Douglas L. Vizard

Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease.

Rat liver nuclei were digested with micrococcal nuclease following incubation with 125I-angiotensin II (AII) or with 125I-AII and excess unlabeled hormone. Chromatin enriched in 125I was solubilized after 3 min and was applied to a BIO-GEL A-5 M column. Labeled hormone was 40-60% displaceable by unlabeled hormone, in nucleoprotein eluting with a V/Vo near 1.9, indicating that these solubilized chromatin fragments contained specific receptors for AII. Furthermore, a discrete AII binding nucleoprotein particle was resolved on DNP gel electrophoresis. Additionally, binding to specific AII nuclear receptors appeared to bring about changes in chromatin structure consistent with the induction of transcriptional activity.

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET‐IRRADIATED CHINESE HAMSTER CELLS

Abstract—As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer‐containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer‐containing DNA is replicated in ultraviolet‐irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.

Saltatory thermal denaturation of double-stranded viral RNAs.

The double-stranded RNAs from bacteriophage phi6 and the replicative form of mengovirus denature upon heating in a series of abrupt steps which resemble the subtransitions (thermalites) observed within the high resolution profiles of small, naturally occurring DNA molecules. Such RNA thermalites are approximately an order of magnitude narrower than typical thermal subtransitions of nominally single-stranded RNA. We conclude that the same features of nucleotide sequence that give rise to cooperative denaturation in DNA genomes are to be found also in RNA genomes. Thus, high resolution thermal denaturation profiles are useful for characterizing double-stranded RNA molecules as well as native DNA in the size range of common viruses. A medium containing dimethylsulfoxide was required to lower the Tm of the RNA samples to a satisfactory temperature range. For double-stranded RNA in 50% dimethylsulfoxide, the dependence of Tm on G . C composition was greater than that of DNA in the same medium and also greater than that of double-stranded RNA in an aqueous medium. The fact that RNA thermalites are broader than DNA thermalites and that the melting temperature of double-stranded RNA has a greater dependence on base composition than that of DNA, indicates that at least one of the thermodynamic parameters for double helix formation in RNA is different from that in DNA.

Temporal replication of an interspersed repeated sequence of mouse DNA

The temporal replication profile of an interspersed repeated DNA sequence (variously named MIF-1, Bam and L1Md) of mouse was determined by isotope analysis of a resolvable restriction fragment differentially labeled in pre- and post-synchrony cultures. While the temporal replication profile of the fragment was similar to that of total nuclear DNA, an average time lag of about 20 min was evident for this interspersed repeated family (called Bam in this paper). In addition, the sequence organisation of Bam homologues were examined for the separable early- and late-replication domains of the hamster genome. The data suggest that late-replicating domains of the rodent genome are slightly enriched in Bam homologous sequences. Furthermore, this repeated sequence family has different sequence organisations in the separable replication domains of hamster.

Preliminary characterization of chelation-sensitive nucleoprotein particles

Abstract Nucleoprotein particles (B2), isolated following digestion of calf thymus chromatin with micrococcal nuclease, are resolved on a non-chelating Bio-Gel A-5m column. B2 protein electrophoresis showed the presence of several H1 species and several nonhistone proteins but was depleted in core histones. DNA electrophoresis demonstrated that native B2 DNA has a length of about 46 base pairs. On DNA sequencing gels, the length distribution of denatured B2 DNA ranged from 12 to 35 bases with a weighted average chain length of about 26 bases. Depletion of a 20 base band in B2 DNA suggested specific protection of internucleosomal DNA sites during the nuclease digestion.

Dual-mode computer processing for high resolution DNA thermal denaturation experiments

Two modes of data processing are appropriate in conducting high resolution thermal denaturation experiments (thermal increments of 0.05 degrees or closer). In the first mode, a general purpose microcomputer provides on-line services important to the control and monitoring of the initial experiment, including control of the spectrophotometer and heater, the recording of data, and the display of current hyperchromicities and approximate first derivatives. A subsequent microcomputer program then reads the recorded data files and carries out accurate calculations of derivative denaturation profiles and the estimate of the statistical error of the first derivative at each point. The data collection program handles three samples at a time and was designed to provide optimal results in thermal denaturation experiments with a single-beam spectrophotometer.