Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S₁ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K⁺ at 2–20 mmol/l, for Mn²⁺ at 0.8 mmol/l, for Mg²⁺ at 4.0 mmol/l, the pH optimum is 8.0. Its K_m is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

Electron paramagnetic resonance of single crystal deoxycobaltohemoglobin

Single crystals of horse ^CoHb were obtained by reduction of ^CoHb⁺ crystals with dithionite. Epr measurements showed that the g? and ^Coà tensors are both axial and share the same principal axis systems. Of the four subunits, the “heme” normals of C? and d? subunits ãb?plane 29 ± 1° from b?; they have the same orientation as the hemes in methemoglobin. The normals of “hemes” à and B? are 47 above the ãb? plane as compared to 16° in methemoglobin.     

Nucleic Acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating > 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of >48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes.     

Site-directed mutagenesis of the T4 endonuclease V gene: mutations which enhance enzyme specific activity at low salt concentrations

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well in E. coli and not only significantly enhanced the UV survival of repair-deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129----Trp and Tyr-129----Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129----Phe and Tyr-129----Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129----Phe mutant and to 20% that of wild type for the Tyr-129----Trp mutant.     

The nuclei of cellular organelles and the formation of daughter organelles by the “plastid-dividing ring”

It has been established that organelles, such as mitochondria and plastids, contain organelle-specific DNA and arise from the division of pre-existing organelles (e.g., Possingham and Lawrence, 1983). We propose that organelle DNAs, such as mitochondrial DNA and plastid DNA are not naked in organellesin situ but are organized in each case to form an “organelle nucleus” with basic proteins (Kuroiwa, 1982). The concept of organelle nuclei has changed our ideas about the division of organelles. Thus, the process of organelle division must be composed of two main events: division of the organelle nucleus and organellekinesis (division of the other components of the mitochondrion or plastid). The latter term has been adopted as an appropriate analogue of cytokinesis. We were the first to identify the plastid-dividing ring (PD-ring), which is located in the cytoplasm close to the outer envelope membrane at the constricted isthmus of dividing chloroplasts in the red algaCyanidium caldirum. The PD-ring is about 60 nm in width and 25 nm in thickness, and is a circular bundle of actin-like, fine filaments, each about 4–5 nm in diameter. Since cytochalasin B, an inhibitor of polymerization of actin filaments, inhibits the formation of the PD-ring and, thus, prevents subsequent division of chloroplasts, the PD-ring is thought to be a structure that is essential for the division of plastids (plastidkinesis). The behavior of the PD-ring during a cycle of chloroplast division can be classified into the following four stages on the basis of morphological and temporal differences. The chloroplast growth stage: the small, spherical chloroplast increases in volume and becomes a football-like structure, while the PD-ring from the previous division disappears. Formation of the PD-ring: the somewhat electron-dense body (see below) is fragmented into many, somewhat electron-dense granules, which are aligned along the equatorial region of the chloroplast and fine filaments are formed from the somewhat electron-dense granules in the equatorial region. The fine filaments of the PD-ring align themselves according to the longest axis of their overall domain, i.e., circumferentially. Contraction stage: a bundle of fine filaments begins to contract and generates a deep furrow. Conversion stage: after chloroplast division, the remnants of the PD-ring are converted into somewhat electron-dense bodies. Similar events occur during the second cycle of chloroplast division. Since similar structures are observed extensively in the plastids of algae, moss and higher plants, the PD-ring appears to be an essential structure for the division of plastids in plants.     

Molecular Diagnosis of Meloidogyne Species

Genetic variation within nuclear and mitochondrial DNA of Meloidogyne species and host races has been evaluated for the development of root-knot nematode molecular diagnostics. This review summarizes the distinctive features of several useful DNA-based assays for plant-parasitic nematodes, focusing upon the direct application of these procedures for Meloidogyne detection, identification, and systematics.     

Intraspecific evolution of a gene family coding for urinary proteins

Summary The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1–4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5 to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.