Biomedical subjects
Publications and source records attributed to R Reeves.
A multiple-baseline design across participants was used to demonstrate the effectiveness of contact desensitization and contingent social approval in decreasing the avoidance behavior of three institutionalized mentally retarded adults towards escalators, measured by a 25-item Behavior Avoidance Test.
Eukaryotic chromatin has a dynamic, complex hierarchical structure. Active gene transcription takes place on only a small proportion of it at a time. While many workers have tried to characterize active chromatin, we are still far from understanding all the biochemical, morphological and compositional features that distinguish it from inactive nuclear material. Active genes are apparently packaged in an altered nucleosome structure and are associated with domains of chromatin that are less condensed or more open than inactive domains. Active genes are more sensitive to nuclease digestions and probably contain specific nonhistone proteins which may establish and/or maintain the active state. Variant or modified histones as well as altered configurations or modifications of the DNA itself may likewise be involved. Practically nothing is known about the mechanisms that control these nuclear characteristics. However, controlled accessibility to regions of chromatin and specific sequences of DNA may be one of the primary regulatory mechanisms by which higher cells establish potentially active chromatin domains. Another control mechanism may be compartmentalization of active chromatin to certain regions within the nucleus, perhaps to the nuclear matrix. Topological constraints and DNA supercoiling may influence the active regions of chromatin and be involved in eukaryotic genomic functions. Further, the chromatin structure of various DNA regulatory sequences, such as promoters, terminators and enhancers, appears to partially regulate transcriptional activity.
We have studied the effects of sodium butyrate on DNA-mediated gene transfer in an effort to investigate interrelationships between chromatin structure and expression of recombinant plasmids. Our results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels. First, the number of cells able to express foreign DNA increases from 10% to up to 40%. Second, there is an increase in enhancer-dependent transcription, approximately 30 fold in HeLa cells, involving the SV40 early promoter. Stable transformation efficiencies increase to 4% and 10% in HeLa S3 and monkey kidney CV-1 cells, respectively. Finally, expression of integrated recombinant plasmid genes is reinducible by a second treatment five weeks after initial exposure to this agent.
Hybrid prokaryotic/eukaryotic expression vectors have been used to introduce Drosophila histone genes into CV-1 African green monkey tissue culture cells. Transfection of CV-1 cells with Drosophila genes under the control of insect DNA promoter sequences results in low level expression of histone genes. On the other hand, when the Drosophila H2a gene is juxtaposed downstream from the long terminal repeat sequence of Rous sarcoma virus (RSV) expression of the insect gene is considerably more efficient; both 3' polyadenylated insect histone messenger RNA and putative Drosophila H2a histone protein can be readily detected in the transduced cells. Using this RSV/H2a vector, we have been able to demonstrate the presence of Drosophila H2a histone in monomer nucleosome preparations isolated from transfected CV-1 cells. These results suggest the feasibility of 'remodeling' cellular chromatin in vivo in precisely defined ways. The techniques described may be generally applicable to other genes coding for chromosomal proteins.
Techniques are described for selectively removing the carbohydrate moieties from the high mobility group (HMG) 14 and 17 proteins of Friend erythroleukemia and calf thymocyte cells without causing degradation of these nonhistone proteins. Investigations were therefore undertaken to investigate the possible significance of this secondary biochemical modification by comparing the activity of the HMGs with and without their glycosyl side chains in various functional assays of the proteins. For example, these HMGs have been found to be equally effective in the partial inhibition of the histone deacetylase enzymes of mammalian cell nuclei whether or not they contain covalently bound carbohydrate residues. We also investigated the possibility that the glycosyl modifications might be involved in the reported ability of these HMGs to specifically cause reconstitution of the DNase I-sensitive structure of "active" genes in salt-depleted chromatin. Unexpectedly, in experiments with both the active beta-globin gene of Friend cells and the total complement of active genes in these same mouse cells (which can be preferentially labeled by nuclear nick translation) we have been unable, using purified HMG 14 and 17 preparations, to reconstitute the active DNase I structure of genes in salt-depleted chromatin preparations. Therefore, the possible role, if any, played by HMG glycosylation in such reconstitution experiments remains unknown. However, in the course of these experiments we did find that HMG 14 and 17 proteins covalently linked to carbohydrate side chains bind preferentially to the nuclear protein matrix of mammalian cells. Furthermore, this association appears to be mediated through the glycosyl side chains since enzymatic removal of these modifications from the HMGs greatly reduced their binding to the nuclear matrix. Since numerous workers have implicated the nuclear matrix as the site of both RNA transcription and DNA replication in eukaryotic cells, the finding of HMG association with this nuclear structure may have significance for our understanding of the overall architectural organization of the active domains of chromatin in cells.
Histone acetylation, DNA replicative synthesis, UV-induced DNA repair synthesis, and UV-induced endonuclease-sensitive sites were measured in normal human fibroblasts and xeroderma pigmentosum fibroblasts (complementation groups A, C, and D) following exposure to sodium butyrate. In all four cell types, treatment with millimolar concentrations of sodium butyrate resulted in a hyperacetylation of the core histones. Furthermore, following an exposure of 20 mM sodium butyrate for 48 h, the extent of hyperacetylation was the same in each cell type. In agreement with previous reports, we observed a marked decrease in DNA replicative synthesis in each cell type following increasing times of exposure to sodium butyrate. On the other hand, we observed a marked increase in DNA repair synthesis occurring during early times after UV irradiation in normal cells and in two of the xeroderma pigmentosum cell strains (groups C and D). This increase appeared to correlate with the increase in the highest acetylated form of histone H4. Furthermore, the total number of endonuclease-sensitive sites (i.e. prior to the onset of repair) induced by UV radiation was the same in both butyrated-treated and untreated normal cells over the dose range of 0-20 J/m2. However, the initial rate of removal of these sites increased in butyrate-treated normal cells. These results indicate that sodium butyrate stimulates the initial rate of nucleotide excision repair in both normal and (partially) repair-deficient human cells at concentrations where the histones are maximally hyperacetylated.
This paper reports the results of numerous biochemical analyses which indicate that the "high mobility group" proteins (HMGs) of mouse and bovine cells are bona fide glycoproteins and can, in addition, be modified by poly(ADP-ribose) addition in vitro. The sugars N-acetylglucosamine, mannose, galactose, glucose, fucose, and one unknown sugar (possibly xylose) have been identified in purified preparations of HMGs 14 and 17. Furthermore, the fucose-specific lectin Ulex europeus agglutinin I bound both to the isolated HMGs and to monomer nucleosomes containing HMGs released from "active chromatin" by micrococcal nuclease digestion. Selective alkaline borohydride reductive cleavages of the HMGs suggested that the oligosaccharide prosthetic groups are primarily bound to these proteins by N-glycosidic linkages. The unexpected finding that the HMGs contain covalently bound complex carbohydrate moieties allows for a potentially great amount of variability and specificity in these proteins that may have important biological implications.
Digestion of isolated Friend erythroleukemic cell nuclei with DNase I under conditions which selectively destroy the DNA of transcriptionally "active" genes releases into the supernatant fraction proteins of the non-histone "High Mobility Group" (HMGs). Two of these, HMG-14 and HMG-17(identified by solubility in trichloroacetic acid, electrophoretic mobility on SDS-polyacrylamide gels and by amino acid composition) will partially inhibit the endogenous mouse cell histone deacetylase enzymes when added to in vitro assay mixtures. Other closely related proteins do not share this inhibitory ability and thus the reaction with the enzymes appears to be specific. Since these two HMG proteins appear to be preferentially associated with the "active" fraction of chromatin, these findings have important implications for possible models of eukaryotic gene regulatory mechanisms.
The strain of Drosophila melanogaster carrying the inversion of In(1)wm4, which juxtaposes the normal w+ gene to the centromeric heterochromatin, variegages for pigmentation in the eye. This strain was treated with various concentrations of n-butyrate and n-proprionate during the embryonic and larval stages. Concentrations as low as 70 mM markedly suppress the variegated eye phenotype. This suggests that non-acetylated histones play a major role in the phenomenon of position-effect variegation.
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The chromatin subunit or nucleosome structure of the amplified, extrachromosomal, ribosomal genes of oocytes of the amphibian Xenopus laevis has been investigated during stages of growth when these genes are markedly changing their rates of transcriptional activity. Nucleic acid hybridization studies involving micrococcal nuclease derived monomer nucleosome DNA fragments and purified ribosomal RNAs indicate that the apparent degree of accessibility of the ribosomal genes to short-term nuclease hydrolysis varies as a function of the rate of ribosomal RNA (rRNA) transcription. However, at no stage during oocyte development are all of the amplified ribosomal genes completely accessible to nuclease hydrolysis, even in those stages with maximal rates of rRNA transcriptional activity. These results suggest that the transcriptionally active ribosomal genes of oocytes are partially, or perhaps transiently, associated with histones in the form of nuclease releasable nucleosomes but that the degree of this association may change with varying rates of rRNA synthesis. Additionally, the present data indicate that the average size of the double-stranded ribosomal DNA associated with monomer nucleosomes is the same (about 200 base pairs) in all of the oocyte stages examined regardless of the rates of rRNA synthesis in these stages.
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Sodium butyrate in millimolar concentrations causes an accumulation of acetylated histone species in a variety of vertebrate cell lines. In all lines tested, butyrate caused hyperacetylation of H3 and H4, and in rat IRC8 cells, H2A and H2B were also affected. In Friend erythroleukemic cells, butyrate also induces the synthesis of a nonhistone chromosomal protein, IP25. butyrate does not affect the rate of histone acetylation in cell-free extracts of nuclei of Friend cells. Rather, this fatty acid inhibits histone deacetylation. Cell-free extracts of either control cells or butyrate-grown cells contain comparable levels of histone-deacetylating activity. This in vitro activity is inhibited by the addition of butyrate to the extracts. Thus butyrate appears to be an inhibitor of histone deacetylases both in vivo and in vitro.