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Biomedical subjects

R Waters

Publications and source records attributed to R Waters.

At least 19 recordsLinked to original sources
PubMed2006

Roles for Gcn5p and Ada2p in transcription and nucleotide excision repair at the Saccharomyces cerevisiae MET16 gene.

Chromatin structure, transcription and repair of cyclobutane pyrimidine dimers at the MET16 gene of wild type, gcn5Delta and ada2Delta Saccharomyces cerevisiae cells were studied under repressing or derepressing conditions. These two components of the SAGA/ADA chromatin remodelling complexes are expendable for the basal transcription of MET16 but are mandatory for its full transcription induction. Despite their influence on transcription neither protein induces major changes in MET16 chromatin structure, but some minor ones occur. Repair at the coding region of the transcribed strand is faster than repair at non-transcribed regions in all strains and either growth condition. Moreover, the more MET16 is transcribed the faster the repair. The data show that by changing the transcription extent the rate of repair at each DNA strand is altered in a different way, confirming that repair at this locus is strongly modulated by its chromatin structure and transcription level. Deletion of GCN5 or ADA2 reduces repair at MET16. The results are discussed in light of the current understanding of Gcn5p and Ada2p functions, and they are the first to report a role for Ada2p in the nucleotide excision repair of the regulatory and transcribed regions of a gene.

Chromatin↗
PubMed2006

Breast reconstruction using deep inferior epigastric perforator flaps in EEC syndrome.

Ectrodactyly, ectodermal dysplasia, cleft lip/palate (EEC) syndrome is a rare condition that may result in failure of breast development. Breast reconstruction in these patients may pose a challenging problem, as they are young and reconstructive options should have minimal long-term complications. The use of deep inferior epigastric perforator (DIEP) flaps in breast reconstruction following breast cancer has been well described with good results. The use of DIEP flaps in breast augmentation, however, is far less common. We present the case of a young patient with EEC syndrome and mammary hypoplasia who underwent DIEP flap reconstruction for breast augmentation. The outcome was satisfactory to both patient and surgeon.

Adolescent↗
PubMed2006

Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Animals↗
PubMed2004

Cbf1p modulates chromatin structure, transcription and repair at the Saccharomyces cerevisiae MET16 locus.

The presence of damage in the transcribed strand (TS) of active genes and its position in relation to nucleosomes influence nucleotide excision repair (NER) efficiency. We examined chromatin structure, transcription and repair at the MET16 gene of wild-type and cbf1Delta Saccharomyces cerevisiae cells under repressing or derepressing conditions. Cbf1p is a sequence-specific DNA binding protein required for MET16 chromatin remodelling. Irrespective of the level of transcription, repair at the MspI restriction fragment of MET16 exhibits periodicity in line with nucleosome positions in both strands of the regulatory region and the non-transcribed strand of the coding region. However, repair in the coding region of the TS is always faster, but exhibits periodicity only when MET16 is repressed. In general, absence of Cbf1p decreased repair in the sequences examined, although the effects were more dramatic in the Cbf1p remodelled area, with repair being reduced to the lowest levels within the nucleosome cores of this region. Our results indicate that repair at the promoter and coding regions of this lowly transcribed gene are dependent on both chromatin structure and the level of transcription. The data are discussed in light of current models relating NER and chromatin structure.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2002

Dermatofibrosarcoma protuberans treated by micrographic surgery.

Dermatofibrosarcoma protuberans is an uncommon cutaneous tumour which rarely metastasises. However, local recurrence following apparently adequate surgical excision is well recognised, presumably as a result of sub-clinical contiguous growth, for which micrographically controlled excision would be a logical treatment. A retrospective study of all patients treated by micrographic surgery, from April 1995-March 2000, at a tertiary skin oncology centre. Twenty-one patients (11 males), age 14 to 71 years with dermatofibrosarcoma protuberans on the trunk (10 patients), groin (four), head and neck (four), and limbs (three) were treated. In 15 patients one micrographic layer cleared the tumour, and four were cleared with two layers. For one patient the second stage was completed by conventional excision guided by positive margins. Another patient with a multiply recurrent perineal dermatofibrosarcoma protuberans, not cleared in one area after two layers, died from a pulmonary embolus before total clearance could be achieved. There was no correlation between tumour size and lateral excision margin. No recurrence was observed during the follow-up, from 21 to 80 months, median 47 months. The study provides further support for micrographic surgery as the treatment of choice for dermatofibrosarcoma protuberans.

Adolescent↗
PubMed2002

Adverse alteration of wound flora with topical negative-pressure therapy: a case report.

Topical negative pressure (TNP) has achieved widespread use in the treatment of problematic wounds. We report the case of a patient treated with TNP for groin-wound dehiscence following inguinal block dissection. During treatment, clinical signs of sepsis developed, in association with a progressively worsening anaerobic wound infection. This infection settled with antibiotic therapy and cessation of TNP treatment. We postulate that the air-free environment created by TNP potentiated the growth of the anaerobic bacteria, resulting in significant sepsis, and therefore recommend close surveillance of bacterial flora while using this therapy, particularly in susceptible patients.

Aged↗
PubMed2001

The mapping of nucleosomes and regulatory protein binding sites at the Saccharomyces cerevisiae MFA2 gene: a high resolution approach.

We have developed an end-labelling approach to map the positions of nucleosomes and protein binding sites at nucleotide resolution by footprinting micrococcal nuclease (MNase)-sensitive sites. Using this approach we determined that the MFA2 gene and its upstream control regions have four positioned nucleosomes when transcription is repressed in mating type alpha cells and that the nucleosomes lose their positioning when the gene became transcriptionally active in mating type a cells. We also detected MNase-hypersensitive sites in the alpha2 operator region of MFA2 in alpha cells but not in a cells. These probably result from the change in the local DNA conformation due to protein(s) binding in this region that governs MFA2 transcription.

Base Sequence↗
PubMed2001

RAD9, RAD24, RAD16 and RAD26 are required for the inducible nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers from the transcribed and non-transcribed regions of the Saccharomyces cerevisiae MFA2 gene.

In this study, the effect of a prior UV irradiation on the removal of cyclobutane pyrimidine dimers (CPDs) from the transcribed strand (TS) and non-transcribed strand (NTS) of the MFA2 gene in haploid Saccharomyces cerevisiae (S. cerevisiae) cells was investigated. In NER competent cells, the pre-irradiation with a dose of 20J/m2 enhances the removal of CPDs induced by a second UV dose of 100J/m2 in the TS and the NTS of MFA2 gene except for the CPDs in the region +258 to +298 in the NTS, where the enhanced repair was absent. No inducible repair was observed in rad9, rad24, rad16 and rad26 cells, indicating two checkpoint genes RAD9 and RAD24, the global repair gene RAD16 and the transcription coupled repair gene RAD26 are essential for inducible NER.

Cell Cycle Proteins↗
PubMed2000

Excision repair at the level of the nucleotide in the upstream control region, the coding sequence and in the region where transcription terminates of the Saccharomyces cerevisiae MFA2 gene and the role of RAD26.

RAD26, the yeast homologue of human CSB, has an essential role in transcription-coupled repair (TCR). We have mapped the requisite of Rad26 for nucleotide excision repair (NER) within the different regions of the yeast Saccharomyces cerevisiae MFA2 gene at nucleotide resolution. Our results show that Rad26 is dispensable for enhanced NER in both the MFA2 upstream promoter, except in the TATA box region, and for enhanced NER in both strands of the active gene at a site close to the transcription termination region. As expected, it is not needed for repair of regions downstream of where transcription terminates. However, it is required for TCR in the transcription initiation and elongation regions. Our data support the hypothesis that Rad26 is required for the interchange between holo-TFIIH and a putative repairosome containing core TFIIH and other NER proteins. Close to the end of transcription, hotspots for the repair of CPDs in both the transcribed strand and the non-transcribed strand occur. This enhanced repair is independent of Rad26. Hence, TFIIH may take a form favourable for forming a repairosome without Rad26 assistance; here the organisation of the DNA during the termination of transcription may facilitate access of a repair complex to enable enhanced repair of both strands.

Cell Cycle Proteins↗
PubMed2000

Biomonitoring the human population exposed to pollution from the oil fires in Kuwait: analysis of placental tissue using (32)P-postlabeling.

The placenta is a readily available source of material for molecular epidemiological investigations. As such, DNA damage in this tissue can be indicative of maternal exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs). Previous reports have demonstrated that (32)P-postlabeling (PPL) is able to detect the presence of aromatic adducts in human placenta that are associated with maternal smoking during pregnancy. Using PPL we have assayed the DNA damage in placental samples from Kuwaiti mothers who were exposed to environmental pollution during pregnancy. This pollution arose in the aftermath of the Iraqi invasion of Kuwait, which left hundreds of oil wells burning. For comparison, further Kuwaiti samples were obtained approximately 1 year after the oil well fires and, as such, are from individuals unexposed to the airborne pollution from the oil well fires during pregnancy. In addition, placental samples were obtained from subjects in the United Kingdom. Adduct levels were measured in all samples using both the nuclease P1 and butanol extraction enhancement procedures. No elevation of adduct levels was observed in the placenta of mothers exposed to the oil well fires (n = 40) with either procedure (144 +/- 30 attomol/microg DNA for nuclease P1 enrichment, 245 +/- 50 attomol/microg DNA for butanol extraction), when compared with the nonexposed Kuwaiti mothers (180 +/- 32 and 281 +/- 39 attomol/microg DNA, respectively, n = 24). Similar adduct levels were observed in UK mothers who smoked cigarettes (178 +/- 30 and 284 +/- 52 attomol/microg DNA, n = 30), which in turn were approximately twice those observed in nonsmoking mothers (90 +/- 14 and 141 +/- 15 attomol/microg DNA, n = 12), although there is no significant difference in the distribution of adduct levels when statistical analysis is performed. Comprehensive interpretation of the Kuwaiti data is difficult as precise information on PAH levels is unavailable, although the data do seem to indicate that exposure to PAHs was not biologically significant.

Air Pollutants↗
PubMed2000

Low- and high-resolution mapping of DNA damage at specific sites.

Measurement of DNA damage and repair at the nucleotide level in intact cells has provided compelling evidence for the molecular details of these events as they occur in intact organisms. Furthermore, these measurements give the most accurate picture of the rates of repair in different structural domains of DNA in chromatin. In this report, we describe two methods currently used in our laboratories to map DNA lesions at (or near) nucleotide resolution in yeast cells. The low-resolution method couples damage-specific strand breaks in DNA with indirect end-labeling to measure DNA lesions over a span of 1.5 to 2 kb of DNA sequence. The resolution of this method is limited by the resolution of DNA length measurements on alkaline agarose gels (about +/-20 bp on average). The high-resolution method uses streptavidin magnetic beads and special biotinylated oligonucleotides to facilitate end-labeling of DNA fragments specifically cleaved at damage sites. The latter method maps DNA damage sites at nucleotide resolution over a shorter distance (<500 bp), and is constrained to the length of DNA resolvable on DNA sequencing gels. These methods are used in tandem for answering questions regarding DNA damage and repair in different chromatin domains and states of gene expression.

Biotinylation↗
PubMed2000

Quality-of-life study on four patients who underwent esophageal resection and delayed reconstruction for Boerhaave's syndrome.

Boerhaave's syndrome is the condition of spontaneous rupture of the esophagus as a consequence of the strain of emesis with or without predisposing esophageal disease. It is a condition with high mortality. We describe four patients who underwent a transthoracic esophagectomy to remove the rupture of the intrathoracic esophagus, closure of the esophageal gastric junction, fashioning of a feeding gastrostomy, and formation of a left cervical esophagostomy. Three patients underwent reconstruction with subcutaneous colon. We suggest that this method of management may be considered where primary repair is impossible in those patients too ill for prolonged reconstruction or as a salvage procedure where other methods have failed. The poor quality of life after esophagectomy is improved by reconstruction. Other surgical options include covering the repaired opening with a circumferential wrap of pleura, chest wall muscle, or omentum or closing the repair around a T-tube of large caliber. Esophageal exclusion using absorbable staples is another approach.

Aged↗
PubMed2000

Compartment syndrome complicating metastatic malignant melanoma.

Compartment syndrome is well documented in the literature. Neoplasia as a cause is a rare. We report a patient with known metastatic malignant melanoma presenting with a compartment syndrome of the arm caused by a relatively slow growing, non-invasive metastatic deposit. This was excised and the patient made an uneventful recovery.

Compartment Syndromes↗
PubMed1999

Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae.

The bifunctional alkylating anticancer drug nitrogen mustard forms a variety of DNA lesions, including monoadducts and intrastrand and interstrand crosslinks. Although it is known that nucleotide excision repair (NER) is important in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined. Using isogenic Saccharomyces cerevisiae strains disrupted for a variety of NER and BER genes we have examined the relative importance of the two pathways in the repair of nitrogen mustard adducts. As expected, NER defective cells (rad4 and rad14 strains) are extremely sensitive to the drug. One of the BER mutants, a 3-methyladenine glycosylase defective (mag1) strain also shows significant hypersensitivity. Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity. Furthermore, both rad14 and mag1 disruptants show elevated levels of nitrogen mustard-induced forward mutation. Measurements of repair rates of nitrogen mustard N-alkylpurine adducts in the highly transcribed RPB2 gene demonstrate defects in the processing of mono-adducts in rad4, rad14 and mag1 strains. However, there are differences in the kinetics of adduct removal in the NER mutants compared to the mag1 strain. In the mag1 strain significant repair occurs within 1 h with evidence of enhanced repair on the transcribed strand. Adducts however accumulate at later times in this strain. In contrast, in the NER mutants repair is only evident at times greater than 1 h. In a mag1/rad4 double mutant damage accumulates with no evidence of repair. Comparison of the rates of repair in this gene with those in a different genomic region indicate that the contributions of NER and BER to the repair of nitrogen mustard adducts may not be the same genome wide.

Base Sequence↗
PubMed1999

Spontaneous and photosensitiser-induced DNA single-strand breaks and formamidopyrimidine-DNA glycosylase sensitive sites at nucleotide resolutionin the nuclear and mitochondrial DNA of Saccharomyces cerevisiae.

A system is described for mapping oxidative DNA damage (sites sensitive to formamidopyrimidine-DNA glycosylase and single-strand breaks) at nucleotide resolution in the nuclear and mitochondrial DNA of Saccharomyces cerevisiae. Our 3' end labelling method is sensitive and was first developed using the well-studied inducer of oxidative DNA damage, methylene blue (MB) plus light. We treated yeast DNA in vitro with this so as to maximise levels of damage for assay development. Unfortunately, MB does not remain in yeast cells and yeast DNA repair mutants sensitive to active oxygen species are not sensitive to this agent, thus for in vivo experiments we turned to a polycyclic aromatic, RO 19-8022 (RO). This resulted in oxidative DNA damage when light was applied to yeast cells in its presence. The spectra of enzyme-sensitive sites and single-strand breaks induced by MB in vitro or by RO plus light in vivo or in vitro were examined in two yeast reporter genes: the nuclear MFA2 and the mitochondrial OLI1. The experiments revealed that most of the enzyme-sensitive sites and single-strand breaks induced by MB or RO plus light are at the same positions in these sequences, and that these are guanines.

Binding Sites↗
PubMed1999

Spontaneous mutation, oxidative DNA damage, and the roles of base and nucleotide excision repair in the yeast Saccharomyces cerevisiae.

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG). When compared to wild-type, ogg1 mutants show an increase in the frequency of GC to TA transversions, indicating a role for Ogg1 in the repair of 8-OxoG. Here we report an increased frequency of forward mutation to canavanine resistance in mutants defective in the nucleotide excision repair (NER) gene RAD14. This was not increased further in strains additionally defective in OGG1. However, when compared to strains solely defective in OGG1, ogg1radl4 mutants displayed an increase in spontaneous GC to TA transversions. Intriguingly, reversion of the lys1-1 ochre allele was not increased in rad14 mutants, suggesting that oxidative base damage may only represent a substrate for NER in certain regions of the genome. We also examined repair of oxidative DNA damage by transforming mutant strains with plasmid DNA treated with methylene blue plus visible light. Mutants defective in OGG1 showed no significant reduction in transformation efficiency compared with wild-type strains. In contrast, disruption of RAD14 reduced the efficiency of transformation, yet there was no further decrease in an ogg1rad14 mutant. This strongly supports a role for NER in the repair of oxidative base damage in yeast, and differs from similar experiments carried out in E. coli, where transformation efficiency is only reduced in mutants defective in both fpg and uvrA. Finally, the repair of Fpg-sensitive sites was examined at the MATalpha and HMLalpha mating type loci, and NER was found to play a role in their removal.

Canavanine↗