Conditions Of DNA Excess Research Articles

Affinity of interactions between human glucocorticoid receptors and DNA: at physiologic ionic strength, stable binding occurs only with DNAs containing partially symmetric glucocorticoid response elements

Sucrose density gradient shift assays were adapted to permit determination of the affinity of interaction between human glucocorticoid receptors (GR) and DNA under conditions of DNA excess. Saturation analyses were performed to ascertain dissociation constants for the interaction of activated human GR with each of five DNA fragments. Centrifugation of GR-DNA complexes on sucrose gradients under nearly isotonic salt conditions revealed similar affinities with dissociation constants in the range of 2-16 nM for GR interaction with DNA fragments containing glucocorticoid response elements (GREs) exhibiting partial dyad symmetry. By contrast, GR exhibited virtually no affinity for non-GRE-containing DNA or for DNA containing only GRE half-sites. Additionally, GR showed evidence of multiple-site interaction with a DNA fragment containing two partially symmetric GREs, but interacted at only one site of an MMTV LTR DNA fragment containing a single partially symmetric GRE along with a cluster of three half-GREs. Together these data indicate that under physiologically relevant conditions, glucocorticoid receptors have high selectivity and affinity only for DNA containing specific partially symmetric GREs and further suggest that this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid-responsive cells.

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA topoisomerase I from rat brain neurons

The DNA topoisomerase I has been isolated from neurons of rat cerebral cortex. The most homogeneous fraction purified contains only one polypeptide of M r approx. 100 000. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg 2+. The optimum monovalent cation concentration for the relaxation of superhelical DNA under conditions of DNA excess is found to be 175–200 mM. The neuron enzyme is similar to other mammalian type I DNA topoisomerases in that it links to the 3′ ends of the broken DNA strands. Like calf thymus DNA topoisomerase I, the neuron topoisomerase can be selectively inhibited by poly(dG) but not by other homopolymerical deoxyribonucleotides.

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450.

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.

The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin

The optimum monovalent cation concentration (Na + or K +) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150–200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalen cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking-closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.

Assembly of SV40 chromatin in a cell-free system from Xenopus eggs

A cell-free system is described which assembles chromatin from purified DNA in 1 hr under physiological incubation conditions. It consists of a 145,000 × g (maximum) supernatant fraction from eggs of Xenopus laevis. It converts SV40 DNA to a nucleoprotein which co-sediments with naturally occurring SV40 chromatin and which can be cleaved by micrococcal nuclease to a highly ordered pattern of DNA fragments resembling those from digestion of liver chromatin. It inserts superhelical turns into relaxed, covalently closed DNA. The assembly process is not cooperative. Under limiting conditions, each DNA molecule becomes partially assembled. Assembly does not require replication of the DNA or protein synthesis, but occurs from a stored histone pool of at least 40 ng per egg. Under conditions of DNA excess, assembly becomes dependent upon the amount of exogenous histones added to the incubation. Apart from histones and a nicking-closing activity, chromatin assembly requires an additional thermolabile factor which is present in the egg supernatant.

No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed.

Base sequence differences between the RNA components of Harvey sarcoma virus

The 50 to 70S RNA of the Harvey sarcoma-Moloney leukemia virus (MLV) complex consists of 30 to 40S RNA subunits of two different size classes and contains sequences homologous to Moloney mouse leukemia virus and to information contained in a C-type rat virus, termed NRK virus. We have isolated by preparative gel electrophoresis the large (component 1) and the small (component 2) 30 to 40S RNA species from the Harvey sarcoma-MLV complex. Harvey RNA component 1 was completely complementary to DNA transcribed from MLV RNA and showed no homology to DNA transcribed from NRK virus when annealed under conditions of DNA excess. Harvey RNA component 2 was about 65% complementary to MLV DNA and about 33% complementary to NRK virus DNA. Approximately 60 to 80% of the MLV-specific sequences in RNA component 2 is either a distinct molecular species or is part of a hydrid molecular including NRK virus- and MLV-specific sequences. The rest of the MLV sequences in component 2 could be accounted for by degraded component 1 co-purifying with component 2. The possible role of these sequences in the ability of the virus to transform cells is discussed.

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60–70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of “laboratory” leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken genome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection. Increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.

Effect of estrogen on gene expression in the chick oviduct. III. Hybridization studies with [ 3H] messenger RNA and [ 3H] complementary DNA under conditions of DNA excess

A kinetic analysis of the hybridization of a pulse-labeled polysomal mRNA fraction and a 3H-complementary DNA copy of ovalbumin mRNA with an excess of chick DNA is described. The 8–18S RNA fraction has previously been characterized (Means et al., 1972) and has been translated in a cell-free system into immunologically-identifiable ovalbumin. The majority of the polysomal mRNA fraction formed hybrids at C ot values greater than 100. This suggests that these molecules were transcribed from unique sequence DNA and are coded for by a limited number of gene copies. The pulse-labeled mRNA fraction was also incubated with an excess of fractionated chick unique sequence DNA. The extent of hybridization with both the isolated unique sequences and total chick DNA was quite similar at a C ot value of 10,000. The mean thermal stability of these hybrids also indicates little base mismatching, as would be expected for unique sequence hybrids. Purification of a specific mRNA coding for ovalbumin from a hen magnum total nucleic acid extract was accomplished by a combination of Millipore filtration and sucrose gradient centrifugation. The resulting mRNA fraction had approximately 110 times more ovalbumin mRNA activity than the initial nucleic acid preparation in the heterologous rabbit reticulocyte cell-free translation system, and a corresponding 56-fold increase in poly A content. A [ 3H] DNA copy of the enriched mRNA fraction was synthesized using a partially purified RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Approximately 70% of the [ 3H] DNA copy reannealed to chick DNA under conditions of vast DNA excess with an apparent C ot 1 2 of 480, characteristic of chick unique sequence DNA. Therefore, our data suggest that ovalbumin mRNA may be coded by as few as a single gene copy.

Evidence for single copies of globin genes in the mouse genome.

IT is crucial to all our ideas about the structure of eukaryotic chromosomes that we should have estimates of the numbers of copies of typical genes in the genome. Genetic evidence argues for a single copy of each gene but this is difficult to reconcile with experimental evidence that some genes, for example those specifying histones1 and ribosomal RNA2, are reiterated some hundreds or thousands of times without significant divergencies. In a previous report from this laboratory3, it was estimated that globin mRNA hybridized with an amount of DNA corresponding to 50,000 copies of globin genes. These experiments were performed in conditions of RNA excess and the hybrids were of poor quality; hence they were probably relatively non-specific and the results are readily explained in terms of hybridization of a component of the RNA with a family of repetitive sequences4. Similar considerations also apply to the estimate of 60,000 copies of globin genes obtained by hybridization of avian blood 10S RNA5. More recently, in experiments performed in conditions of DNA excess, Bishop et al.6 obtained a much lower estimate, of about five globin genes per genome, when duck 9S RNA was hybridized to excess duck DNA. These experiments have been criticized7 because the relationship between the rate constants for RNA–DNA and DNA–DNA hybridization reactions is uncertain and could be changed by partial degradation of RNA; moreover, a considerable fraction of the RNA remained unreacted in the presence of excess DNA even at very high C0t values (the product of concentration of nucleic acid and incubation time expressed as mole nucleotide per litre × s).

Virus-coded origin of a low molecular weight RNA from KB cells infected with adenovirus 2

After infection with adenoviruses, KB cells synthesize a unique species of low molecular weight RNA (VA RNA). The primary structure of this RNA has been studied and reported previously, but its coding by DNA was in question because it annealed with both adenovirus DNA and KB cell DNA. In the experiments to be reported, most of the nonspecific interaction of VA RNA with KB cell DNA was eliminated by the use of formamide and by further purification of the DNA and RNA. Of VA RNA, 0.74 copy was detected in adenovirus 2 DNA by saturation of the DNA with increasing amounts of VA RNA; 66% of VA RNA added into the incubation mixture was hybridized by saturation with increasing amounts of adenovirus DNA fixed on the cellulose nitrate filter. No increase of the hybridized count was observed when ribonuclease treatment of the hybrids, formed under conditions of DNA excess, was omitted. From these results, it was concluded that VA RNA is coded by the infecting viral genome.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: I. In vitro studies with haemophilus influenzae transforming DNA and yeast photoreactivating enzyme

The photoenzymatic repair of ultraviolet (UV) lesions in DNA is a photochemical reaction which occurs in an enzyme-substrate complex between these lesions and photoreactivating enzyme (PRE). It is shown here that high intensity flash illumination (duration of the order of 1 millisecond) causes this repair in essentially all enzyme-substrate complexes present at the time. This fact allows several kinds of studies. (1) By applying the flash at timed intervals after mixing PRE and UV-irradiated bacterial transforming DNA, the formation of enzyme-substrate complex can be observed directly. The process takes of the order of minutes for its completion at the usual reaction concentrations, and the effect of changing concentration shows that most of this time is required for the extremely dilute reactants (∼ 10 −9 M) to encounter each other in solution. (2) When a sequence of flashes is applied with UV lesions in excess, the resulting stepwise repair permits complex formation to be studied at each level of recovery, from the first set of lesions erased to the last. The result shows that the lesions are heterogeneous with respect to rate of complex formation. (3) The rate of complex formation depends on temperature, but once a comples has been formed at 37° it does not rapidly dissociate on shifting the temperature to 2°. Repeating the experiments at lower light intensity, where not all the complexes present are repaired at each flash, then shows that the actual photochemical process in the complex is temperature-independent over the 2–37° range—at least for the most rapidly complexing lesions. (4) When PRE is allowed to complex with irradiated transforming DNA in the dark, and irradiated non-transforming DNA is subsequently added flashes applied at various times after this addition allow the dark dissociation of enzyme-substrate complexes to be followed. Comparison with the converse experiment, in which PRE is first complexed with lesions on non-transforming DNA, shows that some complexes are extremely stable, the enzyme preferentially remaining on the first DNA with which it forms a complex even after 2 h. Complexes with UV lesions on the synthetic polydeoxynucleotides dA:dT and dG:dC are more stable than most of complexes with natural DNA. (5) Measurement of complex formation under conditions of DNA excess, where essentially all the enzyme is bound, permits determination of the number of enzyme molecules relative to the number of UV lesions. The reasonable assumption that the number of lesions equals the number of pyrimidine dimers recoverable from the DNA then gives an absolute count. Provided the molecular weight of 3·10 4 given by Muhammed is approximately correct, the proportion of pure enzyme to totat protein in crude yeast extract is about 2·10 −6.