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R Axel

Publications and source records attributed to R Axel.

At least 127 records · Page 7Linked to original sources
PubMed1977

Genes transcribed at diverse rates have a similar conformation in chromatin.

We have analyzed the DNA generated upon treatment of oviduct nuclei with pancreatic DNase I (deoxyribonucleate 3'-oligonucleotidohydrolase; EC 3.1.4.6), with cDNA copies of specific mRNA sequences to study the structure and organization of transcriptionally active genes in chromatin. In this report we examine the kinetics of digestion of three classes of genes in the oviduct which are transcribed at significantly different rates. Our results indicate that the ovalbumin genes appear to be organized by chromatin proteins in such a way that they are rendered exceedingly sensitive to digestion by DNase I. This sensitivity is not observed in the liver, a tissue in which these genes are transcriptionally inert. Furthermore, the transcriptionally inactive globin genes in the oviduct are not selectively sensitive to nuclease attack and are digested 5 times more slowly in the ovalbumin genes in this tissue. In addition, we have examined the accessibility of a complex subset of genes that are rarely represented in the mRNA and are likely to be transcribed at a frequency orders of magnitude below that of the ovalbumin gene. Comparison of the accessibility of these sequences with that of the ovalbumin gene indicates that these two subsets of genes are recognized and cleaved by DNase I at similar rates. These results suggest that the maintenance of an active conformation about specific genes does not reflect the polymerase distribution about these genes. This active conformation is therefore not confined to sequences actively engaged in the transcription process and may reflect the structure about a subpopulation of the genome which represents the transcriptional potential of a given cell type.

Animals↗
PubMed1976

Analysis of the complexity and diversity of mRNA from chicken liver and oviduct.

We have analyzed the sequence complexity and diversity of poly(A)-containing mRNA derived from two highly differentiated chicken tissues. Two independent approaches were used in our analyses. The first involves the annealing of cDNA copies of mRNA to a vast excess of the template RNA; the second procedure uses hybridization between highly radioactive single-copy genomic DNA and mRNA. The results obtained using these two experimental approaches are in good accord and reveal the presence of 12,000-15,000 diverse mRNA species in both chicken liver and oviduct. In both cell types, the kinetics of annealing of cDNA to its template mRNA demonstrate discrete frequency classes with most of the different mRNA species present in fewer than 10 copies per cell. 70% of oviduct mRNA, however, consists of about 10 abundant RNA species, which probably are responsible for the synthesis of the egg white proteins. The diversity of mRNA species in chicken liver and oviduct was further studied by heterologous annealing reactions between cDNA or singlecopy genomic DNA and a vast excess of mRNA. These studies demonstrate that 85% of the different mRNA sequences detected are present in both liver and oviduct, and suggest that the vast majority of the information expressed as mRNA is required for the maintenance of cellular functions common to all tissues.

Animals↗
PubMed1976

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei.

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array. Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA. Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.

Animals↗
PubMed1976

Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei.

Analysis of the DNA of isolated nucleosomes suggests that virtually all genomic DNA sequences are organized in this basic chromatin subunit. In this report, we demonstrate that although histones reside on the transcriptionally active ovalbumin genes in the oviduct, the organization of proteins about this gene renders it highly sensitive to deoxyribonuclease I (deoxyribonucleate 5'-oligonucleotidohydrolase, EC 3.1.4.5). Treatment of oviduct nuclei from the laying hen with pancreatic deoxyribonuclease I results in the preferential digestion of over 70% of the ovalbumin sequences when only 10% of the total nuclear DNA has been solubilized. Treatment of liver nuclei does not reveal selective sensitivity of these genes to DNase I. Furthermore, regions of DNA not actively transcribed, such as the endogenous leukosis virus genes in the oviduct, are not selectively degraded by this enzyme. Similar digestions with micrococcal nuclease, however, reveal no specific digestion of transcriptionally active chromatin. These data confirm the observations of H. Weintraub and M. Groudine [(1976) Science 193, 848-856] and suggest we are dealing with an aspect of structure that may be necessary to permit transcription of the chromatin complex.

Animals↗
PubMed1976

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.

Cell Nucleus↗
PubMed1975

The structure of the globin genes in chromatin.

The distribution of proteins in the neighborhood of the globin genes of duck reticulocyte chromatin has been studied. This chromatin is first shown to be an active template for transcription in vitro of globin messenger-like RNA. The chromatin is then treated with staphylococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated (open "DNA"). In order to determine the distribution of globin gene sequences in open and covered DNA, these two fractions are annealed to globin cDNA (globin probe). It is found that while all globin gene sequences are represented in covered DNA, a specific portion of the globin gene is missing from open DNA, corresponding to about 20% of the gene length. It is concluded that specific regions of the globin genes of reticulocyte chromatin are partly covered by proteins in such a way as to render them in accessible to polylysine. In contrast, no difference is observed in the annealing properties of open and covered regions to globin probe using DNA isolated from erythrocyte chromatin, which is a poor templete in vitro for production of globin message. The annealing of open and covered DNA to each other has also been studied. It is found that open and covered DNA have identical sequence populations. Thus, in contrast to the special arrangement of proteins in the neighborhood of the globin gene, there does not appear to be any sequence-specific arrangement of the bulk of the chromatin proteins on chromatin DNA.

Adenosine Triphosphate↗
PubMed1975

Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease.

Treatment of either rat liver chromatin or intact nuclei with the enzyme staphylococcal nuclease results in the conversion of about half of the DNA to acid-soluble oligonucleotides. As previously described, mild digestion of nuclei results in the liberation of a series of nucleoprotein particles containing DNA fragments which are all integral multiples of a unit length DNA 185 base pairs in length. Analysis of the kinetics of appearance of these fragments suggests that at least 85% of the nuclear DNA is involved in the formation of the repeating subunit profile. More extensive digestion of nuclei however results in the generation of a series of eight unique DNA fragments containing 160 to 50 base pairs. The series of smaller molecular weight DNA is virtually identical with the profile obtained upon limit digestion of isolated chromatin. By velocity centrifugation we have obtained highly purified preparations of the monomeric nucleoprotein particle. Digestion of this monomeric subunit results in the solubilization of 46% of the DNA and analysis of the resistant DNA again reveals the set of eight lower molecular weight fragments. These data suggest that the initial site of nuclease cleavage in chromatin resides within the DNA bridging the repeating monomeric subunits. Further attack results in cleavage at a set of sites within the monomer liberating a pattern of smaller DNA fragments which probably represents the points of intimate contact between the histones and DNA.

Animals↗
PubMed1975

Analysis of DNA of isolated chromatin subunits.

Partial digestion of rat liver nuclei with staphylococcal nuclease results in the liberation of nucleo-protein complexes consisting of one or more upsilon bodies. By velocity centrifugation we have isolated the monomeric subunit in relatively pure form. We find that this subunit contains 185 base pairs of DNA and 240,000 daltons of protein, resulting in a protein to DNA ratio identical to that of unperturbed chromatin. The isolated monomeric particle is further susceptible to internal nuclease attack resulting in the solubilization of 46% of the monomeric DNA. Analysis of the resistant DNA reveals a complex but highly reproducible pattern of DNA fragments ranging from 160 to 60 base pairs in length. Analysis of the reassociation kinetics of the isolated subunit DNA reveals that most, if not all genomic sequences, are involved in this basic subunit structure. No special frequency class of DNA is absent from upsilon bodies. Furthermore, virtually all liver sequences transcribed into mRNA are present in upsilon body DNA. These results indicate that upsilon body formation may be random with respect to DNA sequence and suggest that the mere presence of upsilon bodies over a specific region of DNA is not sufficient to restrict its transcription.

Animals↗
PubMed1974

Specific sites of interaction between histones and DNA in chromatin.

Staphylococcal nuclease digestion of purified chromatin from duck reticulocytes or calf thymus results in the production of a series of double-stranded DNA fragments of discrete molecular size, ranging from about 130 to 45 base pairs, which can be detected by polyacrylamide gel electrophoresis. Similar patterns of protected DNA fragments are obtained from limit digests of chromatin "reconstituted" from purified DNA and chromatin proteins. The results obtained with reconstituted material do not depend upon the origin of the DNA, which may be derived from a bacterial, viral, or homologous source. The specificity of the protective mechanism, therefore, resides in the structure of the bound histones, and probably not in any special nucleotide sequences present in the DNA. Removal of lysine-rich histones from chromatin before digestion results principally in disappearance from the digest of a DNA fragment about 130 base pairs long. Our preliminary results suggest that other elements of the digest pattern can be assigned uniquely to the remaining histone components. These results indicate that the binding of histones to DNA in chromatin involves a limited number of specific and very well defined contacts between protein and nucleic acid, which arise from structural properties of the histones.

Animals↗
PubMed1973

Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro.

The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo. In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.

Animals↗
PubMed1972

Type C virus from cell cultures of chemically induced rat hepatomas.

Type C RNA viruses are present in cell cultures from transplantable and primary hepatomas induced by aromatic amine carcinogens. Virus yield was markedly enhanced by treating the cells with bromodeoxyuridine. Preparations of rat hepatoma-associated virus obtained from cultures treated with this compound were deficient in DNA polymerase activity.

Animals↗