Search PubMed⌕ Search

Biomedical subjects

R D Wells

Publications and source records attributed to R D Wells.

At least 37 records · Page 2Linked to original sources
PubMed1999

Nucleotide excision repair affects the stability of long transcribed (CTG*CAG) tracts in an orientation-dependent manner in Escherichia coli.

The influence of nucleotide excision repair (NER), the principal in vivo repair system for DNA damages, was investigated in Escherichia coli with uvrA, uvrB and uvrAuvrB mutants with the triplet repeat sequences (TRS) involved in myotonic dystrophy, the fragile X syndrome and Friedreich's ataxia. (CTG*CAG)175was more stable when the (CTG) strand was transcribed than when the (CAG) strand was transcribed in the alternate orientation. A lack of the UvrA protein dramatically increases the instability of this TRS in vivo as compared with the stability of the same sequence in uvrB mutant, which produces an intact UvrA protein. We propose that transcription transiently dissociates the triplet repeat complementary strands enabling the non-transcribed strand to fold into a hairpin conformation which is then sufficiently stable that replication bypasses the hairpin to give large deletions. If the TRS was not transcribed, fewer deletions were observed. Alternatively, in the uvrA-mutant, the hairpins existing on the lagging strand will suffer bypass DNA synthesis to generate deleted molecules. Hence, NER, functionally similar in both prokaryotes and eukaryotes, is an important factor in the genetic instabilities of long transcribed TRS implicated in human hereditary neuro-logical diseases.

Adenosine Triphosphatases↗
PubMed1999

Sticky DNA: self-association properties of long GAA.TTC repeats in R.R.Y triplex structures from Friedreich's ataxia.

A novel DNA structure, sticky DNA, is described for lengths of (GAA.TTC)n found in intron 1 of the frataxin gene of Friedreich's ataxia patients. Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) triplexes in negatively supercoiled plasmids at neutral pH. An excellent correlation was found between the lengths of (GAA.TTC) (> 59 repeats): first, in FRDA patients, second, required to inhibit transcription in vivo and in vitro, and third, required to adopt the sticky conformation. Fourth, (GAAGGA.TCCTTC)65, also found in intron 1, does not form sticky DNA, inhibit transcription, or associate with the disease. Hence, R.R.Y triplexes and/or sticky DNA may be involved in the etiology of FRDA.

DNA↗
PubMed1998

Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases.

Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG.CAG, CGG.CCG, and GAA.TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG.CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG.CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG.CAG)175 fall into distinct size classes (1-8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletions versus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients.

DNA Repair↗
PubMed1998

Inhibitory effects of expanded GAA.TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo.

Friedreich ataxia (FRDA) is associated with the expansion of a GAA. TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA.TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA.TTC tracts (approximately 250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA.TTC)9 and (GAA.TTC)90. Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)230 than for the plasmid whose transcript contains (UUC)270. Our in vivo investigation revealed that expanded GAA.TTC repeats from intron I of the FRDA gene inhibit transcription rather than post-transcriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures.

Animals↗
PubMed1998

Comparison of the levels of quality of inpatient care delivered by pediatrics residents and by private, community pediatricians at one hospital.

PURPOSE: To compare the quality and cost of inpatient care provided by pediatrics residents with the quality and cost of that provided by private, practicing pediatricians. METHOD: Valley Children's Hospital--a 175-bed, private, nonprofit, university-affiliated pediatric hospital located in Fresno, California--has both a resident service and a community physician service. From May 1994 to March 1995 a total of 154 pediatric patients (64 from the resident service, 90 from the community service) were selected for the study using incidental sampling. In order to be included in the study, patients had discharge diagnoses of uncomplicated gastroenteritis or asthma. The parent or guardian of each patient completed a satisfaction questionnaire at discharge and agreed to a home visit one month later, when the same questionnaire and another were completed. Patients' charts were studied for treatment procedures, and follow-up data for a year were compiled. Statistical analyses were conducted with analysis of variance for continuous measures and chi-squares for dichotomous variables. RESULTS: The findings indicated no difference between the two services in terms of the parents' and guardians' satisfaction with patient care, hospital charges, and degrees of adherence to follow-up care after discharge. The community physicians were more likely than were the residents to employ non-standard laboratory and management procedures with both the asthma and gastroenteritis patients. The asthma patients cared for by community physicians had significantly more frequent asthma attacks after discharge, with a higher frequency of patients being subsequently treated in the emergency room and readmitted to the hospital, than did the asthma patients cared for by the residents. CONCLUSION: Common perceptions that physicians-in-training (1) overuse medical services and (2) fail, due to their inexperience, to provide high-quality services were not supported in this study.

Adolescent↗
PubMed1997

Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli.

Induction of transcription into long CTG.CAG repeats contained on plasmids in Escherichia coli is shown to increase the frequency of deletions within the repeat sequences. This elevated genetic instability was detected because active transcription into the triplet repeat influenced the growth transitions of the host cell, allowing advantageous growth for cells harboring plasmids with deleted repeat sequences. The variety of deletion products observed in separate cultures suggests that transcription altered the metabolism of the DNA in a manner that produced random length changes in the repeat sequence. For cultures containing plasmids without active transcription into the triplet repeat, or those maintained in exponential growth, deletions occurred within the repeat at a lower frequency (5-20-fold lower). In these incubations the extent of deletions was proportional to the number of cell divisions and many repeat lengths were observed within each culture, suggesting that the decrease in average repeat length at long incubation times was due to multiple small deletions. These observations show that deletions within long CTG.CAG repeats contained on plasmids in E.coli occur via more than one pathway and their level of genetic instability is altered by the enzymatic processes occurring upon the DNA.

Escherichia coli↗
PubMed1997

Flexible DNA: genetically unstable CTG.CAG and CGG.CCG from human hereditary neuromuscular disease genes.

The properties of duplex CTG.CAG and CGG.CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 x 10(-19) erg.cm for CTG and 1.27 x 10(-19) erg.cm for CGG, approximately 40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were approximately 60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 x 10(-19) erg.cm and 10.4 base pairs (bp)/turn for CTG and 2.4 x 10(-19) erg.cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224-245 bp in length (64-71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.

DNA↗
PubMed1997

Triplet repeat instability and DNA topology: an expansion model based on statistical mechanics.

The variance of writhe, the contribution of writhe to supercoiling, and the free energies of supercoiling were calculated for (CTG.CAG)n and (CGG.CCG)n triplet repeat sequences (TRS) by statistical mechanics from the bending and torsional moduli previously determined. Expansions of these sequences are inherited by non-mendelian transmission and are linked with several hereditary neuromuscular diseases. The variance of writhe was greater for the TRS than for random B-DNA. For random B-DNA, (CGG)n, and (CTG)n, the contribution of writhe to supercoiling was 70, 78, and 79%, whereas the free energy of supercoiling at a length of 10 kilobase pairs was 1040.RT, 760.RT, and 685.RT, respectively. These data indicate that the TRS are preferential sites for the partitioning of supercoiling. Calculations of the differences in free energy of supercoiling between the TRS and random B-DNA revealed a local minimum at approximately 520 base pairs. Human medical genetic studies have shown that individuals carrying up to 180-200 copies of TRS (540-600 base pairs, premutations) in the fragile X or myotonic dystrophy gene loci are usually asymptomatic, whereas large expansions (>200 repeats, full mutations), which lead to disease, are observed in their offspring. Therefore, the length corresponding to the local minimum in free energy of supercoiling correlates with the genetic breakpoint between premutation and full mutation. We propose that (a) TRS instability is mediated by DNA mispairing caused by the accumulation of supercoiling within the repeats, and (b) the expansions that take place at the premutation to full mutation threshold are associated with increased mispairing caused by the optimal partitioning of writhe within the TRS at this length.

DNA, Superhelical↗
PubMed1997

Hairpin formation during DNA synthesis primer realignment in vitro in triplet repeat sequences from human hereditary disease genes.

Genetic expansion of DNA triplet repeat sequences (TRS) found in neurogenetic disorders may be due to abnormal DNA replication. We have previously observed strong DNA synthesis pausings at specific loci within the long tracts (> approximately 70 repeats) of CTG.CAG and CGG.CCG as well as GTC.GAC by primer extensions in vitro using DNA polymerases (the Klenow fragment of Escherichia coli DNA polymerase I, the modified T7 DNA polymerase (Sequenase), and human DNA polymerase beta). Herein, we have isolated and analyzed the products of stalled synthesis found at approximately 30-40 triplets from the beginning of the TRS. DNA sequence analyses revealed that the stalled products contained short tracts of homogeneous TRS (6-12 repeats) in the middle of the sequence corresponding to the flanking region of the primer-template sequence. The sequence at the 3'-side terminated at the end of the primer, indicating that the primer molecule had served as a template. In addition, chemical probe and polyacrylamide gel electrophoretic analyses revealed that the stalled products existed in hairpin structures. We postulate that these products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the TRS due to primer realignment. The consequence of these steps is DNA synthesis to the end of the primer and termination. Primer realignment including hairpin formation may play an important intermediate role in the replication of TRS in vivo to elicit genetic expansions.

Betaine↗
PubMed1996

Relationship between Escherichia coli growth and deletions of CTG.CAG triplet repeats in plasmids.

Instabilities that are intrinsic to natural repetitive DNA sequences produce high frequencies of length changes in vivo. Triplet repeats cloned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors. We show that CTG-CAG repeats in plasmids can influence the growth of E. coli, which affects the observed stabilities. At extended growth periods, the observed frequencies of deletions were dramatically increased if the cells passed through stationary phase before subculturing. Deletions were particularly pronounced for a plasmid containing the longest repeat, 525 bp in total, with the CTG sequence as the lagging strand template for replication. Measurements of cell growth showed that the lag phase associated with E. coli growth was increased for cultures containing plasmids with long CTG-CAG repeats, particularly when the CTG-containing strand was the lagging template. High frequencies of deletions were observed because of a growth advantage of cells containing plasmids with deleted triplet repeats. Incubation conditions that reduced the bacterial growth-rate produced a decreased extent of deletions, presumably because they alleviated the growth advantage of cells harboring plasmids with deleted triplet repeats. The experimental observations were simulated by a model in which shorter triplet repeats provided a growth advantage due to a shorter lag phase. We demonstrate that the accumulation of deletions within repeating sequences during growth of E. coli can be prevented, and discuss these findings in relation to the studies of repetitive DNA sequences. These are the first observations to show a direct influence between a plasmid-based DNA sequence or structure and factors controlling bacterial growth.

Base Sequence↗
PubMed1996

Long CCG triplet repeat blocks exclude nucleosomes: a possible mechanism for the nature of fragile sites in chromosomes.

The rare folate-sensitive, fragile sites on chromsomes X, 11, and 16 contain blocks of CCG triplet repeats and large expansions of the CCG block at the FRAXA site produce the fragile X syndrome (FraX). The fragile, poorly staining nature of these sites suggested an altered chromatin structure. Here, repeating CCG DNAs from FraX patients were tested for their ability to assemble into nucleosomes, the basic subunits of chromatin, using in vitro nucleosome reconstitution, electron microscopy and competitive assembly gel retardation assays. CCG blocks of >50 repeats displayed strong nucleosome exclusion, providing a possible explanation for the nature of these sites.

Base Sequence↗
PubMed1996

Cloning, characterization, and properties of seven triplet repeat DNA sequences.

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D.(1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA.TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D.(1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D.(1996) J. Biol. Chem. 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.

Base Sequence↗
PubMed1996

TTA.TAA triplet repeats in plasmids form a non-H bonded structure.

CTG.CAG, CGG.CCG, and AAG.CTT triplet repeats proximal to or in disease genes expand by a non-Mendelian genetic process to cause several human hereditary syndromes. As part of our physical, biological, and genetic studies on the 10 possible triplet repeats, we discovered that the TTA.TAA repeat, isolated from the upstream region of the variant surface glycoprotein gene of Trypanosoma brucei, shows a propensity to adopt a non-H bonded structure under appropriate conditions. The other nine triplet repeat sequences do not exhibit this property. (TTA.TAA)n, where n = 90, 60, 30, and 18, cloned into pUC19 was studied by chemical and enzymatic probes as well as two-dimensional gel electrophoretic analyses under a variety of conditions. The helix opening was observed for all four inserts in supercoiled plasmids as a function of temperature, pH, metal ions, and buffer conditions using OsO4, diethyl pyrocarbonate, and chloroacetaldehyde probes. This unusual property of the TTA.TAA repeat suggests that it plays a different role from the other nine triplet repeats in gene expression.

Base Sequence↗
PubMed1996

CTG triplet repeats from the myotonic dystrophy gene are expanded in Escherichia coli distal to the replication origin as a single large event.

The expansion and contraction of CTG and CGG trinucleotide repeat sequences have been associated with several heritable genetic diseases. We developed a system for investigating the expansion of triplet repeat sequences in Escherichia coli in order to elucidate molecular mechanisms. Analysis of expanded regions using the interrupting CTA triplet sequence as a location marker within the CTG tract revealed that the expansion of large CTG repeats is one event rather than an accumulation of multiple small expansions and that the expansions occur more frequently in the region distal from the replication origin. Also, we showed that a loss of interruptions increases the expansion frequency. Thus, the instability of large triplet repeats in hereditary diseases occurs by a mechanism different from the instability in microsatellite sequences caused by defects in mismatch repair systems for certain sporadic cancers and hereditary non-polyposis colorectal cancers.

Base Sequence↗
PubMed1996

Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene.

The FMR-1 gene for the human fragile-chi syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5' non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG)n inserts (where n = 6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-chi syndrome.

Base Sequence↗