are very divergent.

Amino Acid Sequence↗
PubMed2025

Genetic and chromatin regulation of Pvt1 monoallelic expression.

While most genes are equivalently expressed on both alleles, genes with random monoallelic expression (RME) stably maintain expression from only one allele, but the mechanisms and consequences of RME remain unclear. We performed allele-specific RNA sequencing (RNA-seq) on ∼100 F1 hybrid neural progenitor cell (NPC) clonal lines to reveal the extent of autosomal RME (aRME). Of the 287 aRME genes, Pvt1, an oncogenic long non-coding RNA, is an aRME with a genetic bias. In the absence of genetic differences, Pvt1 undergoes balanced aRME. Pvt1 monoallelic expression is maintained by allele-specific active and repressive histone modifications, opposed to DNA methylation. Additionally, we provide a two-step mechanism for the initiation of aRME and demonstrate that Pvt1 monoallelic expression results in a growth phenotype due to the interplay with Myc. These findings provide insight into how genetic differences can skew a stochastic process, resulting in monoallelic expression with a phenotypic consequence in early development.

Chromatin↗
PubMed1982

Independently evolving chicken histone H2B genes: identification of a ubiquitous H2B-specific 5' element.

The DNA sequence of two chicken histone H2B genes has been determined. Both genes code for the same H2B subtype. Except for conserved "promoter" elements, the sequences 5' to the protein coding regions are completely divergent, indicating that the genes are distantly related and are not evolving in concert. This presents an ideal situation for sequence comparisons. We have discovered a 13 bp, H2B specific homology block, 5' CTCATTTGCATAC 3' located close to the "TATA box". This motif is conserved in all H2B gene leader regions so far sequenced. One of the H2B genes is closely linked, in a divergent arrangement, to an H2A gene, and sequence data suggests that the linked genes share promoter elements.

Amino Acid Sequence↗
PubMed1998

Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome.

The pattern of histone H4 acetylation in different genomic regions has been investigated by immunoprecipitating oligonucleosomes from a human lymphoblastoid cell line with antibodies to H4 acetylated at lysines 5, 8, 12 or 16. DNA from antibody-bound or unbound chromatin was assayed by slot blotting. Pol I and pol II transcribed genes located in euchromatin were shown to have levels of H4 acetylation at lysines 5, 8 and 12 equivalent to those in input chromatin, but to be slightly enriched in H4 acetylated at lysine 16. In no case did the acetylation level correlate with actual or potential transcriptional activity. All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromatin and with the isoform. Two single copy genes that map within or adjacent to blocks of paracentric heterochromatin are depleted in H4 acetylated at lysines 5, 8 and 12, but not 16. Consensus sequences of repetitive elements of the Alu family (SINES, enriched in R bands) were associated with H4 that was more highly acetylated at all four lysines than input chromatin, while H4 associated with Kpn I elements (LINES, enriched in G bands) was significantly underacetylated.

Acetylation↗
PubMed1988

Single copy gene for the chicken non-histone chromosomal protein HMG-17.

A chicken genomic library was screened with the human cDNA encoding the non-histone chromosomal protein HMG-17 and a 4565-base pair fragment containing the entire gene encoding this protein was isolated and characterized. Sequence analysis of the fragment revealed that from the start to end of transcription, the HMG-17 gene is 3293 base pairs long and is comprised of 6 exons ranging in size from 30 to 890 base pairs. Upstream of the putative cap site are both a CAAT box and a TATA box as well as several Sp1 binding sites. The gene has an extremely high content of G and C residues (75%) in a 1150-base pair fragment starting 500 base pairs from the putative cap site. This region satisfies the definition of an HpaII tiny fragment island. Southern analysis indicated that there is a single copy of this gene in chickens, whereas Northern analysis revealed that a single transcript is being synthesized from this gene. A comparison of the chicken and human cDNA and protein sequences and subsequent calculation of the evolutionary rates indicated that HMG-17 is a slowly evolving gene. The present article, which is the first study on the isolation and characterization of a complete gene coding for a high mobility group non-histone protein, indicates that the gene has features characteristic of housekeeping genes.

Amino Acid Sequence↗
PubMed1992

Evolutionary consequences of nonrandom damage and repair of chromatin domains.

Some evolutionary consequences of different rates and trends in DNA damage and repair are explained. Different types of DNA damaging agents cause nonrandom lesions along the DNA. The type of DNA sequence motifs to be preferentially attacked depends upon the chemical or physical nature of the assaulting agent and the DNA base composition. Higher-order chromatin structure, the nonrandom nucleosome positioning along the DNA, the absence of nucleosomes from the promoter regions of active genes, curved DNA, the presence of sequence-specific binding proteins, and the torsional strain on the DNA induced by an increased transcriptional activity all are expected to affect rates of damage of individual genes. Furthermore, potential Z-DNA, H-DNA, slippage, and cruciform structures in the regulatory region of some genes or in other genomic loci induced by torsional strain on the DNA are more prone to modification by genotoxic agents. A specific actively transcribed gene may be preferentially damaged over nontranscribed genes only in specific cell types that maintain this gene in active chromatin fractions because of (1) its decondensed chromatin structure, (2) torsional strain in its DNA, (3) absence of nucleosomes from its regulatory region, and (4) altered nucleosome structure in its coding sequence due to the presence of modified histones and HMG proteins. The situation in this regard of germ cell lineages is, of course, the only one to intervene in evolution. Most lesions in DNA such as those caused by UV or DNA alkylating agents tend to diminish the GC content of genomes. Thus, DNA sequences not bound by selective constraints, such as pseudogenes, will show an increase in their AT content during evolution as evidenced by experimental observations. On the other hand, transcriptionally active parts may be repaired at rates higher than inactive parts of the genome, and proliferating cells may display higher repair activities than quiescent cells. This might arise from a tight coupling of the repair process with both transcription and replication, all these processes taking place on the nuclear matrix. Repair activities differ greatly among species, and there is a good correlation between life span and repair among mammals. It is predicted that genes that are transcriptionally active in germ-cell lineages have a lower mutation rate than bulk DNA, a circumstance that is expected to be reflected in evolution. Exception to this rule might be genes containing potential Z-DNA, H-DNA, or cruciform structures in their coding or regulatory regions that appear to be refractory to repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Evolution↗
PubMed1986

Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences.

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.

Acetyltransferases↗
PubMed1988

The H1 and core histone subtypes: differential gene expression and varied primary structures.

The patterns of chromosomal proteins reflect in many cases the functional state of the respective cell type. The H1 histone group is particularly important in this respect, since these histones are involved in the higher order chromatin organization above the level of chains of nucleosomes. In mammals, the H1 histone family comprises at least five main subtypes (H1a-H1e), a testicular variant (H1t) and, thirdly, a subtype H1(0), which is found only in terminally differentiated cells. The H1(0) variant is structurally related to the avian red blood cell specific histone H5, which was the basis for our recent isolation of the human H1(0) gene. Changes of H1 histone patterns may be crucial events in modulating local chromatin arrangements, since the formation of higher order chromatin structures depends on a cooperative interaction of the H1 histones. Variations in their patterns can be studied in vivo during several developmental processes (such as spermatogenesis, erythropoiesis, maturation of several cell types) or in vitro in several tumor cell lines upon treatment with several inducers or upon inhibition of cell division. The differential regulation of the individual H1 subtypes is reflected in the gene and mRNA structures coding for the respective proteins. The cell cycle regulated histones are mostly encoded by non-polyadenylated mRNAs, whereas H5 as well as H1(0) mRNA shows a poly(A) tail at its 3' end. In conclusion, gene activity may not only be controlled at the level of RNA polymerases and their regulatory transcription factors. The varied patterns of chromosomal proteins at different stages during development and differentiation suggest that the local or overall organization of chromatin plays an additional role in these regulatory programs. Hence, the analysis of variations in patterns of chromosomal proteins is an integral part of the investigation of gene regulation mechanisms.

Amino Acid Sequence↗
PubMed1995

Cloning and sequence analyses of the genes coding for the integration host factor (IHF) and HU proteins of Pseudomonas aeruginosa.

Histone-like proteins, such as HU and the integration host factor (IHF), are small, dimeric, DNA-bending proteins which play a role in maintaining constrained DNA structures and hence in regulating gene expression. Two different strategies were used to isolate the genes coding for Pseudomonas aeruginosa (Pa) HU and IHF, two proteins that we have previously isolated from a mucoid strain. By use of a PCR-based technique with oligodeoxyribonucleotides (oligos) designed from the N-terminal amino acid (aa) sequences of HU and the beta-subunit of IHF, and Southern blot analyses, hupB and himD, encoding HU and IHF beta, respectively, have been cloned. The himA gene of Pa, encoding the alpha-subunit of IHF, was isolated using himA of Escherichia coli (Ec) as a probe in Southern blot analyses. The deduced hupB product (90 aa, 9 kDa) is 79% identical to HU beta and 61% to HU alpha of Ec. The predicted products of himA (100 aa, 11.5 kDa) and of himD (94 aa, 10.6 kDa) share 77 and 70% identity with IHF alpha and IHF beta of Ec, respectively. The promoter region of himD contains an IHF consensus sequence, as is the case for Ec himD.

Amino Acid Sequence↗
PubMed1995

Cis-acting elements and expression pattern of the spinach rps22 gene coding for a plastid-specific ribosomal protein.

In order to study the regulation of nuclear genes coding for plastid ribosomal proteins, we have analysed the promoter region of spinach rps22 using both in vitro and in vivo approaches. By footprinting analyses, we have identified eight DNA elements interacting with spinach leaf nuclear factors in the 300 bp promoter region upstream of the transcription start site. Among these elements, four are short AT-rich sequences and one is identical to the Hex motif characterized initially in wheat histone genes. In transgenic tobacco plants, the reporter gene coding for the beta-glucuronidase (GUS) directed by a 1.2 kb upstream region of rps22 was expressed in several plant organs, with high levels in leaf mesophyll, embryo cotyledons and root meristematic cells and very low levels in other cell types. Interestingly, when deleted to -295, the promoter, which contained all the foot-printed elements, was still able to confer the same expression pattern, although the activity was relatively lower than with the 1.2 kb promoter. When deleted further to -154, the promoter, from which the AT-rich elements were eliminated, loses its activity almost completely, suggesting that these AT-rich elements are important for the rps22 promoter activity. Altogether, our results show that rps22 gene expression is controlled by specific cis elements not present in other nuclear-encoded plastid ribosomal protein genes studied so far.

Base Sequence↗
PubMed1988

Matching nucleotide sequences of human antibodies with other known sequences.

From an evolutionary point of view, the complementarity-determining regions of antibodies are distinct from other proteins including the framework regions of antibodies. A search for identical nucleotide sequences of eighty-four 15 consecutive bp in the complementary-determining regions of human antibody heavy chains with other known sequences yielded four matches: two sequential 15-bp matches, or one 16-bp match, with the coding region of a sea-urchin testis histone H2b-2, one 15-bp match with the promotor region of a cauliflower mosaic virus inclusion body protein, and a 15-bp match with an intron between exons 1 and 2 of human factor IX. As a control, an identical search of eighty-four 15 consecutive bp in the framework regions of human antibody heavy chains yielded no matches with other sequences except those from other antibody framework regions. Since the currently available nucleotide sequence database used in the search consisted of about 1 x 10(7) bp, finding such matches in the complementarity-determining regions might not be random.

Antibodies↗
PubMed1997

Cloning and expression of human NF-YC.

The CCAAT box is an important element in eukaryotic promoters and NF-Y (CBF) is a conserved heterotrimeric protein binding to it. Two subunits, NF-YB and NF-YC, contain a histone-like motif. We cloned the complete cDNA coding for the human NF-YC gene. The ORF codes for a 335 aa protein that shows virtual identity to the rat sequence, confirming the stunning invariance of NF-Y genes across species. We expressed and purified the yeast homology domain of NF-YC in bacteria and performed EMSA together with the corresponding conserved domains of NF-YA and NF-YB, obtaining a CCAAT-binding mini-NF-Y. We evaluated the expression of NF-YC and found that mRNA levels are similar in different human tissues except in testis.

Amino Acid Sequence↗