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Biomedical subjects

H Weintraub

Publications and source records attributed to H Weintraub.

At least 145 records · Page 8Linked to original sources
PubMed1980

The detection of DNA-binding proteins by protein blotting.

A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.

Carrier Proteins↗
PubMed1980

Interaction of HMG 14 and 17 with actively transcribed genes.

The interaction of HMG 14 and 17 with actively transcribed genes was studied by monitoring the sensitivity of specific genes to DNAase I after reconstitution of HMG-depleted chromatin with HMG 14 and 17. Our experiments lead to the following conclusions: most actively transcribed genes become sensitized to DNAase I by HMG 14 and 17; either HMG 14 or HMG 17 can sensitize most genes to DNAase I; genes transcribed at different rates have about the same affinity for HMG 14 and 17; HMG 14 and 17 bind stoichiometrically to actively transcribed nucleosomes; and HMG 14 and 17 can restore DNAase I sensitivity to purified nucleosome core particles depleted to HMGs. This last observation suggests that during reconstitution, low levels of HMG 14 and 17 can associate with the active nucleosomes in the presence of a 10--20 fold excess of inactive nucleosomes. Consequently, we conclude that besides their association with HMGs, active nucleosomes also have at least one other unique feature that distinguishes them from bulk nucleosomes and insures proper HMG binding during reconstitution.

Animals↗
PubMed1980

Activation of cellular genes by avian RNA tumor viruses.

We demonstrated previously that chicken embryo fibroblasts accumulate approximately 100 copies of embryonic globin RNA after transformation by Rous sarcoma virus. Here we demonstrate that the globin gene in chicken embryo fibroblasts is activated by infection with two other oncogenic retroviruses, avian erythroblastosis virus and strain MC-29 of avian myeloblastosis virus, which contain transforming genes unrelated in nucleotide sequence content to each other or to the Rous sarcoma virus src gene. In addition, we have measured the genetic complexity of transformation by using established techniques for determining the number of different RNA sequences in specific populations of cells. Our results indicate that transformation of chicken embryo fibroblasts by Rous sarcoma virus results in the accumulation of RNA from approximately 1000 average-sized new transcription units.

Alpharetrovirus↗
PubMed1979

Assembly of an active chromatin structure during replication.

MSB cells were pulse labeled with 3H-thymidine and the isolated nuclei digested with either staphylococcal nuclease (to about 40% acid solubility) or DNase I (to 15% acid solubility). The purified, nuclease resistant single-copy DNA was then hybridized to nuclear RNA (nRNA). The results of these experiments show that actively transcribed genes are assembled into nucleosome-like structures within 5-10 nucleosomes of the replication fork and that they also acquire a conformation characteristic of actively transcribed nucleosomes (ie, a DNase I sensitive structure) within 20 nucleosomes of the fork. Assuming DNA sequence specific interactions are required for establishing a DNase I sensitive conformation on active genes during each round of replication, our results indicate that a specific recognition event can occur very rapidly and very specifically in eukaryotic cells. The results are discussed in terms of the possible mechanisms responsible for propagating active, chromosomal conformations from mother cells to daughter cells.

Animals↗
PubMed1979

The asymmetric segregation of parental nucleosomes during chrosome replication.

SV40 DNA replicated in the presence of cycloheximide was more sensitive to staphylococcal nuclease digestion and had a lower superhelical density than viral DNA replicated in the absence of this drug. These data indicate that fewer nucleosomes are associated with progeny SV40 DNA molecules after DNA replication in the absence of protein synthesis and that these nucleosomes are derived from the parental histones. We designed an experiment to determine whether these parental SV40 nucleosomes segregate to the leading side of the replication form where DNA synthesis is continuous, the lagging side of the fork where DNA synthesis is discontinuous or randomly to both sides of the fork. The results indicate that the parental histones distributed themselves asymmetrically, preferentially (80-90%) segregating with the leading side of both SV40 DNA replication forks during bidirectional replication in the absence of protein synthesis. In the case of SV40, the same parental DNA strands are the templates for the leading side of DNA replication at both forks as well as the templates for the informational or coding strand of early and late viral mRNA synthesis. Based on this correspondence, we designed an experiment to test whether chicken cells growing in culture and replicating their DNA in the absence of protein synthesis segregated their parental histones asymmetrically to the progeny DNA strand that also coded for stable nuclear RNA transcripts. The results of these experiments indicate that, like SV40, parental cellular histones segregate asymmetrically and are preferentially associated with those DNA template strands that code for stable nuclear RNA species detected by hybridization to single-copy DNA.

Cycloheximide↗
PubMed1979

Conservative segregation of parental histones during replication in the presence of cycloheximide.

Long stretches of protein-free, nonbeaded DNA were observed electron microscopically in nuclear spreads prepared from cells that had replicated their DNA in the absence of protein synthesis. The amount of this DNA increased with increasing time of replication in the presence of cycloheximide and was greatly decreased when replication was inhibited with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside). This DNA is considered to be "free" DNA because it has the same diameter as marker PM2 DNA and it is preferentially sensitive to DNase I digestion. Reversal of the cycloheximide block resulted in a burst of histone synthesis and repair of the depleted chromatin within 5 min. In addition, 26 presumptive replication forks were observed with beaded chromatin on two arms and free DNA on the third. These results suggest that new histones are usually deposited onto new DNA, that the cellular histone pool is very small, that histone migration is minimal in vivo for at least 18 hr, that for most fibers nuclesome assembly and segregation is conservative for stretches of DNA as long as 100 kbases, and that some part of the octameric histone core may remain bound to DNA during the replication process. The regularity we have observed for the assembly and segregation of nucleosomes is likely to be important for our understanding of how chromosomal information is segregated during development.

Cell Line↗
PubMed1979

Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin.

The globin gene is preferentially sensitive to digestion by DNase I in erythrocyte chromatin but not in brain, fibroblast, or oviduct chromatin. Elution of the erythrocyte chromatin with 0.35 M NaCl leads to no detectable change in the gross structure of individual nucleosomes; however, in this depleted chromatin the globin gene is no longer preferentially sensitive to DNase I. Reconstitution of the depleted chromatin with either the entire 0.35 M NaCl fraction or a subclass from this fraction greatly enriched in two high mobility group proteins (nos. 14 and 17) results in the successful reconstitution of DNase I sensitivity of the globin gene. For all of these preparations, the inactive ovalbumin gene exhibited no preferential sensitivity to DNase I. Reconstitution of the erythrocyte 0.35 M NaCl fraction with depleted brain chromatin resulted in no preferential sensitivity of the globin gene in brain chromatin; however, reconstitution of the brain 0.35 M NaCl fraction with depleted erythrocyte chromatin led to successful reconstitution of DNase I sensitivity of the globin gene. Thus, the eluted proteins responsible for conferring DNase I sensitivity are probably not tissue-specific and probably do not recognize specific DNA sequences.

Animals↗
PubMed1978

Nucleosomal DNA is digested to repeats of 10 bases by exonuclease III.

Nucleosomes were treated with increasing concentrations of exonuclease III (Exo III) from E. coli. At low levels of Exo III, the heterogeneous distribution of monomers (with associated DNA fragments ranging in size between 140 and 170 bp) is "trimmed" down to a discrete core of 140 bp. The "trimming" of monomers to 140 bp results from a 3' exonucleolytic digestion accompanied by a 5' clipping activity which is specific for the conformation of internucleosomal DNA. At higher concentrations of Exo III, the enzyme digests the 140 bp "trimmed" nucleosome core from both 3' ends without associated 5' nuclease activity. Most striking is the observation that the fragments produced during such a digestion display discrete single-stranded lengths that are integer multiples of 10 bases. For some dimer nucleosomes, Exo III can digest as many as 200 bases from at least one 3' end and produce a 10 base interval ladder from about 400 bases down to 180 bases. This suggests that the enzyme can traverse the length of an entire nucleosome without destroying whatever structural features are necessary to produce a 10 base DNA ladder.

Chromatin↗
PubMed1978

Regulation of expression and chromosomal subunit conformation of avian retrovirus genomes.

We have investigated the copy number, chromosomal subunit conformation and regulation of expression of integrated avian retrovirus genomes. Our results indicate that there are approximately two copies of the endogenous viral genomes (RAV-O) per haploid cell genome in uninfected chick embryo fibroblasts (CEF) and red blood cells (RBC). The copy number and subunit conformation (as measured by DNAasel sensitivity) of the RAV-O genomes are independent of the level of expression of these viral DNA sequences. In cells isolated from embryos of the V+, gs-chf- and gs+chf+ phenotypes, approximately one of the two viral genomes is in a DNAase l-sensitive conformation. Upon infection with an exogenous Rous sarcoma virus (PR-RSV-C), one new viral genome is integrated per haploid CEF genome. The newly integrated RSV genome is completely sensitive to DNAase l, and the subunit conformation of the endogenous viral genomes is not altered by the integration of additional exogenous proviruses. Both the endogenous and newly integrated exogenous viral genomes are present in "nu-body" structures, and the selective sensitivity of these proviral DNA sequences to DNAase l is maintained in isolated nucleosomes. Our experiments revealing the DNAase l sensitivity of one of the two RAV-O genomes in gs-chf-CEF led us to reexamine the level of viral specific RNA in CEF of various GS genotypes. We find that GS/GS CEF contain approximately 100 copies of viral RNA per cell, gs/gs CEF contain no detectable viral RNA, and the heterozygote GS/gs CEF contain approximately 50 copies of viral specific RNA per cell. These results suggest that the GS gene controls production of RAV-O RNA sequences in CEF in a "cis" fashion. In RBCs, however, the expression of the RAV-O genome is independent of the GS gene, with both GS/GS and gs/gs RBCs containing roughly equivalent amounts of viral specific RNA. Our results suggest that the chromosomal structure of the endogenous viral genes is independent of the GS gene, and that the GS gene is cis-acting and tissue-specific.

Avian Leukosis Virus↗
PubMed1978

The nucleosome repeat length increases during erythropoiesis in the chick.

During erythropoiesis in the chick, the nucleosome repeat length increases from 190 base pairs to 212 base pairs. This increase is correlated with a dramatic increase in the concentration of the red cell specific histone H5 (from 0.2 molecules per nucleosome to 1 molecule per nucleosome) and with no change in the concentration of H1 (1 molecule per nucleosome).

Aging↗
PubMed1977

Histones H2a, H2b, H3, and H4 are present in equimolar amounts in chick erythroblasts.

A quantitative labeling method for stoichiometric analysis has been applied to an investigation of the histone composition of chick erythroblast chromatin. The necessity of long fractionation procedures and the dangers of preferential extraction and staining are eliminated through the use of this simple procedure which involves the direct analysis of nuclear proteins on sodium dodecyl sulfate gels. Only a knowledge of the amino acid compositions of the histones is required. H2a, H2b, H3, and H4 are found to be present in nearly equimolar quantities.

Animals↗
PubMed1977

Conservative assembly and segregation of nucleosomal histones.

The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3-4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2-3 generations.

Animals↗
PubMed1976

Chromosomal subunits in active genes have an altered conformation.

Ten percent digestion of isolated nuclei by pancreatic deoxyribonuclease I preferentially removes globin DNA sequences from nuclei obtained from chick red blood cells but not from nuclei obtained from fibroblasts, from brain, or from a population of red blood cell precursors. Moreover, the nontranscribed ovalbumin sequences in nuclei isolated from red blood cells and fibroblasts are retained after mild deoxyribonuclease I digestion. This suggests that active genes are preferentially digested by deoxyribonuclease I. In contrast, treatment of red cell nuclei with staphylococcal nuclease results in no preferential digestion of active globin genes. When the 11S monomers obtained after staphylococcal nuclease digestion of nuclei are then digested with deoxyribonuclease I, the active globin genes are again preferentially digested. The results indicate that active genes are probably associated with histones in a subunit conformation in which the associated DNA is particularly sensitive to digestion by deoxyribonuclease I.

Age Factors↗