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J M Gottesfeld

Publications and source records attributed to J M Gottesfeld.

69 records · Page 4Linked to original sources
PubMed1980

Nonrandom alignment of nucleosomes on 5S RNA genes of X. laevis.

Mild digestion of Xenopus nuclei with micrococcal nuclease results in the cleavage of oocyte-type 5S RNA genes once every four nucleosomes, or about once per tandem repeating unit of 5S DNA. This specific cleavage pattern is observed with nuclei from somatic cells where oocyte-type 5S genes are never transcribed (blood and liver) and with cultured cell nuclei where these genes are in a DNAase I-sensitive chromatin conformation and low level transcription is observed. Cleavage of protein-free DNA with micrococcal nuclease does not result in a specific digestion pattern. The similarity of the nuclease-generated repeat length and the sequence repeat length of oocyte-type 5S genes suggested a sequence-specific arrangement of nucleosomes on these DNA sequences. Restriction endonuclease analysis indicates that micrococcal nuclease preferentially cleaves in a restricted region within the 5S repeating unit, about 200 bp from the single Hind III site. Using specific end-labeled DNA probes derived from cloned 5S DNA we can recognize at least four possible modes of organization of the nucleosomes on 5S DNA. In each of these phase arrangements, functionally significant regions of the 5S gene (start of transcription, middle control region and transcription termination site) are found in or near nucleosome linkers.

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PubMed1978

Organization of transcribed regions of chromatin.

The endonuclease DNase II preferentially attacks a limited and tissue-specific portion of chromosomal DNA. This material may be separated from the bulk of chromatin DNA by virtue of its solubility in 2 mM MgCl2. The Mg2+ soluble fraction forms a specific subset of DNA sequences and is enriched four to sevenfold in sequences coding for cytoplasmic poly(A)-containing RNA and globin messenger RNA (in globin-producing cells). The bulk (70--90%) of rapidly labelled RNA is found associated with the Mg2+-soluble fraction. Transcriptionally active, Mc2+-soluble chromatin is organized into repeating subunits of DNA (200 +/- 5 base pairs) and histone. Mc2+-soluble active subunits differ from the subunits or nucleosomes of non-transcribed regions in many respects: namely, chemical composition (non-histone protein and RNA), sedimentation properties, differential sensitivity to DNase I and the single-strand-specific nuclease S1, and optical melting behaviour. These results suggest that chromatin subunits adopt a new configuration during the process of transcription.

Animals↗
PubMed1977

Structure of transcriptionally-active chromatin subunits.

Rat liver chromatin is organized into regions of DNA which differ in degree of susceptibility to attack by the endonucleases DNase I and DNase II. The most nuclease-sensitive portion of chromatin DNA is enriched in transcribed sequences. This fraction may be separated from the bulk of chromatin by virtue of its solubility in solutions containing 2 mM MgCl2. Both transcribed and nontranscribed regions of chromatin are organized into repeating units of DNA and histone, which appear as 100 A beads in the electron microscope. The length of DNA in the repeat unit is the same for these two classes of chromatin (198 +/- 6 base pairs in rat liver); however, the subunits of active, Mg++-soluble chromatin differ from the nucleosomes of inactive regions of chromatin in several respects. Active subunits are enriched in nascent RNA and nonhistone protein and exhibit higher sedimentation values than the corresponding subunits of inactive chromatin.

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PubMed1976

Sequence composition of the template-active fraction of rat liver chromatin.

Rat liver chromatin has been separated into nuclease-sensitive and -resistant fractions after mild digestion with DNAase II. The nuclease-sensitive material is further fractionated into Mg2+ -soluble and -insoluble chromatin fractions. The kinetics of production of these chromatin fractions have been investigated. After a brief enzyme treatment (5 min at 10 enzyme units/A260 unit of chromatin at pH 6.6), 11% of the input chromatin DNA is found in the Mg2+ -soluble fraction. This DNA has a weight-average single-strand length of about 400 nucleotides and, as determined by renaturation kinetics, comprises a subset of nonrepetitive DNA sequences and a subset of families of middle repetitive sequences. This demonstrates the nonrandom distribution of repetitive and single copy sequences in the Mg2+ -soluble fraction of chromatin. Previous studies have shown that the Mg2+ -soluble fraction is enriched in nonrepeated sequences which are transcribed in vivo (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F., and Bonner, J. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). We now report that the Mg2+ -soluble fraction of liver chromatin contains a low proportion of sequences in common with the Mg2+ -soluble fraction of brain chromatin. Thus, fractionation does not depend on some general property of chromatin but is specific with regard to the template activity of the tissue from which the chromatin was obtained.

Animals↗
PubMed1975

Structure of transcriptionally active chromatin.

Rat-liver chromatin has bee fractionated into transcriptionally active and inactive regions [Gottesfeld et al. (1974) Proc. Nat. Acad. Sci. USA 71, 2193-2197] and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by tne endonuclease DNase II. The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nuclease-resistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition.

Animals↗
PubMed1974

Partial purification of the template-active fraction of chromatin: a preliminary report.

A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl(2). The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA.RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA.

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PubMedDate not supplied

Identifier sequences are transcribed specifically in brain.

'Identifier' or ID sequences are present in 62% of the RNA polymerase II and III transcripts made in vitro from brain nuclei but in fewer than 4% of the transcripts made from the nuclei of other tissues. An homologous 160-nucleotide cytoplasmic poly(A)+ RNA species, BC1, and a smaller species, BC2, are located in vivo exclusively in neural tissues. Cloned ID sequences are polymerase III templates in vitro. Our data suggest a model in which brain-specific polymerase III transcription of ID sequences located in introns of brain genes activates those genes in a primary manner for polymerase III transcription.

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PubMedDate not supplied

Nuclear extracts from globin-synthesizing cells enhance globin transcription in vitro.

In vitro transcription studies of cloned messenger RNA-coding genes have yielded considerable information regarding the sequence elements and protein factors involved in transcription initiation and RNA processing. Fractionation of whole-cell, S-100 protein and nuclear extracts reveals the existence of both general class II and gene-specific transcription initiation factors. Because the soluble in vitro transcription systems prepared from cells in culture are largely nonspecific for the origin of the template DNA, they are highly suited to searching for tissue-specific and gene-specific transcription regulatory factors. In the experiments reported here, we have added a nuclear extract prepared from human erythroleukaemia-like cells (K562, which can be induced to synthesize epsilon- and gamma-globin mRNA and protein) to several deproteinized DNA templates, and monitored transcription levels in a HeLa cell-free transcription system. The K562 nuclear extract enhanced transcription of beta-, epsilon- and gamma-globin genes by as much as 30-fold compared with control non-globin templates. These results suggest the presence of a globin gene regulatory factor in erythroleukaemia cell nuclei.

Cell Extracts↗
PubMedDate not supplied

The 5S gene internal control region is B-form both free in solution and in a complex with TFIIIA.

Rhodes and Klug have recently proposed that the internal control region of the Xenopus 5S RNA gene adopts an A-type DNA structure in solution. This suggestion was based on a Fourier analysis of both the spacing of DNase I cutting sites and on the distribution of G residues in the DNA sequence. Both revealed a approximately 5.6-5.7-base periodicity which the authors interpreted as a structural repeat every half helical turn of A-type DNA. This contention was strengthened by the finding that a 9-base-pair (bp) double-stranded deoxyoligonucleotide corresponding to residues +81 to +89 of the 5S gene exhibits an A' RNA-like crystal structure. This region of DNA is of special interest as it forms the binding site for the 5S gene-specific transcription factor IIIA (TFIIIA). TFIIIA is a Zn2+-binding protein which interacts with both the internal control region of the gene and the 5S transcript. As base-paired regions of RNA are of the A type, it was reasonable to postulate that 5S DNA might also adopt this conformation. We report here that the circular dichroism (CD) spectrum of a synthetic 54-bp deoxyoligonucleotide corresponding to the TFIIIA binding site is similar to the CD spectrum of B-form DNA in solution. Further, DNA-TFIIIA complexes show an unaltered DNA CD component indicating no gross alteration in DNA structure on protein binding.

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