Search PubMed⌕ Search

Biomedical subjects

J H Waterborg

Publications and source records attributed to J H Waterborg.

At least 19 recordsLinked to original sources
PubMed2001

Dynamics of histone acetylation in Saccharomyces cerevisiae.

Rates of turnover for the posttranslational acetylation of core histones were measured in logarithmically growing yeast cells by radioactive acetate labeling to near steady-state conditions. On average, acetylation half-lives were approximately 15 min for histone H4, 10 min for histone H3, 4 min for histone H2B, and 5 min for histone H2A. These rates were much faster than the several hours that have previously been reported for the rate of general histone acetylation and deacetylation in yeast. The current estimates are in line with changes in histone acetylation detected directly at specific chromatin locations and the speed of changes in gene expression that can be observed. These results emphasize that histone acetylation within chromatin is subject to constant flux. Detailed analysis revealed that the turnover rates for acetylation of histone H3 are the same from mono- through penta-acetylated forms. A large fraction of acetylated histone H3, including possibly all tetra- and penta-acetylated forms, appears subject to acetylation turnover. In contrast, the rate of acetylation turnover for mono- and di-acetylated forms of histones H4 and H2B, and the fraction subject to acetylation turnover, was lower than for multi-acetylated forms of these histones. This difference may reflect the difference in location of these histones within the nucleosome, a difference in the spectrum of histone-specific acetylating and deacetylating enzymes, and a difference in the role of acetylation in different histones.

Acetic Acid↗
PubMed2001

Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis.

Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5'-untranslated regions (5'-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.

Arabidopsis↗
PubMed2000

Steady-state levels of histone acetylation in Saccharomyces cerevisiae.

The importance of control of the levels of histone acetylation for the control of gene expression in eukaryotic chromatin is being elucidated, and the yeast Saccharomyces cerevisiae has proven to be an important model system. The level of histone acetylation in yeast is the highest known. However, only acetylation of H4 has been quantified, and reports reveal loss of acetylation in histone preparations. A chaotropic guanidine-based method for histone isolation from intact wild-type cells or from a single-step nuclear preparation with butyrate preserves acetylation of all core histones. Histone H4 has an average of more than 2 acetylated lysines per molecule, distributed over 4 sites. Histones H2A, H3, and H2B have 0. 2, approximately 2, and >2 acetylated lysines per molecule, respectively, distributed across 2, 5, and 6 sites. Thus, yeast nucleosomes carry, on average, 13 acetylated lysines per octamer, i. e. just above the threshold of 10-12 deduced for transcriptionally activated chromatin of animals, plants, and algae. Following M(r) 100,000 ultrafiltration in 2.5% acetic acid, yeast histone H3 was purified to homogeneity by reversed-phase high pressure liquid chromatography. Other core histones were obtained at 80-95% purity.

Acetylation↗
PubMed1998

Dynamics of histone acetylation in Chlamydomonas reinhardtii.

The dynamic character of core histone post-translational acetylation in the unicellular green alga Chlamydomonas reinhardtii was studied by tritiated acetate incorporation. Histone H3 is the major target of acetylation, steady state, and in pulse and pulse-chase analyses. Acetylation turnover rates were measured by tracer labeling under steady-state conditions. Half-lives of 1.5-3 min were found for penta- to mono-acetylation of H3, dynamically acetylated to the 30% level. Twenty percent of H3 was multi-acetylated, on average with 3. 2 acetyl-lysines, all with rapid turnover. Deacetylase inhibitor trichostatin A (TSA) caused doubling of average acetylation levels, primarily as penta-acetylated H3, but half of H3 was not acetylated at all. The level of histone H4 acetylation was only half that of H3 and a major fraction of mono- and di-acetylated forms appeared static. The dynamic fraction had an average half-life of 3.5 min with higher turnover rates for more highly acetylated H4 forms. TSA, inhibiting less effectively deacetylases active on H4, strongly increased multi-acetylated H4 levels and doubled average acetylation. As for H3, half of histone H4 remained unacetylated. Acetylation of histone H2B was low and of H2A was barely measurable. Despite turnover with half-lives of approximately 2 min, no increase beyond di-acetylation was seen upon TSA treatment.

Acetates↗
PubMed1997

A cell cycle-regulated histone H3 gene of alfalfa with an atypical promoter structure.

The control of cell cycle expression of histone genes in plants is incompletely understood. A new histone H3 gene was cloned from alfalfa (Medicago sativa) that codes for the replication-dependent histone H3.1 variant protein. Despite lacking all promoter sequence motifs that have been associated with cell cycle-dependent histone gene expression in plants, northern analysis of synchronized cells clearly linked gene expression to DNA replication. TTAATNA was recognized as a new sequence element in the 3' untranslated regions of this and all other cell cycle-dependent histone H3 genes of dicotyledonous plants. It is not found in the replication-independent histone H3 genes.

Amino Acid Sequence↗
PubMed1996

Common features of analogous replacement histone H3 genes in animals and plants.

Phylogenetic analysis of histone H3 protein sequences demonstrates the independent origin of the replacement histone H3 genes in animals and in plants. Multiple introns in the replacement histone H3 genes of animals in a pattern distinct from that in plant replacement H3 genes supports this conclusion. It is suggested that replacement H3 genes arose at the same time that, independently, multicellular forms of animals and of plants evolved. Judged by the degree of invariant and functionally constrained amino acid positions, histones H3 and H4, which form together the tetramer kernel of the nucleosome, have co-evolved with equal rates of sequence divergence. Residues 31 and 87 in histone H3 are the only residues that consistently changed across each gene duplication event that created functional replacement histone H3 variant forms. Once changed, these residues have remained invariant across divergent speciation. This suggests that they are required to allow replacement histone H3 to participate in the assembly of nucleosomes in non-S-phase cells. The abundant occurrence of polypyrimidine sequences in the introns of all replacement H3 genes, and the replacement of an intron by a polypyrimidine motif upstream of the alfalfa replacement H3 gene, suggests a function. It is speculated that they may contribute to the characteristic cell-cycle-independent pattern of replacement histone H3 genes by binding nucleosome-excluding proteins.

Amino Acid Sequence↗
PubMed1996

Identification of three highly expressed replacement histone H3 genes of alfalfa.

One genomic and six cDNA clones for the replacement histone H3.2 protein of alfalfa (Medicago sativa) were isolated and sequenced. By gene organization they represent 3 distinct genes. PCR methods were used to confirm that only three intron-bearing histone H3.2 genes of this type exist per haploid genome. They co-exist with approximately 56 copies of the previously characterized replication-dependent, intronless histone H3.1 variant gene. Comparison of the relative expression of few constitutive H3.2 genes with the high S phase expression of the abundant cell cycle-dependent H3.1 genes by mRNA levels and protein synthesis measurements revealed that the replacement histone H3.2 genes are very highly expressed. Structural analysis of the genomic replacement H3.2 gene revealed a unique feature. A repeated polypyrimidine sequence motif in the 5' untranslated region of this gene replaces the ubiquitous intron present in all known replacement H3 genes. A hypothesis is presented that this motif and other, non-randomly distributed polypyrimidine sequences in the introns of replacement histone H3 genes of alfalfa and Arabidopsis, may affect nucleosome assembly. Chromatin repression of these replacement genes would be avoided, consistent with the high, constitutive expression of replacement H3 histone genes in plants.

Amino Acid Sequence↗
PubMed1995

Histone H3 transcript stability in alfalfa.

The stability of histone H3 transcripts in alfalfa for replication-dependent and -independent gene variants was measured by northern analysis under conditions of inhibition of transcription and/or translation. Replication-dependent histone H3.1 transcripts were about three-fold less stable than the equally polyadenylated mRNA for replacement variant H3.2 histone. In actively growing suspension cultures treated with dactinomycin half-lives of 2 and 7 h were observed for H3.1 and H3.2 mRNAs, respectively. mRNA stabilities were also measured indirectly by histone protein synthesis. The translation inhibitor cycloheximide strongly increased mRNA levels for both histone H3 variants. The dependence of histone mRNA turnover on translation in animals also appears to exist in plants. The combination of inhibition of transcription and translation by dactinomycin and cycloheximide was used in an indirect assessment of H3 mRNA stability throughout the cell cycle in partially synchronized and cycle-arrested cultures. Destabilization of replication-dependent histone H3.1 mRNA was detected in non-S phase cells.

Cell Cycle↗
PubMed1995

Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation.

Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a single threonine as residue 28 instead of the serine28-alanine29 sequence, present in all other known plant and animal H3 histones.

Amino Acid Sequence↗
PubMed1994

A simple method to make better probes from short DNA fragments.

A detailed method is presented for the creation of head-to-tail multimers of short blunt restriction fragments, ligated into a plasmid vector in a single-tube reaction. Random priming of the concatemer insert readily yields hybridization probes of high specificity, unattainable from the short monomer fragments.

DNA Ligases↗
PubMed1993

Efficient large-scale purification of restriction fragments by solute-displacement ion-exchange HPLC.

Extreme overloading of HPLC columns with sample can create a condition of binding site saturation causing competition and displacement among solutes during column elution. This has been termed solute-displacement chromatography (SD-HPLC). We present an example of this phenomenon for the preparative fractionation and purification of restriction fragments of almost identical size (1337 and 1388 bp) which cannot be resolved by agarose gel electrophoresis. Standard analytical ion-exchange HPLC chromatography failed to separate these fragments from each other and from an unexpectedly early eluting pUC-derived vector fragment of 2.7 kbp. We demonstrate that by intentional overloading of the small (4.6 x 35 mm) non-porous TSK-DEAE HPLC column, hundreds of micrograms of DNA restriction fragments could be resolved and purified in a single HPLC run of less than 30 minutes.

Binding Sites↗
PubMed1993

Histone synthesis and turnover in alfalfa. Fast loss of highly acetylated replacement histone variant H3.2.

Histone synthesis in alfalfa tissue culture cells was studied by labeling with tritiated lysine, purification of histone proteins by reversed-phase high pressure liquid chromatography, and fluorography of acid/urea/Triton X-100 polyacrylamide gels. Minor histone variant H3.2 was synthesized twice as fast as major variant H3.1. The predicted difference in histone H3 variant turnover was examined during continued growth. More than 50% of newly synthesized histone H3.2 and 20% of new H3.1 were lost from chromatin over a period of 100 h. This produced a ratio between the stable remaining portions of each new histone H3 variant protein identical to that of the steady-state histone H3 variants. The labile portion of new histone H3.2 (half-life of 20 h) was rapidly lost specifically from transcriptionally active chromatin as judged by the acetylation level of nearly 1.5 acetylated lysines/histone molecule, a level 50% higher than the acetylation in histone H3.2 overall and three times that of histone H3.1. These results and the constitutive level of H3.2 gene expression identify histone H3.2 of alfalfa as a functional replacement histone variant. The extent of its preferential assembly into active chromatin nucleosomes and the rapid rate of its subsequent loss indicate significant dissolution of plant nucleosomes during gene transcription.

Acetylation↗
PubMed1993

Dynamic methylation of alfalfa histone H3.

Dynamic lysine methylation of histone H3 in alfalfa tissue culture cells was studied by labeling with tritiated methionine, purification of variants H3.1 and H3.2 by reversed-phase high pressure liquid chromatography and amino acid analysis. Mono- and dimethyl-L-lysine were the major labeled amino acids. Within 100 h of continued growth conversion from N epsilon-monomethyl-L-lysine (MML) to N epsilon-dimethyl-L-lysine (DML) and N epsilon-trimethyl-L-lysine (TML) was observed, consistent with steady-state histone methylation. During the same time 20% of the methylation label was lost from major variant H3.1 protein and more than 50% from the more highly labeled minor variant H3.2. A similar pattern of label incorporation and loss was observed during a study of histone synthesis and turnover. This conforms with the general observation in animal cells that lysine methylation is limited to newly synthesized histone. Increased methylation of the more highly acetylated forms of histone H3 protein indicates limited accessibility of chromatin for histone methylation. After loss of the labile fraction of newly synthesized H3 variants, stably methylated proteins with 30% of the label in MML, 40% in DML, and 25% in TML remain. Turnover of methyl modification groups independent of histone turnover was not detected.

Cells, Cultured↗
PubMed1992

Identification of five sites of acetylation in alfalfa histone H4.

Radioactive acetylation in vivo of plant histone H4 of alfalfa, Arabidopsis, tobacco, and carrot revealed five distinct forms of radioactive, acetylated histone. In histone H4 of eukaryotes ranging from fungi to man, acetylation is restricted to four lysines (residues 5, 8, 12, and 16) possibly caused by a quantitative methylation of lysine-20. Chemical and proteolytic fragmentation of the amino terminally blocked alfalfa H4 protein, dynamically acetylated by radioactive acetate in vivo, allowed protein sequencing and identification of selected peptides. Peptide identification was facilitated by analyzing fully characterized calf histone H4 in parallel. Acetylation in vivo of alfalfa histone H4 was restricted to the lysines in the amino-terminal domain of the protein, residues 1-23. Lysine-20 was shown to be free of methylation, as in pea histone H4. This apparently makes lysine-20 accessible as a novel target for histone acetylation. The in vivo pattern of lysine acetylation (16 greater than 12 greater than 8 greater than or equal to 5 = 20) revealed a preference for lysines -16 and -12 without an apparent strict sequential specificity of acetylation.

Acetylation↗
PubMed1992

Existence of two histone H3 variants in dicotyledonous plants and correlation between their acetylation and plant genome size.

Histone H3 proteins were purified to near homogeneity from callus cultures of dicotyledonous plants alfalfa, soybean, Arabidopsis, carrot and tobacco to determine the number of histone H3 variants. In every species two histone H3 variants were identified by gradient gel electrophoresis and reversed-phase chromatography. They were named H3.1 and H3.2 in order of increasing mobility in acid-urea-Triton gels. Co-electrophoresis of histone H3.2 proteins of all species in this gel system and HPLC co-chromatography suggest that all histone H3.2 variants have a primary protein sequence identical to alfalfa H3.2. Two distinct H3.1 variant forms were identified, represented by alfalfa and Arabidopsis H3.1 proteins which differ only at residue 90. Soybean H3.1 resembles H3.1 of alfalfa. Carrot and tobacco H3.1 appear identical to the Arabidopsis H3.1 histone variant. All H3 proteins were acetylated to multiple levels and in each plant the histone H3.2 forms were more highly acetylated. An inverse relationship was observed between plant genome size and the relative abundance of histone variant H3.2 and also with the level of acetylation of both histone H3 variants. This correlation matches the general tendency that in plants with smaller genomes a larger fraction of the genome is transcriptionally active.

Acetylation↗