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T R Butt

Publications and source records attributed to T R Butt.

49 records · Page 3Linked to original sources
PubMed1985

Sea urchin metallothionein sequence: key to an evolutionary diversity.

The metallothioneins (MTs) constitute a diverse family of proteins, which are enriched in cysteines and bind heavy metals. The amino acid sequence of sea urchin MT has been obtained from its mRNA sequence and compared with MT sequences of various sources. A largely conserved sequence of 10 amino acids, the "central segment," is located near the center of the MT molecules of Neurospora, yeast, and Drosophila and the center of putative domains in mammalian and sea urchin MTs. The sea urchin carboxyl-terminal-half MT resembles the mammalian 9-cysteine amino-terminal MT domain I, both in the presence of this central segment and in the relative placement of cysteine residues. Conversely, the sea urchin amino-terminal-half MT, containing 11 cysteines, resembles the mammalian carboxyl-terminal MT domain II in its exclusive enrichment in vicinal cysteines. The reversed order of these sea urchin and mammalian MT halves appears to be just one aspect of a diversity based on the elaboration of structures containing the central segment. Still another variation in this diversity is the duplication of the central segment, apparent in Drosophila and crab MTs.

Amino Acid Sequence↗
PubMed1984

Cloning and expression of a yeast copper metallothionein gene.

The induction of a copper-binding metallothionein (Cu-MT) was studied in yeast, Saccharomyces cerevisiae, and a relationship between copper resistance and intracellular levels of Cu-MT in these eukaryotes was established. Poly(A)-containing RNA from a copper-resistant (Cur) yeast strain, which synthesized abundant quantities of Cu-MT and in which Cu-MT gene transcription was enhanced 50-fold upon exposure to CuSO4, was used to screen yeast genomic DNA clones. Restriction analysis revealed common XbaI and KpnI sites in five genomic clones isolated. The transcription of these clones was regulated by copper. Transformation of a copper-sensitive (Cus) yeast strain by one of these clones confers copper resistance in yeast. The results suggest that the expression of the Cu-MT gene is, in part, responsible for mediating copper resistance in yeast.

Cloning, Molecular↗
PubMed1984

Copper metallothionein of yeast, structure of the gene, and regulation of expression.

Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.

Amino Acid Sequence↗
PubMed1983

Interferon and sodium butyrate inhibit the stimulation of poly(ADP-ribose) synthetase in mouse cells stimulated to divide.

The activity of poly(ADP-ribose) synthetase, a chromatin-bound enzyme, increases when quiescent 3T3 cells are stimulated to proliferate. The elevation of enzymatic activity requires de novo RNA and protein synthesis. Interferon (IFN) or sodium butyrate, when added to quiescent cells at the time of stimulation, suppressed the rise of enzymatic activity as well as initiation of DNA synthesis in cells. However, other DNA synthesis inhibitors like methotrexate, FudR and hydroxyurea had little effect on the elevation of poly(ADP-ribose) synthetase in quiescent cells.

Animals↗
PubMed1980

Relationship between nicotinamide adenine dinucleotide concentration and in vitro synthesis of poly(adenosine diphosphate ribose) on purified nucleosomes.

When oligonucleosomes (8-10 N) were incubated under conditions favoring poly(ADP-ribosylation) with concentrations of [32P]NAD 10 microM and higher, the labeled chromatin components migrated in 3-8% gradient native chromatin polyacrylamide gels, in positions of far greater size than the starting nucleosomes. Only a small fraction of chromatin and/or oligonucleosome components was found to be involved in this aggregation. This phenomenon could be demonstrated as well by the increased sedimentation of ADP-ribosylated chromatin components in velocity sucrose gradients. In contrast, at [32P]NAD concentrations less than or equal to 1 microM, the modified nuclear proteins were found strictly associated with the original oligonucleosomes. The extent of aggregation was proportional to both substrate concentration and time of incubation with NAD. During this process, progressively longer chains of poly (ADP-Rib) (10-60 N) were generated on chromatin proteins correlating with the level of complex formation. Analysis of protein by either acid-urea or Na-DodS4O-gel electrophoresis indicated that at NAD concentrations favoring aggregation, poly(ADP-Rib) induced ADP-ribosylated complexes of histone H1, and possibly core histones were observed. Poly(ADP-Rib) polymerase requires both DNA and nuclear proteins for activity. The presence of this enzyme in the high molecular weight complexes was demonstrated on sucrose gradients and also by direct enzyme assays, in situ, in regions of 3-8% gradient chromatin gels containing the complexed chromatin components.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

Characterization of poly(ADP-ribose)--histone H1 complex formation in purified polynucleosomes and chromatin.

Poly(ADP-ribose) [poly(ADP-Rib)] polymerase of HeLa nucleosomes has been shown in vitro, to catalyze the synthesis of a complex of histone H1 containing 2 H1 histones and 15-16 units of oligo(ADP-Rib). The synthesis of the H1 complex in vitro was compared in polynucleosome populations of various sizes (3--16 and greater than 30) released from HeLa nuclei following micrococcal nuclease digestion. Poly(ADP-Rib) was synthesized from [32P]NAD and the poly(ADP-ribosyl)ation of H1 was studied by selective H1 extraction, gel electrophoresis and autoradiography. Quantitative differences in H1 complex formation occurred when either chromatin concentration or polynucleosome length was varied. The data indicated that H1 complex formation in vitro was favored in polynucleosomes 16 nucleosomes long as compared to 8 nucleosomes. A series of partially ADP-ribosylated H1 species was also detected. Partially modified H1 species migrate more slowly than pure H1 in dodecylsulfate gels. The reduced mobility is a function of the number of attached ADP-Rib moieties. Thus, molecules containing one molecule of H1 and various numbers of ADP-Rib residues can be separated. When the partially modified H1 species were incubated in alkali to cleave the linkage of ADP-Rib to protein, (ADP-Rib1-15) were detected by chain length analysis on 15% polyacrylamide gels. The intermediate H1 species could be chased, in vitro, into as H1 complex with NAD and thus were determined to be successive precursors in the formation of the H1 complex. Evidence is presented that the H1 complex is synthesized in intact cells permeabilized with lysolecithin.

Chromatin↗
PubMed1979

Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin.

The relationship between poly(adenosine diphosphate) ribosylation of nuclear proteins and functionally different forms of chromatin from mid-S-phase HeLa nuclei was investigated. The major observations emerging from this study were that unique nonhistone proteins were modified in mid-S-phase HeLa nuclei. The major acceptor for poly(adenosine diphosphate-ribose) [poly(ADP-Rib)] was an internucleosomal nonhistone protein (protein C; 125 000 molecular weight). Histones H3, H1, H2b, and H2a but not H4 were ADP-ribosylated in S-phase nuclei. Chromatin fragments preferentially released by micrococcal nuclease were enriched in nonhistone proteins, poly(ADP)-ribosylated nuclear proteins, poly(ADP-Rib) polymerase activity and nascent DNA from the DNA replicating fork. In extended forms of chromatin, contiguous to the DNA replicating fork, poly(ADP-Rib) polymerase was maximally active. However, in chromatin distal to the replicating fork (i.e., more condensed structures), nucleosomal histones and histone H1 were not significantly ADP-ribosylated, and poly(ADP-Rib) polymerase activity was depressed two- to threefold. The data suggest that a subset of nucleosomes in extended regions of chromatin is subject to extensive ADP ribosylation.

Cell Nucleus↗
PubMed1979

Nucleosome periodicity in HeLa cell chromatin as probed by micrococcal nuclease.

When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16 N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. The 16N polynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-10N, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.

Chromatin↗
PubMed1978

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

Animals↗
PubMed1978

A nuclear protein-modifying enzyme is responsive to ordered chromatin structure.

Poly (ADP-ribose) polymerase, a nuclear protein-modifying enzyme, binds to the internucleosomal linker region of chromatin, although it modifies certain core nucleosomal histones in addition to histone H1. The activity per unit of DNA chromatin changes with the nucleosome repeat number. It reaches a maximum on chromatin of 8-10 nucleosomes in length. As the complexity of chromatin with respect to nucleosome repeat number and compactness increases, a decline and stabilization of specific activity is noted. The difference in specific activity is maintained through resedimentation and dialysis of particles. It does not appear due to differences in polymer chain length or differential degradation of poly (ADP-ribose). The data suggest a relationship between ADP-ribosylation and chromatin organization and vice versa.

Binding Sites↗