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W M Bonner

Publications and source records attributed to W M Bonner.

At least 37 records · Page 2Linked to original sources
PubMed1988

Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis.

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.

Animals↗
PubMed1988

Sequence of cDNAs for mammalian H2A.Z, an evolutionarily diverged but highly conserved basal histone H2A isoprotein species.

The nucleotide sequences of cDNAs for the evolutionarily diverged but highly conserved basal H2A isoprotein, H2A.Z, have been determined for the rat, cow, and human. As a basal histone, H2A.Z is synthesized throughout the cell cycle at a constant rate, unlinked to DNA replication, and at a much lower rate in quiescent cells. Each of the cDNA isolates encodes the entire H2A.Z polypeptide. The human isolate is about 1.0 kilobases long. It contains a coding region of 387 nucleotides flanked by 106 nucleotides of 5'UTR and 376 nucleotides of 3'UTR, which contains a polyadenylation signal followed by a poly A tail. The bovine and rat cDNAs have 97 and 94% nucleotide positional identity to the human cDNA in the coding region and 98% in the proximal 376 nucleotides of the 3'UTR which includes the polyadenylation signal. A potential stem-forming sequence imbedded in a direct repeat is found centered at 261 nucleotides into the 3'UTR. Each of the cDNA clones could be transcribed and translated in vitro to yield H2A.Z protein. The mammalian H2A.Z cDNA coding sequences are approximately 80% similar to those in chicken and 75% to those in sea urchin.

Amino Acid Sequence↗
PubMed1987

Direct analysis of RNA in whole cell and cytoplasmic extracts by gel electrophoresis.

Procedures are presented by which whole cell, cytoplasmic, or nuclear extracts can be subjected to gel electrophoresis for the separation of the various RNA species, which are then analyzed by conventional blotting and hybridization techniques. Since the methods for preparing the extracts do not involve precipitation or two-phase extraction steps, the minimum number of cells that can be processed is limited only by the sensitivity of detection for specific RNA species. Multiple small or large aliquots of tissue culture cells can be quickly prepared. Cell preparations with high RNase levels, such as resting human lymphocytes or HL60, can be processed reliably with these procedures.

Animals↗
PubMed1986

H1 variant synthesis in proliferating and quiescent human cells.

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.

Adult↗
PubMed1986

Histones and their modifications.

Histones constitute the protein core around which DNA is coiled to form the basic structural unit of the chromosome known as the nucleosome. Because of the large amount of new histone needed during chromosome replication, the synthesis of histone and DNA is regulated in a complex manner. During RNA transcription and DNA replication, the basic nucleosomal structure as well as interactions between nucleosomes must be greatly altered to allow access to the appropriate enzymes and factors. The presence of extensive and varied post-translational modifications to the otherwise highly conserved histone primary sequences provides obvious opportunities for such structural alterations, but despite concentrated and sustained effort, causal connections between histone modifications and nucleosomal functions are not yet elucidated.

Amino Acid Sequence↗
PubMed1985

Studies on nuclease digestion of chromatin phosphorylated in vivo.

We have previously shown that, by culturing cells in hypertonic media, histone 2A becomes hyperphosphorylated (Pantazis, P., West, M. H. P., and Bonner, W. M. (1984) Mol. Cell. Biol. 4, 1186-1188). In the present study we have probed the effect of this histone modification on the overall chromatin structure by micrococcal nuclease and DNase I digestion. Although no significant quantitative differences in the extent of hydrolysis were observed between control and hyperphosphorylated chromatin by micrococcal nuclease, DNase I digested hyperphosphorylated chromatin at a 3- to 4-fold higher rate than unmodified chromatin.

Animals↗
PubMed1985

Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum. Disappearance of these proteins at metaphase and reappearance at anaphase.

Ubiquitinated histones uH2A.1, uH2A.Z, and uH2B have been identified in the basic nuclear proteins of the slime mold Physarum polycephalum by three methods: peptide mapping, cross-reaction with anti-ubiquitin antibody, and uH2A and uH2B isopeptidase cleavage. In microplasmodia, uH2A amounts to 7% of H2A and uH2B amounts to 6% of H2B. Detailed studies of mitosis in Physarum polycephalum macroplasmodia show that in early prophase, which last 15 min, the uH2As and uH2B are both strongly present, whereas minutes later in metaphase, which lasts 7 min, they disappear. When the nuclei enter anaphase, which lasts 3 min, both the uH2As and uH2B reappear. These precise studies suggest that cleavage of ubiquitin from the uH2As and uH2B is a very late, possibly final event in chromosome condensation to metaphase chromosomes and that ubiquitination is an early event in their decondensation. It is proposed that the uH2A and uH2B mark specific regions of the genome which have to be deubiquitinated prior to packaging into metaphase chromosomes; after metaphase these regions are the first to be decondensed and ubiquitinated. This modification, however, is not thought to be a general factor in chromosome condensation but labels a specific subcomponent of chromatin containing the expressed genes of a particular cell type or an important subset of these genes required by the cell to be available for activation, e.g. stress genes.

Anaphase↗
PubMed1985

Mechanism for differential sensitivity of the chromosome and growth cycles of mammalian cells to the rate of protein synthesis.

It has been documented widely that when the generation times of eucaryotic cells are lengthened by slowing the rate of protein synthesis, the duration of the chromosome cycle (S, G2, and M phases) remains relatively invariant. Paradoxically, when the growth of exponentially growing cultures of CHO cells is partially inhibited with inhibitors of protein synthesis, the immediate effect is a proportionate reduction in the rate of total protein, histone protein, and DNA synthesis. However, on further investigation it was found that over the next 2 h the rates of histone protein and DNA synthesis recover, in some cases completely to the uninhibited rate, while the synthesis rates of other proteins do not recover. We called this process chromosome cycle compensation. The amount of compensation seen in CHO cell cultures can account quantitatively for the relative invariance in the length of the chromosome cycle (S, G2, and M phases) reported for these cells. The mechanism for this compensation involves a specific increase in the levels of histone mRNAs. An invariant chromosome cycle coupled with a lengthening growth cycle must result in a disproportionate lengthening of the G1 phase. Thus, these results suggest that chromosome cycle invariance may be due more to specific cellular compensation mechanisms rather than to the more usual interpretation involving a rate-limiting step for cell cycle progression in the G1 phase.

Animals↗
PubMed1985

Interrelationships of protein and DNA syntheses during replication of mammalian cells.

During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.

Animals↗
PubMed1984

Histone variants of the insect Plodia interpunctella during metamorphosis.

The pattern of histone variants from the meal moth Plodia interpunctella was compared to the mouse histone variant pattern. Plodia contains histones which comigrate on two dimensional gels with H3.2, H3.3, H4 and H2A.Z in mouse. Plodia H2A.1 and H2B.1 migrate somewhat differently from the respective mouse histones. Comparison of the iodinated tryptic peptides of H2A.1 and H2A.Z from mouse and Plodia showed that the H2A.Z proteins have two iodinated peptides that comigrate in the two species and three more that are different. The H2A.1 proteins in the two species have one iodinated peptide which comigrates and two more which migrate very close to each other. The histone variants from three developmental stages, larval, pupal and adult of Plodia interpunctella were also identified and compared. The same histone variant pattern is found through all stages of development. It is concluded that histone gene expression does not change during metamorphosis in Plodia .

Animals↗
PubMed1984

Specific alterations in the pattern of histone-3 synthesis during conversion of human leukemic cells to terminally differentiated cells in culture.

The presence of nano- to micromolar concentrations of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in suspension cultures of human promyelocytic leukemia cells, HL-60, or human monocytic leukemia cells, THP-1, resulted in the appearance of macrophage-like cells attached to the substratum. The terminally TPA-differentiated cells continued to synthesize histones at a low rate even though DNA replication had ceased. The pattern of synthesis of histone variants in differentiated cells differed from that in undifferentiated cells and resembled that of quiescent or density-arrested cells. In undifferentiated cells, all three histone-H3 variants are synthesized, while in quiescent cells, only the H3.3 variant is synthesized. When TPA-differentiated macrophages were placed in normal medium, the pattern of histone synthesis was not altered, thus substantiating previous findings that the differentiation is irreversible. Further, TPA-differentiated macrophages and macrophages isolated from a normal human donor exhibited identical pattern of histone synthesis. Altogether, the results indicate that changes in the synthetic rates of histones during the TPA-induced maturation of human leukemic cells is not directly due to TPA or terminal cell differentiation per se but is due to the cessation of cell proliferation and DNA replication.

Acetamides↗
PubMed1984

Phosphorylation of histones in cells treated with hypertonic and acidic media.

Factors in the extracellular environment, specifically hypertonic or acidic growth media, are shown to alter the modification of histones in several cell lines. For histone 2A, changes in modification were visible in the mass pattern and were found to be primarily changes in phosphorylation. The increased modification of the core histones was quickly reversed when cells were returned to normal medium.

Acetylation↗
PubMed1983

Fate of newly synthesized histones in G1 and G0 cells.

We have shown that quiescent cells as well as those in the G1 phase of the cell cycle synthesize histones at a reduced but significant rate. Now, we show that the histones synthesized during G0 and G1 are stably incorporated into nuclei soon after synthesis. Micrococcal nuclease digestion of nuclei isolated from cells in G0 and G1 revealed that the specific histone variants synthesized in these different physiological states are found associated with DNA as nucleosomes. Nucleosomes were separated by polyacrylamide gel electrophoresis in a reducing buffer so that histone spot morphology, particularly that of the H3s was improved.

Animals↗
PubMed1983

Changes in histone H3 composition and synthesis pattern during lymphocyte activation.

Freshly isolated human lymphocytes were found to synthesize histones at a significant rate even though no DNA was being synthesized. The synthesis pattern of histone variants in resting lymphocytes is similar to that found in other quiescent cells and different from that found in S-phase cells. For this reason, the histone synthesis in resting lymphocytes cannot be attributed to contamination by S-phase cells. Stimulation by the mitogen phytohemagglutinin resulted in a dramatic switch in the histone H3 variant synthesis pattern as well as a readily apparent change in the histone H3 mass pattern. Thus, the chromatin of activated lymphocytes has a different histone H3 variant composition than resting or quiescent lymphocytes. It is suggested that the proportion of H3.3 in the mass pattern of the chromatin of a cell may be related solely to how long that cell has been quiescent. Inducing resting lymphocytes to synthesize DNA by UV irradiation did not qualitatively change the histone variant synthesis pattern. No S-phase H3 variants were induced by the repair process. Furthermore, the quantity of histone synthesized neither increased nor decreased after treatment with UV light.

DNA Replication↗
PubMed1983

Minor histone 2A variants and ubiquinated forms in the native H2A:H2B dimer.

Histone octamers from calf thymus were separated into (H3:H4)2 tetramers and H2A:H2B dimers by chromatography through Sephadex G100. The tetramers and dimers were analyzed for variants, ubiquitin adducts, and proteolyzed forms. The minor histone variants H2A.X and H2A.Z were found to be associated with histone H2B as H2A.X:H2B and H2A.Z:H2B dimers, respectively. Ubiquitin adducts of the H2A's and H2B were also present in H2A:H2B dimers.

Animals↗