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R D Wells

Publications and source records attributed to R D Wells.

At least 55 records · Page 3Linked to original sources
PubMed1996

CTG triplet repeats from human hereditary diseases are dominant genetic expansion products in Escherichia coli.

The relative ability of the 10 triplet repeat sequences to be expanded in Escherichia coli was determined. Surprisingly, CTG tracts are expanded at least 8 times more frequently than any of the other nine triplets. Low levels of expansion were found also for CGG, GTG, and GTC. Thus, the structure of the CTG repeats and/or their utilization by the DNA synthetic systems in vivo must be quite different from the other triplets. These data further validate this genetically defined system for elucidating molecular mechanisms of expansion and may explain why most triplet repeat hereditary neuromuscular and neurodegenerative disease genes contain CTG repeats.

Base Sequence↗
PubMed1995

Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.

Adenosine Triphosphatases↗
PubMed1995

Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes.

Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degrees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausing increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.

Base Sequence↗
PubMed1995

An intramolecular triplex in the human gamma-globin 5'-flanking region is altered by point mutations associated with hereditary persistence of fetal hemoglobin.

The properties of an intramolecular triplex formed in vitro at the 5'-flanking region of the human gamma-globin genes were studied by chemical and physical probes. Chemical modifications performed with osmium tetroxide, chloroacetaldehyde, and diethyl pyrocarbonate revealed the presence of non-paired nucleotides on the "coding strand" at positions -209 through -217. These reactivities were induced by negative supercoiling, low pH, and magnesium ions. Downstream point mutations associated with hereditary persistence of fetal hemoglobin (HPFH) altered the extent of the modifications and some of the patterns. Specifically, C-202-->G and C-202-->T significantly decreased the reactivities, whereas the patterns were increased and altered in the T-198-->C. C-196-->T and C-195-->G caused local decreases in reactivity. Modifications at the upstream flanking duplex were modulated by the composition of the vector sequence. In summary, our data indicates the formation of an intramolecular triplex between nucleotides -209 to -217 of the "non-coding strand" and the downstream sequence containing the HPFH mutations. All of the HPFH point mutations altered the structure. More than one sequence alignment is possible for each of the triplexes. In addition, a consequence of some of the point mutations may be to facilitate slippage of the third strand relative to the Watson-Crick duplex.

Acetaldehyde↗
PubMed1995

Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

Animals↗
PubMed1995

Emotional, behavioral, and physical symptoms reported by parents of sexually abused, nonabused, and allegedly abused prepubescent females.

There have been few studies that have examined emotional, behavioral, and physical symptoms that discriminate between well-defined groups of sexually abused and nonabused children. This study examined the results of a structured parent interview (SASA) completed on three matched samples of prepubescent females: 68 who were selected for nonabuse (NA Group), 68 from a sexual abuse clinic in which a perpetrator confessed (SA Group) and 68 seen at the same clinic who did not have a perpetrator confession (AA Group). Parents of girls in both the SA and AA groups reported increased sleep problems, fearfulness, emotional and behavioral changes, concentration problems, and sexual curiosity and knowledge. When contrasting the known (SA) with the allegedly abused sample (AA), self-consciousness, nightmares, and fearfulness of being left alone emerged significantly more frequently in the SA sample.

Affective Symptoms↗
PubMed1995

Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli.

Several human hereditary neurological and neurodegenerative disease genes are associated with the expansion of CTG repeats. Here we show that the frequency of genetic expansions or deletions in Escherichia coli depends on the direction of replication. Large expansions occur predominantly when the CTGs are in the leading strand template rather than the lagging strand. However, deletions are more prominent when the CTGs are in the opposite orientation. Most deletions generated products of defined size classes. Strand slippage coupled with non-classical DNA structures may account for these observations and relate to expansion-deletion mechanisms in eukaryotic chromosomes for disease genes.

Base Sequence↗
PubMed1994

Left-handed Z-DNA and in vivo supercoil density in the Escherichia coli chromosome.

A system for studying Z-DNA formation in the Escherichia coli chromosome was developed. Prior investigations in recombinant plasmids showed that alternating (Pur-Pyr) sequences can adopt a left-handed Z-DNA conformation both in vitro and in vivo. We constructed mobile, transposon-based cassettes carrying cloned (Pur-Pyr) sequences containing an EcoRI site in the center. These cassettes were subsequently inserted into different locations in the E. coli chromosome in a random fashion. A number of stable insertions were characterized by Southern analysis and pulsed-field gel electrophoresis mapping. A cloned temperature-sensitive MEcoRI methylase was expressed in trans as the probe to study Z-DNA formation in vivo. In this system, the control EcoRI sites were quickly methylated when cells were placed at the permissive temperature. Strong inhibition of the methylation was observed, however, only for the EcoRI sites embedded in a 56-bp run of (C-G). In contrast, the shorter sequence of 32 bp did not show this behavior. Prior in vitro determinations revealed that the longer tract required less energy to stabilize the Z-helix than the shorter block. We conclude that the observed inhibition of methylation is due to Z-DNA formation in the E. coli chromosome. In vitro, these sequences undergo the B- to Z-DNA transition at a supercoil density of -0.026 for the 56-bp insert and -0.032 for the 32-bp block. Since only the longer (C-G) tract but not the shorter run adopted the left-handed conformation in the chromosome, we propose that these densities establish the boundaries in the different chromosomal loci investigated; these boundaries are in good agreement with the extremes found in plasmids.

Base Sequence↗
PubMed1994

Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene.

The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

DNA↗
PubMed1994

Zinc destabilizes DNA Watson-Crick pairs at AGCT.

Zinc, a component of a large number of enzymes and an abundant and essential trace metal, alters the structure of AGCT sites in negatively supercoiled DNA. Related sequences show little or no effect. Cobalt and cadmium show reactivities similar to zinc. The conformational change in DNA was fine mapped with haloacetaldehydes using chemical cleavage or primer extension methods. Since a many fold excess of zinc to AGCT sites is required to saturate the reaction, we propose that the zinc-AGT complex is preferentially accessible to the haloacetaldehyde probes due to a conformational deformation. Hence, zinc is the smallest ligand (by approximately 500-fold) that causes a perturbation of a specific DNA sequence.

Base Composition↗
PubMed1992

Central non-Pur.Pyr sequences in oligo(dG.dC) tracts and metal ions influence the formation of intramolecular DNA triplex isomers.

The effect of the central non-Pur.Pyr sequences in oligo(dG.dC) inserts on determining the type of intramolecular DNA triplex isomers formed in negatively supercoiled plasmids was investigated. Different triplex types (H-r3, H-r5, and H-y3), revealed by a combination of chemical probing and Maxam-Gilbert sequencing reactions, were adopted by the oligo(dG.dC) tracts depending on the length and composition of the central non-Pur.Pyr sequences (0, 3, or 5 base pairs) and the kind of metal ions. The H-r3 triplex conformer, one isomer of a Pur.Pur.Pyr structure, was formed in the (C)20 and (C)10GCG(C)10 inserts in plasmids in the presence of certain metal ions. Interestingly, H-r5, the other isomer of the Pur.Pur-Pyr triplex which had not been detected previously, was formed in a (C)9GAATT(C)9 insert in the presence of either Mg2+ or Ca2+. Alternatively, H-y3, one isomer of a Pyr.Pur.Pyr triplex, was formed in the (C)9GAATT(C)9 insert in the absence of metal ions. Thus, central non-Pur.Pyr sequences and metal ions play a role as determinants of the types of intramolecular triplexes formed; they also reduce the requirement of longer Pur.Pyr repeat sequences to form intramolecular triplexes. Furthermore, the effects of MgCl2 concentration and pH on the formation of triplex isomers were examined. The Pur.Pur.Pyr conformations (H-r3 and H-r5) may be the favored conformations in the cellular milieu, since they are stable at physiological pH and metal ion concentration.

Acetaldehyde↗
PubMed1992

GC-rich flanking tracts decrease the kinetics of intramolecular DNA triplex formation.

The effect of the base composition of flanking sequences on DNA intramolecular triplex formation was investigated in negatively supercoiled plasmids. The rates of triplex formation at two oligopurine.oligopyrimidine inserts with interrupting sequences in plasmids containing AT- or GC-rich flanking sequences were compared as a function of temperature, pH, and negative superhelical density. The kinetics of the transition of linear B-DNA to triplex (also called H-DNA) were influenced by all of these factors; triplexes were formed slower in a GC-rich background than in an AT-rich background. However, at equilibrium, the same amounts of the triplexes in AT- or GC-rich contexts were formed, and the conformations adopted by (GAA)4TTCGC(GAA)4 showed the canonical intramolecular triplex as mapped with chemical probes. We propose that the GC-rich segments caused this effect by thermodynamically clamping the DNA inserts, since the dependence of kinetics on base composition disappeared in tetraalkylammonium ions which eliminate the dependence of helix-coil transitions on base composition. The dependence of the kinetics of intramolecular triplex formation on flanking sequences further strengthens the concept of the role of DNA as a dynamic participant in cellular events.

Base Sequence↗
PubMed1992

Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11.

A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.

Base Sequence↗
PubMed1992

Nodule DNA in the (GA)37.(CT)37 insert in superhelical plasmids.

Di- or trivalent metal ions stabilize a supercoil-dependent transition in pGA37, which contains the (GA)37.(CT)37 insert, at neutral and basic pH. The structure formed is different from the well known protonated triplexes (H-DNA) adopted at low pH by polypurine.polypyrimidine (Pur.Pyr) inserts in plasmids. DNA samples must be preincubated in the presence of multivalent ions at 50 degrees C for the new transition to occur. At neutral pH in the presence of Co hexamine, both strands of the insert have modification maxima situated at one-third of the distance from both ends. We propose the formation of a new structure called nodule DNA which consists of both Pyr.Pur.Pyr and Pur.Pur.Pyr triplexes and does not contain continuous single-stranded regions. At basic pH (greater than 8.5) in the presence of magnesium ions, the modification pattern corresponds to Pur.Pur.Pyr triplex formation in the whole insert. At neutral pH in the presence of magnesium, both nodule DNA and the Pur.Pur.Pyr triplex can be formed in the insert. We also observed a magnesium-dependent transition at neutral pH in the other Pur.Pyr insert containing plasmids. These data demonstrate that Pur.Pyr sequences can adopt several non-B conformations at close to in vivo conditions.

Base Sequence↗
PubMed1992

Metal ions cause the isomerization of certain intramolecular triplexes.

The influence of cations on the capacity of five oligopurine.oligopyrimidine mirror repeat sequences to adopt intramolecular triplexes (the usual H-y3 isomer and/or the rare H-y5 isomer) in recombinant plasmids was investigated. Unexpectedly, the presence of certain metal ions (magnesium, zinc, manganese, and calcium) stabilized the (GAA)4TTCGC(GAA)4 insert in the rare H-y5 when cloned into either of two different sequence backgrounds. Alternatively, either shortening or lengthening the sequence at the central interruption, which becomes the loop of the triplex, led to the formation of the canonical H-y3 under all conditions tested. Similarly, other oligopurine.oligopyrimidine mirror repeat sequences (i.e. (GAA)8 or (GGA)8) formed only the H-y3 under all experimental conditions. All triplexes were stabilized by negative supercoiling at pH 5.0; chemical probe and primer extension analyses served as critical structural tools. Hence, the nature of the central interruption sequence (the loop) of the mirror repeat is important for the stabilization of the H-y5. This region may be important for metal ion binding in the initial stages of triplex formation and thus may play a critical role in determining which isomer is formed. The possible biological role of a DNA sequence adopting three different conformations is discussed.

Cations↗
PubMed1992

Direct evidence for the effect of transcription on local DNA supercoiling in vivo.

The B-to-Z structural transition of varying lengths (74 to 14 base-pairs) of (CG) tracts has been used as a superhelicity probe to examine the local topological changes induced by transcription at defined genetic loci in vivo. The local-topology reporter sequences indicate that under steady-state transcription the region upstream from the promoter experiences an increase in negative supercoiling whereas the region downstream from the terminator displays a decrease in negative superhelicity. This result provides direct in vivo evidence for the notion that the translocation of an RNA polymerase elongation complex along the double-helical DNA generates positive supercoils in front of it and negative supercoils behind it. Also, this twin-supercoiled domain model was tested inside a transcribed region where a high degree of negative supercoiling generated by the passage of each individual RNA polymerase was detected. Hence, these data indicate that the induced supercoils are confined to the vicinity of each RNA polymerase complex in a multipolymerase system.

Base Composition↗