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Chicken histone genes retain nuclear matrix association throughout the cell cycle.

The association between histone genes and the nuclear matrix (NM) during periods of high (S-phase) and low (non-S-phase) transcriptional activity has been investigated with synchronized cells from a chicken erythroid cell line (abbreviated ts34). By DNase I and restriction enzyme analysis, these studies reveal that both core and linker histone genes (represented by H2A and H1 genes respectively) are attached to the NM independent of their transcriptional activity during the cell-cycle. The tissue-specific histone gene H5, expressed constitutively, is nuclear matrix (NM)-associated in ts34 cells but is found in the supernatant (S/N) fractions of a non-erythroid T-cell line. Furthermore, we show that DNA sequences necessary for NM-attachment of the H5 gene lie within a 780 base pair region spanning part of the coding and 5' non-translated region. Of the three non-histone genes investigated, beta-actin sequences are expressed and are NM-attached, feather keratin genes are not expressed and predominate in the S/N, and beta-globin genes although not expressed in the ts34 cell line used were found in the NM fraction. In this case the association may be fortuitous or may reflect an early event prior to transcription of globin genes in differentiating erythroid cells. These results generally support the notion that actively transcribed genes are NM-attached, but that attachment per se is not synonymous with transcription.

Actins↗

An imbalance of HU synthesis induces mucoidy in Escherichia coli.

Mutations in a number of loci, including the lon gene, dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid. The lon gene, which encodes an ATP-dependent protease, is localized at ten minutes on the E. coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU. Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E. coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid. As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically. Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis. Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting. The UV sensitivity of the hup transformed lon+ bacteria is identical to the lon+ parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal. Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.

ATP-Dependent Proteases↗

Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements.

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.

Animals↗

Argyrophilic nucleolar organizer regions (AgNORs) in inflammatory pre-malignant and malignant lesions of the uterine cervix.

Nuclear organizer regions (NORs) code for ribosomal RNA and are associated with non-histone nucleoproteins, which can be identified by silver staining (AgNORs). AgNORs have been correlated to proliferative activity of tumors and hence may be prognosis-related. The present study evaluates AgNOR counts in inflammatory lesions of the uterine cervix, cervical intra-epithelial neoplasia, and invasive cervical squamous carcinoma. Significant variation in AgNOR counts were observed between the three study groups, with invasive carcinoma showing maximum counts. Further, a highly significant positive correlation was observed between AgNOR counts and tumor progression. These results therefore suggest that AgNOR counts may be of significance in the evaluation of cervical carcinogenesis and could elaborate histopathological diagnosis of cervical lesions.

Carcinoma, Squamous Cell↗

A nested polymerase chain reaction for the detection of Borrelia burgdorferi sensu lato based on a multiple sequence analysis of the hbb gene.

A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.

Base Sequence↗

Argyrophilic nuclear organizer regions (AgNORs) in pediatric acute lymphoblastic leukemia.

Nuclear organizer regions (NORs) code for ribosomal RNA and are associated with non-histone nucleoproteins which can be identified by silver staining (AgNORs). AgNORs have been correlated to proliferative activity of tumors and hence may be prognosis-related. The present study evaluates the possible prognostic importance of the AgNORs in tumor cells of patients with pediatric acute lymphoblastic leukemia (ALL). A significant increase in AgNOR counts was observed in ALL patients as compared to normal controls. Further, a significant positive correlation was observed between AgNOR counts and total WBC count at diagnosis. A negative correlation was also observed between AgNOR counts and age of the patients. Logistic regression analysis revealed further correlation between AgNOR counts and disease with an odds ratio of 1.29 (P = 0.03) as compared to normal controls. These results therefore suggest that AgNOR counts may be significant in the evaluation of pediatric ALL.

Case-Control Studies↗

Effect of interferon on the accumulation of RNA transcripts of genes coding for cellular and viral proteins.

Using plasmids containing sequences complementary to the genes that code for oligo-2',5'-adenylate synthetase, actin and H4 histone, we have shown that although interferon does not affect the accumulation of RNA transcripts of the actin and H4 histone genes, it activates the accumulation of RNA transcripts of the oligo-2',5'-adenylate synthetase gene. However, interferon inhibits the accumulation of RNA transcripts of simian virus 40 (SV40) genes in SV40-infected cells.

2',5'-Oligoadenylate Synthetase↗

Genetic codes.

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Amino Acids↗

The asymmetric segregation of parental nucleosomes during chrosome replication.

SV40 DNA replicated in the presence of cycloheximide was more sensitive to staphylococcal nuclease digestion and had a lower superhelical density than viral DNA replicated in the absence of this drug. These data indicate that fewer nucleosomes are associated with progeny SV40 DNA molecules after DNA replication in the absence of protein synthesis and that these nucleosomes are derived from the parental histones. We designed an experiment to determine whether these parental SV40 nucleosomes segregate to the leading side of the replication form where DNA synthesis is continuous, the lagging side of the fork where DNA synthesis is discontinuous or randomly to both sides of the fork. The results indicate that the parental histones distributed themselves asymmetrically, preferentially (80-90%) segregating with the leading side of both SV40 DNA replication forks during bidirectional replication in the absence of protein synthesis. In the case of SV40, the same parental DNA strands are the templates for the leading side of DNA replication at both forks as well as the templates for the informational or coding strand of early and late viral mRNA synthesis. Based on this correspondence, we designed an experiment to test whether chicken cells growing in culture and replicating their DNA in the absence of protein synthesis segregated their parental histones asymmetrically to the progeny DNA strand that also coded for stable nuclear RNA transcripts. The results of these experiments indicate that, like SV40, parental cellular histones segregate asymmetrically and are preferentially associated with those DNA template strands that code for stable nuclear RNA species detected by hybridization to single-copy DNA.

Cycloheximide↗

Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes.

During mammalian spermatogenesis, somatic histones are replaced by testis-specific variants. The synthesis of the variants occurs primarily in the germ cells undergoing meiosis in the absence of DNA replication. We have cloned the genes encoding rat somatic and testis-specific H2A (TH2A) histones. The two genes share 300 bp of 5' upstream region with respective H2B genes: somatic H2A with somatic H2B and testis-specific TH2A with testis-specific TH2B gene. The deduced amino acid sequences show that H2A and TH2A histones have eight amino acid differences in the first half of the molecules and three consecutive changes in the C-terminal region. TH2A gene is expressed only in testis. Although synthesis of TH2A and TH2B histones is independent of DNA replication and insensitive to inhibitors of DNA synthesis in testis, the regulatory region shared by the two genes contain a bi-directional S phase-specific transcription regulatory element. In addition, TH2A gene, like TH2B gene, contains the consensus sequence element in the 3' non-coding region which is involved in the S phase-specific stabilization of histone mRNA.

Amino Acid Sequence↗

The pattern of histone H4 expression in the tomato shoot apex changes during development.

Two histone H4 cDNA clones were isolated from a tomato (Lycopersicon esculentum Mill.) shoot-tip cDNA library using a heterologous probe from barley (Hordeum vulgare L.). Both cDNAs, which are 81% identical in the coding region, are polyadenylated and belong to a small gene family in the tomato genome. Histone H4 message is abundant in young tissues and rare in older tissues. In the shoot apical meristem, the distribution of H4-expressing cells changes during development. In a juvenile vegetative apex, H4 message is detectable in the central region and the peripheral parts of the meristem. In a mature vegetative apical meristem, H4-expressing cells are localized in the peripheral zone extending into the provascular strands and the rib meristem whereas the central zone is almost devoid of H4 mRNA. After floral transition, H4 mRNA is found throughout the floral meristem, indicating a second change in the pattern of H4 expression. The observed changes in H4 expression are indicative of changes in the distribution of mitotic activity in the shoot apical meristem during plant development. In addition, H4-expressing cells were found to occur frequently in clusters, which may indicate a partial synchronization of cell divisions in the shoot apex.

Amino Acid Sequence↗

Developmental and tissue expression patterns of histone macroH2A1 subtypes.

MacroH2A is a novel nucleosomal core histone that contains a large nonhistone region and a region that closely resembles a full length histone H2A. We have cloned a cDNA that contains the entire coding region of macroH2A1.2, one of the two identified subtypes of macroH2A1. MacroH2A1.2 was found to differ from the other known subtype, macroH2A1.1, in a single segment of the nonhistone region. MacroH2A1 specific antibodies revealed relatively high levels of both subtypes in adult liver and kidney. MacroH2A1.1 was much lower in fetal liver and kidney in comparison to their adult counterparts, and was not detected in adult thymus and testis, tissues with active cell division and differentiation. Both subtypes were present at very low levels or absent from mouse embryonic stem cells maintained in an undifferentiated state by growth in the presence of leukemia inhibitory factor. MacroH2A1.2 increased when the embryonic stem cells were induced to differentiate in vitro, while macroH2A1.1 remained undetectable. These results support the idea that macroH2A1.1 and macroH2A1.2 are functionally distinct, and suggest that changes in their expression may play a role in developmentally regulated changes in chromatin structure and function.

Animals↗

Unusual features of transcribed and translated regions of the histone H4 gene family of Tetrahymena thermophila.

The complete DNA sequence is presented of H4-II, the second of the pair of histone H4 genes of the ciliated protozoan, Tetrahymena thermophila. Both H4 genes code for the same protein. Codon usage in these and other Tetrahymena genes is severely restricted and is similar to that in yeast. Flanking regions are AT-rich (greater than or equal to 75%), relative to coding sequences (approximately 45% GC). Except for small, similarly positioned homologies, flanking sequences of the two genes are different. Canonical sequences in higher eukaryotic promoters are not obvious in these genes. Instead, short, localized, base composition eccentricities characterize the 5' flanking sequences of all Tetrahymena genes analyzed. The consensus, P yP u(A)3-4 ATGG initiates translation in these and all other known Tetrahymena genes. Nuclear transcripts and messages of both growing and starved cells begin at multiple sites, mainly at the first or second A residue following a pyrimidine. The palindrome typical of histone message 3' termini in higher organisms is not present. Downstream of both genes are sequences similar to the processing/polyadenylation signal of higher eukaryotes, although the unique 3' ends are not those predicted by the location of the signals.

Amino Acid Sequence↗

Reiterated genes with varying location in intercalary heterochromatin regions of Drosophila melanogaster polytene chromosomes.

The localization of two cloned D. melanogaster DNA fragments in polytene chromosomes was determined by means of in situ hybridization. These different fragments (Dm 225 and Dm 234B) are present in the genome in hundreds copies and contain genes whose transcription yields two different classes in abundant mRNA (Ilyin et al., 1976, 1977; Tchurikov et al., 1978). About 20--30 sites of these genes are demonstrable in the polytene chromosomes of a given stock. There are small but significant variations in the number and localization of these sites among individuals of the same stock. On the other hand, different stocks of D. melanogaster have an utterly different distribution of revealed hybridization sites in the polytene chromosomes. The location of both fragments (Dm 225 and Dm 234) was found to be virtually identical within any given stock of D. melanogaster. 69 sites for localization of Dm 225 or Dm 234 genes were detected in the chromosomes of 11 individuals studied. At least 50 (and up to 62) of them coincide with intercalary heterochromatin regions which are known to be characterized by ectopic pairing, late replication and the presence of "weak spots" in the chromosome. The ability of Dm225 and Dm 234 to code for the "abundant" classes of messenger RNA (Ilyin et al., 1976) and the fact that their location may coincide with the histone and ribosomal genes suggest that intercalary heterochromatin regions are "nests" containing various types of actively transcribable tandem-repeated genes coding for common "household" cell functions.

Animals↗

Active chromatin.

Active genes are packaged into an altered nucleosome structure forming a chromosomal domain defined by increased sensitivity to nucleases. This structure, reflecting a potential for transcription, contains sites hypersensitive to nuclease digestion adjacent to the coding regions and may also be distinguished by specific non-histone proteins, variant or modified histones or modified DNA. Its formation, by unfolding of a tightly packed chromatin fibre by factors which might affect DNA supercoiling, may be the first step in gene activation.

Animals↗

Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity.

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A-GUS fusion genes using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.

Amino Acid Sequence↗

Isolation of a new H3.3 histone variant cDNA of P. lividus sea urchin: sequence and embryonic expression.

A cDNA encoding a new H3 histone variant has been isolated from a Paracentrotus lividus sea urchin embryo cDNA library. The encoded protein is identical to the H3.3 histone subtype identified in other species, with the difference that E replaces D at position 81. The clone corresponds to a transcript of about 1.6 kb, not dependent on DNA replication, present in the unfertilized egg and at all stages of embryonic development. The coding part of the cDNA cross-reacts also with a 0.5 kb H3 late histone mRNA.

Animals↗

Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study.

Semen samples from a fertile patient presenting with influenza and a 1-day fever of 39.9 degrees C were obtained and analyzed at 18-66 days postfever (dpf) for sperm nuclear proteins, DNA stainability, free thiols (-SH), and susceptibility to DNA denaturation in situ. At 18 dpf, 36% of sperm demonstrated denatured DNA as measured by the sperm chromatin structure assay (SCSA), and decreased to 23% by 39 dpf. Samples at 33 and 39 dpf contained 49% and 30%, respectively, of cells with increased DNA stainability (HIGRN). A unique sperm nuclear protein band migrating between histones and protamines on acid-urea gels appeared at 33 and 39 dpf and nearly disappeared by 52 dpf. Amino acid sequencing of the first 8 N-terminal residues identified this protein as the precursor to protamine 2. The protamine P1 and P2 ratio remained normal, whereas the histone to protamine ratio increased slightly at 33 to 39 dpf. Flow cytometric measurements of nuclear -SH groups revealed the greatest reduction in free nuclear thiols at 33 dpf, and returned to normal by 45 dpf. The time of appearance of the unprocessed protamine 2 precursor and the relative increase in histone suggest a fever-related disruption of the synthesis of mRNA that codes for a P2 processing enzyme or enzymes. Increased DNA staining is likely due to the increased histone/protamine ratio. This case study demonstrates that fever/influenza can have latent effects on sperm chromatin structure and may result in transient release of abnormal sperm.

Cell Nucleus↗