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Biomedical subjects

D L Robberson

Publications and source records attributed to D L Robberson.

At least 37 records · Page 2Linked to original sources
PubMed1980

Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures.

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.

Base Sequence↗
PubMed1980

A characterization of mitochondrial and mycoplasmal DNAs associated with cloned HeLa thymidine kinase minus cells.

HeLa S3 cells were cloned, recloned, and then selected for growth in the presence of increasing concentrations of bromodeoxyuridine. Cultures of these cloned thymidine kinase minus (TK-) cells were found to harbor mycoplasma which sedimented with mitochondria in sucrose density step gradients. Examination of mitochondrial DNA (mitDNA) components by restriction enzyme analysis and electron microscopy revealed no gross alterations in size, sequence arrangements, or replicative forms compared with mitDNA of HeLa S3 cells. Restriction enzyme cleavage sites for BamHI (one site), PstI (two sites) and HpaI (three sites) were mapped on this genome relative to the three cleavage sites for each of EcoRI and HindIII, respectively. Analysis of topological complexity revealed similar frequencies of catenated mitDNA molecules in both cloned TK- (22.5 +/- 1.5% of mass) and HeLa S3 cells )25.6 +/- 1.5% of mass). Unicircular dimers comprise 6.7 +/- 0.9% of the mitDNA mass in cloned TK- cells but were not detected in HeLa S3 mitDNA. Examination of the mycoplasmal contaminant of mitochondrial DNA after digestion with various restriction enzymes and agarose gel electrophoresis revealed that most of the DNA was distributed in discretely sized fragments in patterns that can probably be used to unambiguously identify and classify the organism.

Bromodeoxyuridine↗
PubMed1979

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Animals↗
PubMed1979

Site specificity of bleomycin-mediated single-strand scissions and alkali-labile damage in duplex DNA.

Form II PM2 DNA, which contained bleomycin-mediated single-strand breaks, was purified and treated with the extracellular endonuclease from Alteromonas BAL 31. This enzyme cleaves the phosphodiester backbone opposite a single-strand break to yield a double-strand break. The locations of these double-strand breaks were determined relative to the cleavage sites produced by the restriction enzyme HindIII. The experimental procedure was as follows. Form I PM2 DNA was treated with bleomycin to produce alkali-labile bonds. These were hydrolyzed by alkali treatment and the DNA, now containing single-strand breaks, was purified and treated with the BAL 31 enzyme and the HindIII enzyme to determine the positions of the original alkali-labile bonds. It was found that the single-strand breaks and alkali-labile bonds were introduced at preferred sites on the PM2 genome, since electrophoretic analyses of the DNA after the HindIII digestion revealed DNA bands of discrete sizes. The molecular weights of the DNA fragments produced by these treatments indicate that single-strand breaks and alkali-labile bonds occur at the same sites as those previously determined for direct double-strand scissions introduced by bleomycin at neutral pH. Some of the specific sites of double-strand scissions mediated by bleomycin at neutral pH (Lloyd et al., 1978b) are also shown here to be relatively more reactive than other sites when the DNA contains superhelical turns.

Bacteriophages↗
PubMed1979

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.

Bacteriophages↗
PubMed1979

Noncovalent intermolecular crosslinks are produced by bleomycin reaction with duplex DNA.

Reaction of covalently closed circular PM2 bacteriophage DNA with the anticancer drug bleomycin produces nicked circular (form II) and linear duplex (form III) DNA [Lloyd, R.S., Haidle, C.W. & Robberson, D.L. (1978) Biochemistry 17, 1890-1896]. As the reaction proceeds, the frequencies of both form II and form III DNA increase and, concomitantly, an increasing fraction of the DNA mass is found to be in crosslinked structures. Approximately 16% of the PM2 DNA mass is found to be crosslinked after 30 min of reaction with bleomycin at 0.5 microgram/ml. The proportion of each form found in any given crosslinked structure is directly related to the concentration of uncrosslinked (monomeric) forms. Multiple sites of crosslinking occur, and these frequently extend over a region of approximately 500 nucleotide pairs. The intermolecular crosslinked bonds are dissociated by extensive dialysis or by the addition of salt at high concentration (0.8 M NaCl), as would be expected if the bonds were noncovalent. Because intramolecular covalent crosslinks between complementary strands are not detected, it is suggested that intermolecular crosslinks are formed by noncovalent association of bleomycin molecules bound to each of the forms of DNA.

Bacteriophages↗
PubMed1978

Bleomycin-specific fragmentation of double-stranded DNA.

Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.

Bacteriophages↗
PubMed1977

A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic or mutagenic agents.

The highly single strand-specific extracellular nuclease of Pseudomonas BAL 31 is shown to cleave non-supercoiled closed circular duplex PM2 bacteriophage DNA containing regions of altered helix structure produced in vitro by irradiation with ultraviolet light or by treatment with the carcinogen, N-acetoxy-N-2-acetylaminofluorene. Untreated samples of this DNA are affected very little by the nuclease. The unwinding of the DNA helix associated with the above treatment renders the closed circular DNA positively supercoiled compared to untreated samples. The extent of unwinding can be accurately measured and correlated with the average number of lesions per molecule of DNA by monitoring the alterations of the electrophoretic patterns, relative to those observed for untreated DNA, of such DNA in agarose gels. Interstrand cross-links and mismatched base pairs produced by treatment of non-supercoilded circular duplex DNA with the mutagen, nitrous acid, do not detectably unwind the DNA helix. The nitrous acid-treated DNA provides substrates for cleavage by the Pseudomonas nuclease which are likely to be the interstrand cross-links rather than the mismatched base pairs. Use of the Pseudomonas nuclease in conjunction with agarose gel electrophoresis can provide a powerful method for the detection of damage in duplex DNA such as that introduced by carcinogenic and mutagenic agents.

Acetoxyacetylaminofluorene↗
PubMed1977

Microheterogeneity detected in circular dimer mitochondrial DNA.

Exhaustive EcoRI digests of circular dimer mitochondrial DNA (mtDNA) from mouse cell lines LD and LDTK- yield two major fragments whose average lengths are slightly smaller than the corresponding fragments of circular monomer mtDNA from mouse LA9 and LMTK- cells. A third fragment approximately 400 nucleotide pairs in length is frequently produced in less than molar yield. Exhaustive EcoRI digests of circular dimer mtDNA from human acute myelogenous leukemic leucocytes yield three major fragments. The presence of mtDNA resistant to cleavage as well as fragments of intermediate sizes indicatesmicroheterogeneity in the genomic positions of EcoRI recognition sequences in both mouse and human circular dimer mtDNA. Analysis of the distribution averages of circular contour lengths indicates microheterogeneity in the sizes of mouse LD and human mtDNAs. The denatured-renatured EcoRI fragments frequently contain a small loop(s) of single-strand DNA as would occur for deletion(s) or addition(s) of single-strand DNA as would occur for deletion(s) or addition(s) of nucleotide sequences in some of the circular dimer molecules.

Cell Line↗
PubMed1976

The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.

Animals↗
PubMed1975

Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities.

The culture medium of Pseudomonas BAL 31 contains endonuclease activities which are highly specific for single-stranged DNA and for the single-stranded or weakly hydrogen-bonded regions in supercoiled closed circular DNA. Exposure of nicked DNA to the culture medium results in cleavage of the strang opposite the sites of preexisting single-strand scissions. At least some of the linear duplex molecules derived by cleavage of supercoiled closed circular molecules contain short single-stranded ends. Single-strand scissions are not introduced into intact, linear duplex DNA or unsupercoiled covalently closed circular DNA. Under these same reaction conditions, 0X174 phage DNA is extensively degraded and PM2 form I DNA is quantitatively converted to PM2 form III linear duplexes. Prolonged exposure of this linear duplex DNA to the concentrated culture medium reveals the presence of a double-strand exonuclease activity that progressively reduces the average length of the linear duplex. These nuclease activities persist at ionic strengths up to 4 M and are not eliminated in the presence of 5% sodium dodecyl sulfate. Calcium and magnesium ion are both required for optimal activity. Although the absence of magnesium ion reduces the activities, the absence of calcium ion irreversibly eliminates all the activities.

Calcium↗
PubMed1975

Unidirectional replication of a minority of polyoma virus and SV40 DNAs.

Poyoma DNA replication is initiated predominatly at a site which is 29% from the EcoRI cleavage site. Molecules replicating from this site, after digestion with EcoRI appear as linear structures with a double stranded loop centered at the origin of replication. These forms constitute 90% of all replicating intermediates. Approx. 10% of the replicating intermediates of polyoma and SVP40 DNAs occur as Y-forms after treatment with EcoRI. These structures have probably resulted from unidirectional replication initiated at an additional origin of DNA replication which is located near the EcoRI cleavage site on the genomes of these viruses.

Animals↗
PubMed1974

Cleavage of replicating forms of mitochondrial DNA by EcoRI endonuclease.

Digestion of mouse L cell mitochondrial DNA with EcoRI restriction endonuclease produces two linear duplex fragments comprising 86.3 +/- 2.0% and 14.2 +/- 1.0% of the circular genome length (16,000 +/- 470 nucleotide pairs). Digestion of human HeLa cell mitochondrial DNA with EcoRI produces three linear duplex fragments comprising 49.2 +/- 1.0%, 44.4 +/- 0.9%, and 6.4 +/- 0.4% of the circular genome length (16,590 +/- 710 nucleotide pairs). These fragments are shown to be generated by cleavage in unique regions of the mouse and human mitochondrial DNAs. An electron microscopic analysis of partially replicated molecules cleaved by EcoRI establishes a unidirectional mode of DNA replication for L cell mitochondrial DNA. The origin for DNA replication is located on the larger EcoRI fragment at a position that is 1,890 +/- 250 nucleotide pairs (11.8 +/- 1.2% of the circular genome length) from the proximal restriction site. Replication proceeds unidirectionally away from this restriction site throughout the length of the larger EcoRI fragment. Analysis of L cell, D-loop mitochondrial DNA cleaved by EcoRI indicates that a unique sequence is synthesized in formation of the D-loop in these nonreplicating molecules. The origin of D-loop synthesis is located on the larger EcoRI fragment at a position 1,760 +/- 180 nucleotide pairs (11.0 +/- 1.1% of the circular genome length) from the proximal restriction site and is, therefore, the origin for unidirectional displacement replication.

Animals↗
PubMed1974

Sequence arrangements in clonal isolates of polyoma defective DNA.

Five clonal isolates of purified polyoma defective DNA [Fried, M. (1974) J. Virol. 13, 939-946] have been examined by electron microscopy. Four isolates (D-92, D-74, D-50, and D-47) are largely homogeneous in sequence, whereas, the fifth isolate (D-80) is somewhat heterogeneous.Polyoma nondefective DNA is cleaved in a unique region of the genome by the EcoR(I) endonuclease. Hybridization of the resulting linear molecules with randomly nicked defective DNA reveals distinguishable types of heteroduplex structures for each of the different defective DNAs. Although the defective DNAs are shorter than polyoma nondefective DNA, the heteroduplex experiments demonstrate that they are not simply deletion mutants containing only a portion of the viral genome. Three isolates (D-92, D-50, and D-47) contain regions of homology to polyoma DNA covalently linked to non-homologous regions. One isolate (D-74) contained no regions of detectable homology to polyoma DNA. Another isolate (D-80) contained a large proportion of molecules with duplicated-inverted regions. Some of these isolates of defective DNA may contain specific host sequences at the site(s) of integration of the polyoma genome during the lytic cycle in mouse cells. A process we term "abortive replication" may explain the formation of different types of defective DNAs.

Base Sequence↗