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Biomedical subjects

R D Wells

Publications and source records attributed to R D Wells.

At least 199 records · Page 11Linked to original sources
PubMed1977

Action of single-strand specific nucleases on model DNA heteroduplexes of defined size and sequence.

The sensitivity of the model DNAs containing dA-dG and dtg-dG heteroduplex regions of defined length to S1 and mung bean single-strand specific nucleases was tested by polyacrylamide gel electrophoretic analysis of the distribution of product oligonucleotides. Single-base mismatch heteroduplexes were extremely resistant to these nucleases, although low levels of cleavage at the heteroduplex nucleotide were observed at high nuclease concentrations. The nuclease sensitivity of dA-dtg heteroduplex regions increased gradually as the length of the heteroduplex region increased frome one to six nucleotides. The sensitivity of dG-dG heteroduplexes three to five nucleotides long was considerably greater than that of the single dtg-dG mismatch.

DNA, Single-Stranded↗
PubMed1977

High resolution proton nuclear magnetic resonance investigation of the structural and dynamic properties of d(C15A15)-d(T15G15).

The high resolution proton nuclear magnetic resonance (NMR) spectra of the synthetic DNA block polymer d(C15A15)-d(T15G15) were studied in order to more completely understand telestability in DNA, and to provide fundamental NMR data on DNA helices and random coils. Spectra were measured in the spectral region from 0 to 15 ppm downfield from the usual standard, sodium 4,4-dimethyl-4-silapentane-1-sulfonate(DSS), at various temperatures (24-98 degrees C) in solution containing either moderate or high ionic strength. The effect of actinomycin binding to the block polymer also was studied. The major conclusions derived from this study are as follows: (1) The majority of base pairs in the AT helix of the block polymer have the same conformation as in d(A)n-d(T)25 and d(A)21-d(T)21. (2) The conformation of the GC helix in the block polymer is different from the AT helix and this perturbs the conformation of three or four A-T base pairs at the junction of the AT-GC helix. (3) The conformation of the AT helix is unaffected by salt over the range examined (approximately 0.04 - approximately 2 M), but the conformation of the GC helix changes. (4) There are subtle changes in the conformation of the AT helix as the temperature is increased and resonances characteristic of the random coil and the double-helical state can be simultaneously observed. (5) Binding of actinomycin, which is specific for the GC helix, induces quite large (over 1 ppm) upfield shifts of the resonances from the GC base pairs. This is consistent with an intercalation model in which actinomycin D (Am) is assymetrically sandwiched between two GC base pairs in such a manner that overlap with the guanosine residues is greater than with the neighboring cytidines. (6) The presence of the drug may also perturb A-T base pairs located near the AT-GC junction, but it has no effect on the majority of the AT pairs. However, as expected, Am elevated the Tm of the AT helix, even though it binds to the other end of the DNA.

DNA↗
PubMed1977

RNA polymerase binding sites in lambdaplac5 DNA.

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of lambdaplac5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p(R)', the INT promoter (p(I)), several sites in the b2 region, the mis promoter, the oop promoter (or p(O)), and p(rm). Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the cIII-t(L) region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at p(L), at p(R), at a site between R and cos, and at a site at the junction of the gamma and cIII-t(L) fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the lambda genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site).

Binding Sites↗
PubMed1976

Coumerimycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. II. In vivo characterization.

In vitro inhibitions by coumermycin A1 of DNA and RNA synthesis in toluenized cells were studied. In a sensitive strain, 50% inhibitions of replication and transcription were observed at 0.035 and 0.600 mug/ml, respectively. DNA synthesis in a toluenized-resistant mutant was 50% inhibited at 0.140 mug/ml of coumermycin A1, whereas RNA synthesis was unaffected at all concentrations tested. Studies with a mixture of toluenized-sensitive and -resistant bacteria ruled out the presence of a diffusable activator or inhibitor of coumermycin A1 action. Density label studies with toluenized pol A+ and pol A- strains indicated that replicative DNA synthesis was specifically inhibited, in agreement with the in vivo studies in the preceding paper of this issue (Ryan, M. J. (1976), Biochemistry 15). Highly purified Escherichia coli DNA polymerase III and RNA polymerase both were inhibited by this antibiotic. However, the high concentrations necessary for these inhibitions suggest that they are not biologically relevant. No interaction between DNA and coumermycin A1 was observed with the following analytical procedures: ultraviolet difference spectra, DNA absorbance-temperature transitions, equilibrium buoyant density centrifugation, and DNA cross-linking determinations.

Anti-Bacterial Agents↗
PubMed1976

All intact subunit RNAs from Rous sarcoma virus contain poly (A).

The capacity of RNA subunits of Schmidt-Ruppin Rous sarcoma virus to bind (dT)n was studied by cesium sulfate density gradient centrifugation, sucrose gradient centrifugation, or polyacrylamide gel electrophoresis. Two classes of subunits were observed. In agreement with previous reports, 60 to 70% of the purified subunits were capable of binding (dT)n. The remaining, nonbinding RNAs, although indistinguishable in size from whole subunits, were shown to be fragments that had lost the (rA)n terminus by random nucleolytic cleavage. This was deduced from the molecular weight distribution of the nonbinding RNAs from high molecular weight RNA. In addition, virtually all subunit RNA from early harvest virus, which contains a greater proportion of intact subunit, binds (dT)n.

Avian Sarcoma Viruses↗
PubMed1976

Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNAs.

Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100-200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced by Hind II + III cleavage of phi80 plac DNA and phi80 plac DNA containing the Ll deletion (73 nucleotides in length). This heteroduplex incorporated twelve times as much iodine as the parent homoduplex fragments. Hence the technique could reveal the presence of a few (two or more) nonpaired cytosines, if they existed within an otherwise helical DNA fragment 789 base pairs long. Iodination studies were performed on superhelical SV40 DNA and on linear lambdaplac DNA. Analysis of the relative amount of iodine in restriction endonuclease fragments of these DNA's revealed the absence of localized single-stranded regions.

Coliphages↗
PubMed1975

Further studies on telestability in DNA. The synthesis and characterization of the duplex block polymers d(C20A10) - d(T10G20) and d(C20A15) - d(T15G20).

The synthesis and characterization of the duplex block polymers d(C20A10) - d(T10G20) and d(C20A15) - d(T15G20) are described. Thermal denaturation studies on these DNAs in the absence and presence of actinomycin, which binds only to the GC portions of these molecules, have confirmed and extended our previous observation that the properties of one region of a DNA can be influenced (telestabilized) by a remote region. In addition, the large scale synthesis of d(C15A15) - d(T15G15) is described.

Animals↗
PubMed1975

Transmission of stability (telestability) in deoxyribonucleic acid. Physical and enzymatic studies on the duplex block polymer d(C15A15) - d(T15G15).

The properties of the duplex block polymer d(C15A15) - d(T15G15) were examined by thermal denaturation and nuclease susceptibility studies in the absence and presence of drugs (actinomycin and netropsin) which bind specifically to only one end of the block polymer. The nucleotide composition of one region of this synthetic double-helical DNA affected the properties of a contiguous but remote region. Furthermore, the binding of actinomycin influenced the properties of both the binding and nonbinding regions. These findings suggest a mechanism for gene regulation at a distance.

Adenosine Monophosphate↗
PubMed1975

The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid.

The simultaneous binding of netropsin and actinomycin to four natural DNAs was studied to determine the influence of one ligand on the binding of the other. Actinomycin binds specifically to GC sites, whereas netropsin binds specifically to AT sites. Spectral titrations, thermal denaturation, and analytical buoyant density centrifugation were employed to measure the binding interference of these drugs. The binding of actinomycin to DNA was decreased by the presence of netropsin. Increasing the GC content of the DNA resulted in a decreased effect of netropsin on actinomycin binding. Quantitative analysis of the binding parameters indicated that netropsin and actinomycin can bind in close proximity along the DNA chain. Supercoiled DNA gave the same result as linear DNA. These results imply that DNA can absorb alterations in conformation within a short distance.

Anti-Bacterial Agents↗
PubMed1975

Comparative ability of RNA and DNA to prime DNA synthesis in vitro: role of sequence, sugar, and structure of template-primer.

The priming efficiency of oligo(RNA) vs. oligo(DNA) in a homopolymer template-homooligomer primer system was compared using four DNA polymerases. The templates included (dT)n, (dA)n, (dC)n, and (dI)n. Primers were the oligomers of the complementary DNA OR RNA with chain lengths of 6 to 23. The DNA polymerases used were from Micrococcus luteus, avian myeloblastosis virus (AMV), and Escherichia coli (polymerase I and polymerase III). The polymerases demonstrated a preference for the DNA primers with (dC)n, (dA)n, and (dI)n templates. However, when (dT)n was the template, all but the AMV polymerase preferred (rA)11 more than 200-fold better than (dA)12. This preference was due to the physical structure of the initiation complex. The structures of the oligo-polymer complexes were characterized by mixing curves, melting curves, and analytical bouyant density analyses. (rA)11 + (dT)n formed predominatly a duplex structure, whereas (dA)12 + (dT)n formed the three- stranded structure, (dA12-2(dT)n. The Km of the duplex with E. coli Pol III was 2.9 mugM (rA)11. The Ki of the triplex was 2.2 mugM (dA)12, indicating that Pol III could bind to the triplex but would not elongate the (dA)12 primer. The influence of structure on priming also was demonstrated with longer oligomers, (dA)23 and (rA)23, where the (dA)23 formed more duplex-like structures and primed more than the (dA)12.(dT)10 + (dA)n complexes also were shown to form triplex structures that inhibited priming. These results show that template-primer structure has more influence on priming than the sugar moiety or the sequence of the nucleic acid.

Avian Myeloblastosis Virus↗
PubMed1974

Mechanistic independence of avian myeloblastosis virus DNA polymerase and ribonuclease H.

Differential inhibition conditions were established for the DNA polymerase and RNase H activities of avian myeloblastosis virus (AMV) with ether-disrupted AMV and a purified enzyme preparation. The RNase H activity of ether-disrupted AMV with (rA)(n).(dT)(n) and (rA)(n).(dT)(11) as substrates was inhibited 80 to 100% by preincubation with NaF at a final reaction concentration of 27 to 30 mM. Under these conditions, the DNA polymerase activity was inhibited only 0 to 20%. Similar inhibitions were found with exogenous Rous sarcoma virus 35S and 70S RNA.DNA hybrid and phiX174 DNA.RNA hybrid as substrates. Studies were also performed with a purified enzyme preparation, in which the two activities essentially co-purified. The RNase H activity was inhibited >80% by 150 mM KCl with three different hybrid substrates, whereas the DNA polymerase activity was uninhibited. The DNA polymerase was completely inactivated by heat denaturation at 41 C or by omission of the deoxytriphosphates from the reaction mixture; the RNase H remained active. These differential inhibition conditions were used to compare the size of the DNA product synthesized with and without simultaneous RNase H action and to examine the effect of inhibition of the DNA polymerase on the size of the RNase H products. The size of the products of one activity was not affected by inhibition of the other activity. These results suggest that the AMV DNA polymerase and RNase H are not coupled mechanistically.

Avian Leukosis Virus↗