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Biomedical subjects

R Kolodner

Publications and source records attributed to R Kolodner.

67 records · Page 4Linked to original sources
PubMed1978

Gene 4 protein of bacteriophage T7. Characterization of the product synthesized by the T7 DNA polymerase and gene 4 protein in the absence of ribonucleoside 5'-triphosphates.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze extensive DNA synthesis on duplex phage T7 or PM2 DNA templates containing single strand breaks. A variety of physicochemical techniques have been used to characterize the DNA product synthesized in this reaction in the absence of ribonucleoside 5'-triphosphates. Pyknographic and sedimentation analyses reveal that all of the newly synthesized DNA is covalently attached to the template DNA. Analysis by electron microscopy shows the major portion of the product molecules synthesized on duplex T7 DNA templates to consist of a double-stranded branch attached to an intact template molecule. Using PM2 DNA templates, the predominant product consists of a double-stranded branch attached to the circular PM2 DNA template. Analyses of these product molecules indicate that DNA synthesis by the gene 4 protein and T7 DNA polymerase is initiated at single strand breaks in the duplex DNA and that synthesis is accompanied by extensive displacement of one of the parental strands. At later times in the reaction, a portion of the 3'-hydroxyl terminus of the newly synthesized DNA is displaced from the template by branch migration and is used as a primer by the DNA polymerase to copy the displaced 5' single-stranded parental strand to form a duplex branch.

Coliphages↗
PubMed1977

Zea mays chloroplast ribosomal RNA genes are part of a 22,000 base pair inverted repeat.

Zea mays chloroplast rDNA exists in two identical units. Each unit contains one sequence for the 16, 23 and 5S rRNAs in the order given. The 16 and 23S sequences in each unit are separated by a 2100 base pair (bp) spacer. The DNA sequence for 5S RNA is closely linked to that for the 23S RNA. Within the above unit, the three RNAs are transcribed from a single DNA strand. The two rDNA units on the circular chloroplast DNA molecule are separated from each other by 18,500 bp in one direction and by 106,100 bp in the other direction. The two rDNA units have an inverted orientation with respect to each other. Each rDNA unit is part of a 22,000 bp sequence which is repeated with inverted orientation.

Base Sequence↗
PubMed1977

Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates.

Homogeneous preparations of phage T7 gene 4 protein catalyze the hydrolysis of dNTPs and rNTPs to NDPs and Pi in the presence of single-stranded DNA. Synthesis on single-stranded DNA by T7 DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) does not affect the hydrolysis of NTPs by the gene 4 protein. Gene 4 protein does not catalyze the hydrolysis of NTPs in the presence of duplex DNA, nor can T7 DNA polymerase use duplex DNA as a template. However, the two proteins together can replicate duplex DNA and, under these conditions, synthesis is accompanied by hydrolysis of NTPs. During synthesis on duplex templates in the presence of T7 DNA polymerase, gene 4 protein, dNTPs, and rNTPs, 4.2 NTPs are hydrolyzed for each dNMP polymerized. 2'3'-Dideoxy-TTP, an inhibitor of DNA synthesis, inhibits hydrolysis by the gene 4 protein during synthesis on duplex DNA, and beta, gamma-methylene-dTTP, an inhibitor of hydrolysis by the gene 4 protein, stops DNA synthesis on duplex DNA. The multiple activities of gene 4 protein are shown to reside in a single protein molecule.

Coliphages↗
PubMed1976

Physical studies on the size and structure of the covalently closed circular chloroplast DNA from higher plants.

The size and structure of the covalently closed circular chloroplast DNAs (ctDNA) from pea, lettuce, and spinach plants, have been studied by analytical ultracentrifugation. The values of so20,w,Na+ of the native and denatured forms of the open and closed circular DNAs from these plants have been determined. The absolute molecular weight of purified closed circular pea ctDNA monomers has been determined by buoyant equilibrium sedimentation to be 89.1 (S.D. +/- 0.7)-10(6). The value of the so20,w,Na+ of open circular pea ctDNA and its molecular weight, in conjunction with corresponding values for other sizes of circular DNA, has been used to derive an empirical relationship between so20,w,Na+ and molecular weight for open circular DNAs. Using this relationship, the molecular weights of lettuce and spinach ctDNAs have been determined to be 98.2 (S.D. +/- 1.5)-10(6) and 97.2 (S.D. +/- 1.5)-10(6), respectively. At pH values 12.7 and 13, closed circular lettuce and pea ctDNAs have been found to exist as mixtures of reversibly and irreversibly denatured closed circular DNAs.

Animals↗
PubMed1975

Presence of displacement loops in the covalently closed circular chloroplast deoxyribonucleic acid from higher plants.

Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.

Binding Sites↗
PubMed1975

The presence of covalently linked ribonucleotides in the closed circular deoxyribonucleic acid from higher plants.

Single-stranded scissions are induced in the covalently closed circular chloroplast (ct-) DNAs from peas, spinach, and lettuce plants by treatment with alkali or by incubation with a mixture of ribonucleases A and T1. These scissions are due to the presence of covalently linked ribonucleotides in these closed circular DNAs. By comparing the scission rates of these ctDNAs to the scission rate of RNA, it has been estimated that pea and spinach ctDNAs contain a maximum of 18 +/- 2 ribonucleotides/molecule, while lettuce ctDNA contains a maximum of 12 +/- 2 ribonucleotides/molecule. Further studies with pea ctDNA by electron microscopic methods have shown that pea ctDNA contains 19 alkali-labile sites at specific locations. A map of the relative positions of the alkali-labile sites has been constructed. These alkali-labile sites are presumably due to the insertion of individual ribonucleotides.

Alkaline Phosphatase↗
PubMed1975

The molecular size and conformation of the chloroplast DNA from higher plants.

Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...

Chloroplasts↗
PubMed1973

Plasmids of Shigella dysenteriae Y6R: a defective Col factor.

The six plasmids of Shigella dysenteriae Y6R were separated by sucrose gradients into five fractions containing deoxyribonucleic acid (DNA), having contour lengths (expressed in units equal to the fraction of the length of the replicative form of phiX174), respectively, of 0.29, 0.35, 0.74, 1.08, and a mixture of 5.7 and 7.2. DNA-DNA hybridization on nitrocellulose filters between each of the plasmids and between plasmid-free S. dysenteriae Y6R host DNA and plasmids was investigated. There was a high degree of homology between the 0.29- and 0.35-unit plasmids. No significant homology was found between any of the other pairs of plasmids. Homologous DNA to the extent of 2.4 copies of the 1.08-unit plasmid was found in the host genome. Homology between the other plasmids and the host genome is very slight, but appears to be significant. About 0.7 of the 1.08-unit plasmid is homologous to the ColE1 façtor of Escherichia coli JC411 (ColE1). This plasmid may be defective ColE1 factor with the immunity function intact, but with a defect in the gene leading to the production of active colicin. Electron microscope examination of heteroduplexes formed between the two smallest plasmids and between the 1.08-unit plasmid and the ColE1 factor yielded independent determinations of the extent of homology in agreement with the values determined by hybridization. In the latter case, two nonhomologous regions of substitution of DNA were detected.

Centrifugation, Density Gradient↗
PubMed1972

Physicochemical characterization of mitochondrial DNA from pea leaves.

The mitochondrial DNA from pea leaves exists in a circular conformation. 25% of the circular molecules exist as supercoils, and 10% of the molecules are dimers. The molecular weight of mitochondrial DNA is about 66 to 70 x 10(6) by electron microscopy, and 74 x 10(6) from its renaturation kinetics. No evidence for inter- and intramolecular heterogeneity is found.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Isolation of genetic elements that increase frequencies of plasmid recombinants.

Some plasmid DNAs, when maintained in wild-type Escherichia coli strains, form high levels of oligomeric species while others remain primarily monomers. One explanation of this observation is that the plasmids that do not form circular oligomers lack a DNA sequence necessary for the formation or maintenance of circular oligomeric species. Here we describe the isolation of segments of DNA from the E. coli genome and other sources that through a recA+ -dependent process: (1) stimulate the conversion of monomeric plasmids to different oligomeric forms, (2) stimulate the conversion of an oligomeric plasmid to a mixture of monomeric and different oligomeric forms, and (3) increase the frequency of recovery of figure-8 molecules. Both cis-acting and trans-acting elements were found. These elements seen to act by stimulating either the frequency of the recombination events that lead to the interconversion of different oligomeric plasmid DNA molecules or some process involved in the maintenance of newly-formed recombinant molecules.

DNA, Bacterial↗