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W T Garrard

Publications and source records attributed to W T Garrard.

At least 55 records · Page 3Linked to original sources
PubMed1987

Sharp boundaries demarcate the chromatin structure of a yeast heat-shock gene.

In both induced and basally transcribed states, the chromatin structure of the yeast HSP82 heat-shock locus exhibits a remarkable degree of organization with respect to DNA sequence. The promoter region contains a constitutive DNase I hypersensitive site. The transcription unit is markedly sensitive to DNase I, and exhibits a sharp transition from a phased half- to a whole nucleosomal cleavage periodicity at the 3' end. Distant upstream and downstream regions are also organized into distinct arrays of phased nucleosomes. Each array is demarcated by DNase I hypersensitive sites that display internal protected regions, suggesting the presence of DNA binding proteins. In addition, since these sites are of mononucleosomal DNA length, they may acquire a nucleosomal structure under certain environmental conditions without disrupting flanking nucleosomal phasing frames. Thus, the HSP82 locus is organized into specific, phased, chromatin structures that appear to function in transcriptional initiation, RNA polymerase passage, transcriptional termination, and the establishment of chromatin-domain microenvironments.

Chromatin↗
PubMed1986

Chromosomal loop anchorage sites appear to be evolutionarily conserved.

We have previously identified a class of DNA sequence elements, termed matrix association regions (MARs), which specifically bind to nuclear matrices in vitro and are believed to be at the bases of chromosomal loops in vivo. Here we demonstrate that nuclear matrices prepared from the yeast Saccharomyces cerevisiae will specifically bind an MAR sequence derived from the mouse kappa light chain immunoglobulin gene. This suggests that both MAR sequences and their binding sites have been strongly evolutionarily conserved.

Animals↗
PubMed1986

Temperature-dependent cleavage of chromatin by micrococcal nuclease near the nucleosome center.

Digestion of nuclei at 4 degrees C with micrococcal nuclease results in significant intranucleosomal cleavage compared to digestion conducted at 37 degrees C. Employing nucleoprotein gel electrophoresis in one dimension followed by DNA electrophoresis in a second dimension, we demonstrate that such temperature-sensitive, internal cleavage predominantly occurs about 20 bp from the nucleosome center. We suggest that lower temperatures reduce the stability of hydrophobic interactions within the histone octamer and lead to a conformational alteration in nucleosomes that is detected by micrococcal nuclease.

Cells, Cultured↗
PubMed1986

Transcription termination and chromatin structure of the active immunoglobulin kappa gene locus.

To investigate the chromatin surrounding an active gene, we have determined the distribution of RNA polymerase molecules, the intactness of nucleosomal structure, and the subnuclear compartmentalization along 15 kilobase pairs (kb) of the mouse kappa immunoglobulin locus of MPC-11 plasmacytoma cells. Hybridization of in vitro nuclear transcripts to probes specific for the template strand reveals that transcription terminates within the region between 1.1 and 2.3 kb downstream from the poly(A) addition site. Ten different short sequences (8-13 base pairs) reside within 460 base pairs of this termination region that exhibit homology with sequences found in the termination regions of mouse beta-globin and chicken ovalbumin genes. Transcription of the nontemplate strand occurs on either side of this termination region. We find that both within the transcription unit and 6.5 kb downstream of the termination region of the kappa gene, the canonical nucleosomal structure is perturbed, the chromatin exhibits pronounced insolubility, and the nucleosomes liberated by micrococcal nuclease appear to lack histone H1. The insolubility is characterized by interactions that are disrupted by 0.3 to 0.6 M NaCl treatment. We conclude that the active chromatin phenotype spreads a considerable distance along the kappa locus, well beyond the region of transcription termination.

Animals↗
PubMed1986

Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites.

Introduction of torsional stress into active chromatin domains requires that linear DNA molecules be anchored in vivo to impede free rotation. While searching for these anchorage elements, we have localized a nuclear matrix association region (MAR) within the mouse immunoglobulin kappa gene that contains two topoisomerase II sites and is adjacent to the tissue-specific enhancer. The same matrix contact occurs when the kappa locus is in germ-line (inactive) or rear-ranged (transcribed) configurations. This constitutive anchorage site partitions the gene into V-J and C region chromatin domains. We demonstrate that at least 10,000 similar and evolutionarily conserved MAR binding sites exist in the nucleus. We propose that these sites, in association with topoisomerase II and possibly in conjunction with enhancers, play fundamental roles in the functional organization of chromatin loop domains.

Animals↗
PubMed1986

The active immunoglobulin kappa chain gene is packaged by non-ubiquitin-conjugated nucleosomes.

To elucidate the molecular features of active chromatin, we have mapped, by two-dimensional electrophoresis, the protein composition of nucleosomes that package the immunoglobulin kappa chain gene of mouse plasmacytoma cells. Nucleoprotein particles that possess the active kappa chain gene comigrate with bulk mononucleosomes that contain high mobility group proteins HMG-14 or -17 but lack histone H1. High electrophoretic resolution of the underlying core particles, after removal of ubiquitin by isopeptidase treatment, reveals that these nucleosomes are nonubiquitinated, even though they coincidently migrate with bulk ubiquitinated particles. This distinctive electrophoretic behavior may be correlated with the presence of histone H2A.X. Nucleosomes exhibiting these unusual properties appear to span at least 10 kilobases, in both transcribed and nontranscribed regions, suggesting that mechanisms independent of transcription exist to initiate, maintain, and propagate a common chromatin phenotype over long distances along the kappa chain locus.

Animals↗
PubMed1986

The ubiquitous potential Z-forming sequence of eucaryotes, (dT-dG)n . (dC-dA)n, is not detectable in the genomes of eubacteria, archaebacteria, or mitochondria.

The potential Z-forming sequence (dT-dG)n . (dC-dA)n is an abundant, interspersed repeat element that is ubiquitous in eucaryotic nuclear genomes. We report that in contrast to eucaryotic nuclear DNA, the genomes of eubacteria, archaebacteria, and mitochondria lack this sequence, since even a single tract of greater than or equal to 14 base pairs in length is not detectable through either hybridization or sequence analysis. Interestingly, the phylogenetic distribution of the (dT-dG)n . (dC-dA)n repeat exhibits a striking parallel to that of (dT-dC)n . (dG-dA)n, but not to other homocopolymeric sequences such as (dC-dG)n . (dC-dG)n or (dT-dA)n . (dT-dA)n.

Archaea↗
PubMed1985

Chromatin structure of the potential Z-forming sequence (dT-dG)n X (dC-dA)n. Evidence for an "alternating-B" conformation.

The sequence (dT-dG)n X (dC-dA)n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG)n X (dC-dA)n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG)n X (dC-dA)n elements instead adopt an "alternating-B" conformation on the nucleosomal surface.

Animals↗
PubMed1984

Differentiation-dependent chromatin alterations precede and accompany transcription of immunoglobulin light chain genes.

We have studied the nature of chromatin alterations along immunoglobulin light chain (IgL) genes during B cell development using cultured murine cell lines. Employing a chromatin fractionation procedure on micrococcal nuclease-treated nuclei, we demonstrate that transcriptionally active k IgL chromatin lacks a canonical nucleosomal repeat and exhibits a pronounced association with insoluble nuclear material but is processed by nuclease to a soluble nucleosomal component that apparently lacks histone H1 and is enriched in high mobility group proteins. Of particular significance, utilizing a variant plasmacytoma cell line that has transcriptionally inactivated one k allele via a promoter deletion, we demonstrate that transcription per se is not responsible for these novel alterations. Furthermore, we show that the chromatin encompassing germline (unrearranged) and transcriptionally silent lambda IgL alleles in k-producing plasmacytomas exhibit some of the same unusual properties that are displayed by k alleles. Finally, we demonstrate that these alterations only occur in cell lines of the lymphocyte lineage that have progressed past the early pre-B cell stage; when inactive, both k and lambda IgL genes possess typical nucleosomal packaging and co-fractionate with histone H1-containing chromatin. These findings lead us to propose a model that predicts B cell stage-specific alterations in IgL chromatin prior to gene rearrangement and transcription.

Animals↗
PubMed1983

Varigated chromatin structures of mouse ribosomal RNA genes.

We have employed a chromatin fractionation procedure on micrococcal nuclease-digested nuclei to examine the chromatin structure of mouse ribosomal RNA genes in two systems that differ by at least 14-fold in the level of ribosomal RNA transcription. In a cultured cell line enriched in transcriptionally active ribosomal chromatin, most ribosomal sequences are preferentially sensitive to digestion by micrococcal nuclease, reside in an insoluble chromatin fraction, and lack typical nucleosomal packaging; only minor amounts of ribosomal sequences are packaged into soluble, nucleosomal chromatin. By contrast, in adult liver, which is enriched in transcriptionally inactive ribosomal chromatin, the majority of ribosomal genes are packaged into soluble, nucleosomal chromatin. However, a significant fraction of liver ribosomal chromatin is insoluble and possesses a non-nucleosomal structure. Therefore, within a single cell population or tissue, mouse ribosomal RNA genes are organized into both nucleosomal and non-nucleosomal chromatin structures. We suggest that these structures have functional significance.

Animals↗
PubMed1983

Amino acid sequence of the N-terminal domain of calf thymus histone H2A.Z.

The minor histone H2A subtype, H2A.Z, has been purified to homogeneity from calf thymus and subjected to automated Edman degradation. The sequence of the first 30 amino acids possesses only 60% homology with major H2A subtypes of the same tissue. This sequence difference is more extreme than that exhibited between evolutionarily distant major H2A subtypes. However, an analysis of secondary structure reveals that H2A.Z and major H2A subtypes exhibit the same general topographical features within their N-terminal domains.

Amino Acid Sequence↗
PubMed1983

5-methylcytosine is localized in nucleosomes that contain histone H1.

We have developed a procedure for purifying highly specific polyclonal antibodies against 5-methylcytosine. These antibodies were used to probe the distribution of 5-methylcytosine among fractionated nucleosomes of mouse cell chromatin. Our results demonstrate that at least 80% of the 5-methylcytosine is localized in nucleosomes that contain histone H1. Native nucleosomes that lack histone H1 or possess high mobility group proteins package DNA that is 1.6- to 2.3-fold undermethylated. We suggest that the preferential association of methylated sequences with histone H1 has functional significance because DNA methylation has been linked to gene inactivation and histone H1 is known to promote chromatin condensation.

5-Methylcytosine↗
PubMed1982

Transferring DNA from electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose: properties of nucleosomes along mouse satellite DNA.

Electrophoresis fractionates nucleosomes which possess different protein compositions. We report here a procedure for transferring the DNA components of electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose (DBM-paper). Histones are first removed from nucleosome components by electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB), leaving DNA fragments fixed within the original gel as the CTAB salts. The DNA is then converted to the sodium salt, denatured, and electrophoretically transferred to DBM-paper. The overall pattern of DNA on the resulting blot is visualized either by fluorography or by immunoautoradiography. This DNA pattern is then compared with autoradiograms obtained after hybridizing the same blot with specific 32P-labeled probes. Using mouse satellite DNA as a hybridization probe, we illustrate the above techniques and demonstrate that nucleosomes carrying satellite sequences are compositionally heterogeneous. The procedures described here should also be useful in the analysis of the nucleic acid components associated with other nucleoprotein complexes.

Animals↗
PubMed1980

A nonamer of histones in chromatin.

The results of dimethyl suberimidate cross-linking experiments demonstrate that histone H1 can be cross-linked quantitatively to the histone octamer of mononucleosomes to yield a histone nonamer. By combining our cross-linking procedure with two-dimensional electrophoretic techniques, we show further that it is possible to trace the nucleosomal origins of a spectrum of cross-linked chromatin products.

Animals↗
PubMed1980

Subunit structures of different electrophoretic forms of nucleosomes.

We have reported previously that five different electrophoretic forms of mononucleosomes (MI to MV) are produced upon treatment of mammalian chromatin with micrococcal nuclease. We show here that each of these mononucleosome classes possesses internal heterogeneity due to the presence of a variety of minor protein species. Defined subsets of mononucleosome classes MII to MV have been reconstituted by reassociating stripped nucleosomes with histone H1 and non-histone protein HMG-17. This procedure leads to the generation of the same five major electrophoretic forms of mononucleosomes found in native chromatin. From the results of one- and two-dimensional electrophoretic analyses on reconstituted samples, it is concluded that different mononucleosome classes possess the following subunit structures: MI, core histone octamer (8-mer); MII, 8-mer plus one copy of HMG-17; MIIIA, 8-mer plus one copy of histone H1; MIIIB, 8-mer plus two copies of HMG-17; MIV, 8-mer plus one copy each of histone H1 and HMG-17; and MV, 8-mer plus one copy of histone H1 and two copies of HMG-17. Equal numbers of HMG-14 molecules can substitute for HMG-17 and generate the same nucleosome components. Thus, mononucleosomes possess independent binding sites for at least 1 histone H1 molecule and 2 nonhistone chromosomal protein molecules. We show further that reassociated HMG-17 molecules can exhibit a rapid interchange between binding sites, even under conditions of low ionic strength.

Animals↗
PubMed1980

Points of contact between histone H1 and the histone octamer.

The topography of the interaction between histone H1 and the histone octamer has been investigated. Bovine thymus nuclei or enzymatically fragmented chromatin were treated 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, which catalyzes the formation of covalent bonds between residues of proteins in electrostatic contact. Histone H1-core histone dimers were identified and the segments of molecules participating in crosslinking were elucidated. The results demonstrate that the major histone H1-core histone dimer generated upon carbodiimide crosslinking of intact nuclei, chromatin, or mononucleosomes consists of the segment of histone H1 containing amino acids 74-106 crosslinked to the segment of histone H2A containing amino acids 58-129. Thus, the central globular region of histone H1 intimately contacts the histone octamer. Besides histone H1-H2 dimers, two other histone H1-containing crosslinked products were detected. In these instances, the segments of histone H1 molecules containing amino acids 1-72 were shown to participate in crosslinking. The histone H1 contact points defined here all occur within mononucleosomes and not between nucleosomes. These results permit the formulation of a testable model for the arrangement of histone H1 along polynucleosome chains.

Amino Acid Sequence↗
PubMed1979

Reassociation of histone H1 with nucleosomes.

The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.

Animals↗