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Biomedical subjects

R D Wells

Publications and source records attributed to R D Wells.

At least 109 records · Page 6Linked to original sources
PubMed1987

Left-handed DNA in vivo.

Left-handed DNA is shown to exist and elicit a biological response in Escherichia coli. A plasmid encoding the gene for a temperature-sensitive Eco RI methylase (MEco RI) was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target Eco RI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed (presumably Z) helices. In vitro methylation with the purified MEco RI agreed with the results in vivo. Supercoil-induced changes in the structure of the primary helix in vitro provided confirmation that left-handed helices were responsible for this behavior. The presence in vivo of left-handed inserts elicits specific deletions and plasmid incompatibilities in certain instances.

Cloning, Molecular↗
PubMed1987

Influence of negative supercoiling and of the proximity of left-handed Z-DNA on the Escherichia coli lactose repressor-operator interaction.

The influence of negative supercoiling and of flanking (dC-dG) tracts in either right-handed B- or left-handed Z-structures on the interaction of the Escherichia coli lac repressor was investigated. The operator was embodied within the lac control sequence, which was 95, 59, or 29 base pairs in length. Thus, the (dC-dG) regions (in either B- or Z-conformations) were at different distances from the repressor-binding site. Surprisingly, the presence of the promoter sequence (-59 to -20 relative to the +1 transcription start site) of the lac operator region increases the binding affinity of lactose repressor to the operator at high negative supercoil densities. This influence of the promoter region on the binding was abolished when the flanking (dC-dG) tracts were in the left-handed Z-DNA conformation. In contrast, minimal differences in the binding affinities were observed between plasmids containing shorter operator fragments (59 or 29 base pairs), whether the flanking (dC-dG) tracts were in right-handed B- or left-handed Z-forms. The promoter region may be directly involved in the repressor-operator complex in a previously unrecognized manner or may exert structural influence on the operator region. In general, increasing the amount of negative supercoiling increases the binding affinity and decreases the dissociation rate constants for the three operator-containing fragments, both with and without flanking Z-DNA tracts. Thus, the lac operator region possesses a previously unrecognized structural pliability, as influenced by negative supercoiling and neighboring sequences and/or conformations, which modulates its biological properties.

DNA, Superhelical↗
PubMed1987

Influence of sequence and distance between two operators on interaction with the lac repressor.

The influence of additional operator or pseudooperator sequences on the lactose repressor-operator interaction has been investigated. Results of kinetic and equilibrium binding measurements suggest an important in vivo role for the Z-gene pseudooperator in repressor-operator binding; the formation of a ternary, looped complex is indicated by the influence of secondary operator sites on binding parameters. Although the binding affinity of the Z-gene pseudooperator [Oz] is only approximately 1/30 that observed for the primary operator [O], the binding affinity to DNA containing both Oz and O is significantly higher than either sequence alone or the sum of the two. This synergistic effect is enhanced further by replacing the pseudooperator sequence [Oz] with the primary operator sequence and results in an even stronger ternary complex in plasmids with duplicate primary sites. The distance between the center of the two primary operators affects the formation of a ternary complex in the linear DNA molecules. Decreased dissociation rate constants were observed with spacing of operator-like sequences between 300 and 500 base pairs (bp). Minimal influence of the second operator on repressor binding is observed when the operators are separated by approximately 4000 or approximately 100 bp. The significant influence of distance on kinetic and equilibrium parameters was demonstrated by measurements on plasmid pRW1511 [Oi-O-PL-Oz] cleaved with restriction enzymes either in the polylinker region to place Oi-O and Oz on opposite ends of the linear plasmid or outside this region to maintain the sites within 500 bp. These results are consistent with the formation of operator-repressor-pseudooperator ternary complex to generate a looped DNA structure.

Base Sequence↗
PubMed1987

Influence of supercoiling and sequence context on operator DNA binding with lac repressor.

The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Krämer, H., Niemöller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.

Base Sequence↗
PubMed1987

Reaction conditions affect the specificity of bromoacetaldehyde as a probe for DNA cruciforms and B-Z junctions.

The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.

Acetaldehyde↗
PubMed1987

Specificity of monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice.

Antibodies reactive with left-handed Z-DNA arise spontaneously in the sera of patients with SLE and rheumatoid arthritis and in autoimmune MRL mice. However, the precise specificity of these autoantibodies has not been established. In this report, we have characterized four monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice that do not cross-react with B-DNA and can discriminate between different types of left-handed helices. Two of the monoclonal antibodies (Za and Zi) behaved similarly in that they bound to two forms of Z-DNA (Br-poly(dG-dC).poly(dG-dC) and AAF-poly(dG-dC).poly(dG-dC) but not to two other Z-form DNA (poly(dG-5BrdC).poly(dG-5BrdC) or poly(dG-5MedC).poly(dG-5MedC)). Neither antibody (Za or Zi) bound significantly to B-DNA or to denatured DNA. A third antibody (Ze) exhibited similar binding characteristics for the Z-DNA preparations, but also recognized denatured DNA. In contrast, a fourth antibody (3-7.3) bound preferentially to poly(dG-5BrC).poly(dG-5BrdC) in Z conformation. These results provide the first evidence for anti-Z-DNA autoantibodies in autoimmune mice that do not cross-react with native or denatured DNA and indicate that these antibodies exhibit considerable heterogeneity in their fine binding specificity.

Animals↗
PubMed1987

The segment inversion site of herpes simplex virus type 1 adopts a novel DNA structure.

The 12-base pair (bp) tandem direct repeat sequences (DR2) at the joint region (a sequence) of herpes simplex virus type 1 (strain F) adopt a new type of DNA conformation under the influence of negative supercoiling. The novel conformation is dependent on the number of the DR2 repeats; the 19 mer (228 bp total) and the 14 mer (168 bp) readily form the alternate structure whereas pentamer, trimer, and dimer repeats show somewhat different properties. S1 and P1 nuclease studies reveal that the new conformation has a major structural aberration at its center and conformational periodicities which are not identical on the complementary strands. Also, the effect of salt and pH, the location of reaction with bromo- and chloroacetaldehyde, the type of sequence (direct repeat) involved, and the nature and extent of supercoil-induced relaxations demonstrate that this structure differs from previously recognized conformations including left-handed Z helices, cruciforms, bent DNA, and slipped structures. We propose the existence of a novel conformation, anisomorphic DNA, with different structures on the complementary strands which elicit a structural aberration at the physical center of the tandem sequences. Since the oligopurine X oligopyrimidine sequence may be inherently inflexible, this supercoil-induced structural change and the physical stress on these inserts in recombinant plasmids tend to deform (crack) the DR2 sequences at their centers. Possible roles for anisomorphic DNA in the functions of this segment of intense biological activity are proposed.

Acetaldehyde↗
PubMed1987

Left-handed Z-DNA binding by the recA protein of Escherichia coli.

recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids. Two to 7-fold preferential binding of recA to Z-DNA polymers was observed. Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding. Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better. recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed. Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed. In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions. recA contains a left-handed Z-DNA-stimulated ATPase activity. This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH. The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA. Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination.

DNA↗
PubMed1987

Supercoiling facilitates lac operator-repressor-pseudooperator interactions.

The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density. However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities. Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined. These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction. The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA. Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h). These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.

DNA, Bacterial↗
PubMed1987

Cloning, sequencing, in vivo promoter mapping, and expression in Escherichia coli of the gene for the HhaI methyltransferase.

A 1476-base pair DNA fragment from Haemophilus haemolyticus containing the HhaI methyltransferase gene was isolated from a cell library and cloned into pBR322. The nucleotide sequence of this fragment was determined. The structural gene is 981 nucleotides in length coding for a protein of 327 amino acids (Mr 37,000). The translational start signal (ATG) is preceded by the putative ribosome-binding site (TAAG). Recombinant plasmids containing the 1476-basepair fragment are completely methylated when isolated from Escherichia coli, as judged by their insusceptibility to the HhaI restriction endonuclease. However, the presence of an active HhaI methylase gene in certain E. coli strains results in a very poor yield of transformants and/or in vivo-originated deletions due to the Rg1 functions of these hosts. The in vivo transcription initiation sites have been identified by S1 protection and primer-extension experiments using specific probes with total RNA prepared from E. coli cells (HB101 or RR1) which tolerate the expression of MHhaI.

Amino Acid Sequence↗
PubMed1987

Test of cure for gonorrhea in teenagers. Who complies and does continuity of care help?

This study evaluates factors influencing test of cure (TOC) compliance for gonorrhea in teenagers attending a public sexually transmitted disease (STD) clinic. The efficacy of providing continuity of care to improve the TOC rate is also evaluated. Overall, females complied better (54%) than males (33%), and younger males better than older males (greater than 16 years of age). Of patients with a prior history of gonorrhea, the TOC rates were significantly higher for those who had previously kept their TOC appointment. Continuity of care did not improve the TOC rate, but initial contact with a clinician appeared to influence the return rate of female adolescents. Male adolescents will apparently require a different intervention strategy to improve their appointment keeping.

Adolescent↗
PubMed1987

Enzymatic probes for left-handed Z-DNA.

In conclusion, one of the aspects of the DNA polymorphism observed is the formation of Z-DNA under a variety of conditions. Left-handed DNA stretches not only represent alternate structures, but also exert long-range effects due to their influence on superhelical properties on an entire supercoiled DNA as first shown six years ago [49, 50]. The examples given in this review emphasize the site-specificity of enzymes due to structural features rather than sequence itself. In this fashion, the reversible transition from B to Z DNA could modulate site-specific events on many levels of biological regulation. Considering all of the enzymes studied to date (S1, mung bean, BAL31, P1 nucleases, Hha I, BssH II, MHha I, BamH I, EcoR I, RNA polymerase, recl, recA, DNA glycosylase, O6-methylguanine-DNA methyltransferase), only the recl (and possibly the recA) protein seems to recognize and utilize left-handed DNA. A large number of questions related to the biology of Z-DNA are unanswered including: what is the DNA structure (B or Z or other) which is in physical contact with proteins; is Z-DNA recognized by proteins or are junctions the important features; do proteins revert the Z structure to B or to some other right-handed conformation; what other cofactors (perhaps chiral in nature) may be involved; what are the alternate forms of left-handed DNA; does left-handed DNA exist in vivo; what is the biological role(s) of left-handed DNA? The future of this field of investigation will be exciting indeed.

Computer Simulation↗
PubMed1987

Chronically ill children and their mothers: predictors of resilience and vulnerability to hospitalization and surgical stress.

Although the responses of normal children to hospitalization and surgery are well documented, an understanding of base rates of upset, the mediating factors involved, and the efficacy of interventions with chronically ill children has not yet been achieved. Using self-report and observational measures, we investigated the effects of hospitalization and surgery on 40 physically handicapped children and their mothers. The results suggest that, compared with normal children, chronically ill children as a group are not more likely to become disturbed. With regard to individuals, extremely high and low parenting stress, over-involved parent-child relationships, fewer previous surgeries, and younger age predicted greater disturbance. Mothers of developmentally delayed children reported feeling more anxious than mothers of children with normal mental ages, but no differences were found on child vulnerability.

Adolescent↗
PubMed1986

Thermodynamic parameters are sequence-dependent for the supercoil-induced B to Z transition in recombinant plasmids.

The entropy and enthalpy changes which contribute to the thermodynamics of the B to Z transition were determined for three recombinant plasmids containing a (dC-dG)16 tract and for a plasmid containing a pair of (dT-dG)20 regions. For each base pair which adopts a left-handed conformation in the plasmids with (dC-dG)16 sequences, the delta HBZ and delta SBZ are -2.1 kcal/mol bp and -8.8 cal/K-mol bp, respectively. In the plasmid containing the (dT-dG)20 tracts, however, the delta HBZ and delta SBZ values are 0.58 kcal/mol bp and -0.76 cal/K-mol bp, respectively. Also, these determinations show that for each B-Z junction that forms in the plasmids containing the (dC-dG), the enthalpy and entropy changes are 24 kcal/mol junction and 65 cal/K-mol junction, whereas for the (dT-dG) plasmid, the enthalpy and entropy changes are -1.8 kcal/mol junction and -22 cal/K-mol junction, respectively. Those values for the enthalpy and entropy changes for the formation of a BZ junction in (dC-dG) and (dT-dG) plasmids suggest that the properties and possibly the structures of the junctions are different. Calculations using the enthalpy and entropy changes determined in this study reveal that the B to Z transition in plasmids containing (dC-dG) blocks are more temperature-dependent than the transitions in plasmids with (dT-dG) blocks. Surprisingly, at temperatures above 60 degrees C, calculations indicate that the B to Z transitions in (dT-dG) plasmids should be energetically favored over that transition in (dC-dG) plasmids.

Base Sequence↗
PubMed1986

A highly bent fragment of Crithidia fasciculata kinetoplast DNA.

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.

Animals↗
PubMed1986

Unusual DNA structures in the adenovirus genome.

More than 80% (approximately 29 kilobase pairs) of the adenovirus serotype 2 genome was surveyed for the presence of unusual DNA conformations. Seven recombinant DNAs containing the largest HindIII fragments of AD2 DNA were analyzed for the presence of negative supercoil-dependent S1 nuclease-sensitive sites. Four plasmids each contained a specific site of S1 nuclease sensitivity whereas the other three showed no reaction. Further investigation was focused on a plasmid containing one of the positively reacting fragments (fragment C) which contained the major late promoter at coordinate 16.4 on the genome; three serotypes (Ad2, Ad7, Ad12) were studied. Fine mapping studies revealed the S1-sensitive sites to be a small region (approximately 6 base pairs) located at the TATA box of the major late promoter in all three cases. Other determinations (supercoil relaxation, T7 gene 3 product sensitivity, bromoacetaldehyde reactivity, anomalous gel mobility, the influence of negative superhelical density on nuclease sensitivity) led to the conclusion that the B-helix deformation was not due to a previously recognized DNA conformation (left-handed Z-DNA, cruciform, bent DNA), but may be accounted for by the homopurine X homopyrimidine nature of this region.

Acetaldehyde↗
PubMed1986

Site-specific OsO4 modification of the B-Z junctions formed at the (dA-dC)32 region in supercoiled DNA.

OsO4 in the presence of pyridine specifically modifies the structural distortions of the primary helix of supercoiled pRW777 near the (dA-dC)32 sequence. Modification occurs at the same negative superhelix density value as required for formation of the Z-helix within the polymer block. Fine mapping of the distorted regions, which are probably the B-Z junctions, is presented. OsO4 reactions provide a powerful and sensitive chemical approach to study DNA polymorphism in solution.

Base Sequence↗