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W H Strätling

Publications and source records attributed to W H Strätling.

At least 37 records · Page 2Linked to original sources
PubMed1991

A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding.

Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops.

Animals↗
PubMed1990

Genomic footprinting of proteins interacting with the chicken lysozyme promoter.

The functions of the promoter of the chicken-lysozyme-encoding gene (Lsz) are complex, since, on the one hand, the promoter serves to mediate the effects of five upstream regulatory elements and, on the other, it contains itself important regulatory sequences. To analyze this interplay we first monitored protein-DNA interactions at the Lsz promoter in hormonally regulated oviducts and in constitutively expressing HD11 cells by in vivo genomic footprinting. Two regions of strong protein-DNA interactions shared by both HD11 and oviduct cells were localized between nucleotides (nt) -105 and -119 and between nt -61 and -74, respectively. A third region of protein-DNA contacts was found selectively in HD11 cells from nt -199 to -204; this region corresponds in position with a previously identified HD11 cell-specific enhancer element. Furthermore, we determined the sites hypersensitive to an endogenous endonuclease in isolated nuclei using the genomic sequencing protocol. These sites fall into two regions: one encompasses the in vivo localized protein-DNA contacts between nt -105 and -119 and between nt -61 and -74; the other was found in a sequence largely devoid of in vivo protein-DNA interactions. We hypothesize that hypersensitive cleavage sites are generated by protein-induced changes in the DNA conformation.

Animals↗
PubMed1990

A non-curved chicken lysozyme 5' matrix attachment site is 3' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.

Animals↗
PubMed1990

The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.

Animals↗
PubMed1989

A nuclear DNA attachment element mediates elevated and position-independent gene activity.

The transcriptional activity of genes that have randomly integrated into the genomes of transfected cells and transgenic organisms is in general unpredictable, varying with the chromosomal site of the insertion. This effect of chromosomal position on gene expression may reflect the organization of chromosomes into topologically constrained loops and functional domains. To assess the biological significance of these loop domains, the anchorage of DNA to the nuclear scaffold has been studied at specific gene loci. We have previously defined cis-acting regions flanking the chicken lysozyme-gene domain that mediate the attachment of the chromatin to the nuclear scaffold. These 'A-elements' map to the 5' and 3' boundaries of the region of general DNase sensitivity in the active chromatin, which contains the lysozyme gene and its cis-regulatory elements. Here we report that when a reporter gene is flanked by 5' A-elements from the lysozyme gene, its expression in stably transfected cells is significantly elevated and is independent of chromosomal position.

Animals↗
PubMed1989

Non-random spontaneous chain breakages occur in DNA methylated with dimethyl sulfate.

The method of in vivo footprinting uses partial methylation of the DNA in living cells with dimethyl sulfate (DMS), cleavage of the DNA chains at modified guanosines with piperidine, and mapping of the site of cleavage by the genomic sequencing technique of Church and Gilbert. Here we report on a spontaneous breakage reaction of DMS-methylated DNA at guanosines as well as adenosines, which is highly non-random with respect to the DNA sequence. In our in vivo genomic footprinting studies at the chicken lysozyme promoter this reaction gave rise to additional adenosine-derived bands in the guanosine sequence ladder.

Alkylating Agents↗
PubMed1988

The matrix attachment regions of the chicken lysozyme gene co-map with the boundaries of the chromatin domain.

The matrix attachment regions of the chicken lysozyme domain were studied in an in vitro DNA binding assay by incubating oviduct nuclear matrices with labeled restriction fragments. A strong attachment region was localized between 11.1 and 8.85 kb upstream of the transcription start site and a weaker one between 1.3 and 5.0 kb downstream of the poly(A)+ addition site. Both attachment regions co-map with the previously established boundaries of the chromatin domain. The upstream matrix attachment region is distinguishable from known enhancers and is composed of multiple binding sites. We find specific but weaker binding of the same restriction fragments to matrix preparations from transcriptionally inactive chicken erythrocytes indicating a cell-type and transcription-independent conservation of the sites for specific binding of matrix attachment sequences. We also demonstrate that the matrix attachment regions are located at the base of a chromosomal loop in histone-extracted nuclei. Thus, the lysozyme domain represents a topologically-sequestered functional unit containing the coding region and all known lysozyme-specific, cis-acting regulatory elements.

Animals↗
PubMed1988

Lysozyme gene specific transcription in isolated hen oviduct nuclei.

1. Transcription from the chicken lysozyme gene domain and the density of RNA polymerase molecules was studied by incubating isolated hen oviduct nuclei in an in vitro transcription system with [alpha-32P]UTP and hybridizing the purified [32P]RNA with immobilized restriction fragments. 2. The labeled RNA hybridized most prominently to coding and flanking restriction fragments that contain repetitive sequences. 3. Relatively weak hybridization signals with a coding, single-copy, genomic fragment and cDNA fragments were detected using 5000 microCi [alpha-32P]UTP per ml of incubation mixture. 4. Flanking, non-repetitive fragments did not hybridize to the [32P]RNA. 5. The results show that transcription from repetitive sequences dominates over lysozyme gene specific transcription.

Animals↗
PubMed1988

Differences among chicken erythrocyte histones H1 and H5 in associating with H1-depleted polynucleosomes.

1. Chicken erythrocyte histones H1a, H1b and H5 were associated to H1-depleted polynucleosomes from rat liver and the products were probed by digestion with micrococcal nuclease. 2. The digestion response of complexes with purified H1a or H1b resembled that of native polynucleosomes, while the digestion response of complexes with purified H5 exhibited specific different features--but none of these complexes resembled those with unfractionated histones H1a, H1b and H5. 3. However, after mixing purified histones H1a, H1b and H5 in the proportions (0.15:0.25:0.9) as these occur in erythrocyte nuclei and associating this mixture, the digestion response of the complexes was similar to that of the complexes with unfractionated histones.

Animals↗
PubMed1987

Gene-specific differences in the supranucleosomal organization of rat liver chromatin.

Rat liver chromatin was separated into a solubilized portion and insoluble nuclear material, and the solubilized portion was fractionated by sucrose gradient sedimentation. The chromatin encompassing three transcribed genes (albumin, tryptophan oxygenase, and alpha-fetoprotein), which are expressed at very different levels, partitions preferentially with insoluble nuclear material and possesses a disrupted nucleosome structure. On the contrary, the chromatin encompassing three inactive genes fractionates into the solubilized chromatin portion and exhibits a canonical nucleosome repeat structure. By sucrose gradient sedimentation, all size classes of inactive chromatin particles are found to contain internal cleavages in the linker region between nucleosomes; they are probably held together by histone H1 and mono- and divalent cations. When the chromatin encompassing two flanking sequences of the tyrosine aminotransferase gene is studied, the 0.35-kilobase upstream-located chromatin exhibits features of active genes, while the 2.55-kilobase upstream-located chromatin partitions preferentially with solubilized chromatin and sediments in internally cleaved particles.

Animals↗
PubMed1986

Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion.

The chromatin structure encompassing the lysozyme gene domain in hen oviduct nuclei was studied by measuring the partitioning of coding and flanking sequences during chromatin fractionation and by analyzing the nucleosome repeat in response to micrococcal nuclease digestion. Following micrococcal nuclease digestion, nuclei were sedimented to obtain a chromatin fraction released during digestion (S1) and then lysed in tris(hydroxymethyl)aminomethane-(ethylenedinitrilo)tetraacetic acid-[ethylenebis(oxyethylenenitrilo)]tetraacetic acid and centrifuged again to yield a second solubilized chromatin fraction (S2) and a pelleted fraction (P2). By dot-blot hybridization with 14 specific probes, it is found that the fractionation procedure defines three classes of sequences within the lysozyme gene domain. The coding sequences, which partition with fraction P2, are flanked by class I flanking sequences, which partition with fractions S1 and P2 and which extend over 11 kilobases (kb) on the 5'side and probably over about 4 kb on the 3' side. The partitioning of class II flanking sequences, which are located distal of class I flanking sequences, is different from that of class I flanking sequences. Coding sequences lack a canonical nucleosome repeat, class I flanking sequences possess a disturbed nucleosome repeat, and class II flanking sequences generate an extended nucleosomal ladder. Coding and class I flanking sequences are more readily digested by micrococcal nuclease than class II flanking sequences and the inactive beta A-globin gene. In hen liver, where the lysozyme gene is inactive, coding and class I flanking sequences fractionate into fractions S2 and P2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Cross-linking of histones with dimethyl 3,3'-dithiobispropionimidate. Interference by a one-end reaction modifying histones at lysine amino groups.

We have studied the HClO4-solubility of histones H1 and H5 in hen erythrocyte nuclei after treatment with the cross-linker dimethyl 3,3'-dithiobispropionimidate (DTPI). The amount of acid-soluble, non-cross-linked, H1 and H5 histones was drastically decreased, and that of acid-soluble H1/H5 histone dimers went through an optimum as the DTPI concentration was raised. Incubation of the HClO4-insoluble fraction with 2-mercaptoethanol regenerated the acid-solubility of H1/H5 histones in this fraction. When purified H1/H5 histones were treated with increasing concentrations of DTPI under non-cross-linking conditions, the amount of HClO4-soluble histones also greatly decreased, but to a much lesser extent if the DTPI treatment was followed by reduction with 2-mercaptoethanol. This decrease was inversely correlated to the proportion of amino groups modified. It is concluded that, when the cross-linker was used in large excess, the cross-linking reaction competed with a one-end reaction modifying the histones at lysine amino groups by cross-linker molecules, of which the imidoester groups that had not reacted were hydrolysed. It is suggested that this modification produced the changes in acid-solubility.

Animals↗
PubMed1984

Cross-linking of histone H5 in hen erythrocyte nuclei.

Following treatment of hen erythrocyte nuclei with dimethyl 3,3'-dithiobispropionimidate, dimers between histones H1a, H1b, and H5 were extracted with 5% perchloric acid. They resolved electrophoretically into four sub-bands and these were identified by non-reducing/reducing gel electrophoresis. The H5-H5 homodimer species was purified by gel electrophoresis and was treated sequentially with BrCN and dithiothreitol. The pattern of resulting fragments indicates that cross-links were mainly formed between the COOH-terminal portions and at a significantly lower frequency between the COOH-terminal and the NH2-terminal portions.

Animals↗
PubMed1984

Ca/Mg-dependent endonuclease from porcine liver. Purification, properties, and sequence specificity.

A rapid and reproducible method for the purification of the Ca/Mg-endonuclease from porcine and rat liver and for the stabilization of the enzyme activity is presented. The optimum conditions for enzyme activity were determined. The enzyme degrades double-stranded DNA endonucleolytically. In the course of digestion of form I closed circular SV 40 DNA, the form II nicked circular DNA is the prominent intermediate product. Digestion of hen erythrocyte nuclei with added endonuclease produces a ladder of mono- and oligonucleosomal fragments similar to that generated by micrococcal nuclease digestion. Determination of the 5'-terminal nucleotides indicates the absence of a significant base specificity. Analyzing the cleavage pattern of end-labeled pBR322 restriction fragments on sequencing gels shows that the enzyme exhibits a weak preference for dinucleotides with A in the 5'-position; dinucleotides with 5%-C are less readily cleaved. Digestion of end-labeled pBR322 DNA, followed by electrophoresis in agarose gels produces a "smear"-like fragmentation pattern with weak superimposed bands, while micrococcal nuclease generates a different and much more distinct pattern. These data show that the sequence specificity of the enzyme is less pronounced than that of micrococcal nuclease and that the sites preferentially cleaved are not the same.

Animals↗
PubMed1983

Neutron diffraction of chromatin in interphase nuclei and metaphase chromosomes.

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.

Animals↗
PubMed1982

Reassociation of histone H1 to H1-depleted polynucleosomes.

A new procedure is described for the reassociation of histone H1 to rat liver polynucleosomes selectively depleted of H1 and nonhistone proteins. The fidelity of reconstitution was scrutinized by nuclease digestion and sedimentation studies monitoring the regeneration of structural features which disappear following removal of H1. The results demonstrate that the 166-base pair barrier of digestion with micrococcal nuclease was restored in polynucleosomes reconstituted at the same ratio of H1 per octamer as that found in native nuclei, while association of twice that amount of H1 produced a barrier of digestion at about 178-180 base pairs. Polynucleosomes associated with polylysine exhibited a similar barrier as H1-depleted polynucleosomes. Digestion of H1-reconstituted polynucleosomes with DNase I produced preferentially dinucleosomal DNA fragments in the same manner as that of untreated polynucleosomes. The rate of digestion of H1-reconstituted polynucleosomes by micrococcal nuclease and DNase I was also comparable to that of untreated polynucleosomes. Sedimentation of H1-reconstituted polynucleosomes in a quasi-physiological ionic milieu revealed the regeneration of disassembly-refractory particles, a structural feature of untreated polynucleosomes. We conclude that the nucleosomal and supra-nucleosomal structure of polynucleosomes was reconstituted with fidelity.

Animals↗
PubMed1981

Mono- and poly-ADP-ribosylation of proteins in mouse kidney after castration and testosterone treatment.

Protein-bound mono(ADP-ribose) and poly(ADP-ribose) residues were determined in mouse kidney after castration and testosterone substitution. After these treatments, the mouse kidney undergoes significant alterations in the extent and pattern of transcription without changes in the amount of DNA and nuclear protein. The amount of mono(ADP-ribose)--protein conjugates (the hydroxylamine-sensitive and -resistant subfractions) decreased by 40% after castration, and returned to normal within 1 week after daily testosterone injections. Polymeric ADP-ribose residues, which amounted to less than 0.3% of the total protein-bound monomeric ADP-ribose, increased after castration and rapidly decreased on testosterone administration. The magnitude of these effects indicates that the decrease in mono(ADP-ribose) was not caused by a shift of monomeric residues into the polymer form. Nuclear ADP-ribosyltransferase activity showed a retarded decrease after castration, reaching 60% of the control value by day 20. After testosterone injections, enzyme activity rose to normal within 3-4 days. The amounts of the substrate NAD+ as well as of NAD+ + NADH also declined after castration, and rapidly returned to values slightly above normal when the androgen was substituted. The differential response of monomeric and polymeric ADP-ribose residues to castration and testosterone treatment suggests that the two modifications serve different functions.

ADP Ribose Transferases↗