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J Westmoreland

Publications and source records attributed to J Westmoreland.

10 recordsLinked to original sources
PubMed1997

Highly mismatched molecules resembling recombination intermediates efficiently transform mismatch repair proficient Escherichia coli.

The ability of related DNAs to undergo recombination decreases with increased sequence divergence. Mismatch repair has been proposed to be a key factor in preventing homeologous recombination; however, the contribution of mismatch repair is not universal. Although mismatch repair has been proposed to act by preventing strand exchange and/or inactivating multiply mismatched heteroduplexes, there has been no systematic study to determine at what step(s) in recombination mismatch repair acts in vivo. Since heteroduplex is a commonly proposed intermediate in many models of recombination, we have investigated the consequences of mismatch repair on plasmids that are multiply mismatched in heteroduplex structures that are similar to those that might arise during recombination. Plasmids containing multiply mismatched regions were transformed into wild-type and Mut+ Escherichia coli mutants. There was only a 30-40% reduction in transformation of Mut+ as compared to mutS and mutL strains for DNAs containing an 18% mismatched heteroduplex. The products obtained from mutS hosts differed from those obtained from Mut+ hosts in that there were many more colonies containing mixtures of two plasmids, due to survival of both strands of the heteroduplex. There were nearly 10 times more recombinants obtained from the mutS as compared to the wild-type host. Based on these results and those from other studies with E. coli and yeast, we propose that the prevention of recombination between highly diverged DNAs may be at a step earlier than heteroduplex formation.

DNA Repair↗
PubMed1996

Homologous and homeologous intermolecular gene conversion are not differentially affected by mutations in the DNA damage or the mismatch repair genes RAD1, RAD50, RAD51, RAD52, RAD54, PMS1 and MSH2.

Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad+ vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts.

Amino Acid Sequence↗
PubMed1994

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair.

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.

Base Sequence↗
PubMed1993

Laparoscopic appendectomy: treatment of choice for suspected appendicitis.

Results and complications in 100 patients treated over a 3-year period with the laparoscopic approach for clinically diagnosed acute appendicitis are evaluated. They are compared with results and complications in 100 patients with the same diagnosis who had been treated with the open technique performed by the same surgeon during the same 3 years. The results suggest that laparoscopy provides excellent exposure of the appendix regardless of its position. In the absence of pathology of the appendix, laparoscopy allows for a thorough examination of the entire abdomen and pelvis and good exposure and definitive treatment of most surgical conditions encountered. In the event of appendicitis, regardless of its severity, laparoscopic appendectomy results in less postoperative pain, shorter hospital stays, faster return to normal activities, fewer postoperative complications, and superior cosmetic results. Our experience suggests that the laparoscopic approach is the best approach to diagnosis and treatment of the conditions encountered in patients with suspected appendicitis.

Abdominal Pain↗
PubMed1992

Recombinant repair of diverged DNAs: a study of homoeologous chromosomes and mammalian YACs in yeast.

Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15-20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms+ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.

Aneuploidy↗
PubMed1992

Laparoscopic management of enlarged cystic duct.

After laparoscopic exploration of the common bile duct, or when a patient has acute cholecystitis, the cystic duct is sometimes edematous and too large to be ligated safely with an Endoclip. In such cases, ligation of the cystic duct with an Endoloop offers a solution to the problem. The standard technique for application of an Endoloop consists of dividing the cystic duct and then applying the Endoloop. This becomes more difficult if, after the cystic duct is divided, loss of traction on the common bile duct results in retraction of the divided cystic stump outside of the laparoscopic field of view. To avoid this difficulty, the authors apply an Endoloop with the grasping forceps on the cystic duct before the duct is divided so that it cannot retract from operative view and for this task developed an instrument that allows simultaneous introduction of both grasping forceps and the Endoloop through a single port.

Acute Disease↗
PubMed1991

Laparoscopic appendectomy for acute appendicitis: indications and current use.

Laparoscopic evaluation was performed in 43 consecutive patients with right lower abdominal pain and preoperative diagnosis of possible appendicitis. Patients with generalized peritonitis and evidence of perforation of the appendix were not considered for laparoscopy. Visualization was sufficient for making a diagnosis in 97.7% of the cases. In 95%, laparoscopic findings were compatible with the pathology report. Thirty-five patients underwent successful laparoscopic appendectomy with neither intraoperative nor postoperative complications. No further surgery was required; slightly elevated temperatures in 6 patients responded to treatment with antibiotics, and there were no wound infections. Laparoscopic appendectomy is minimally invasive and results in less postoperative pain and morbidity and fewer adhesions and other long-term sequelae than conventional laparotomy. It is associated with superior cosmetic results, a shorter hospital stay, and faster return to normal activities. This experience suggests that if there is no evidence that the appendix is perforated or that generalized peritonitis exists and if qualified physicians and adequate facilities are available, patients presenting with right lower quadrant abdominal pain and possible appendicitis are best evaluated and treated with laparoscopic technique.

Acute Disease↗
PubMed1990

Heterogeneity and maintenance of centromere plasmid copy number in Saccharomyces cerevisiae.

We developed a novel approach to quantitate the heterogeneity of centromere number in yeast, and the cellular capacity for excess centromeres. Small circular plasmids were constructed to contain the CUP1 metallothionein gene. ARS1 (autonomously replicating sequence) and a conditionally functional centromere (GAL1-GAL10 promoter controlled centromere). The CUP1 gene provided a gene dosage marker, and therefore a genetic determinant of plasmid copy number. Growth of cells on glucose is permissive for centromere function, while growth on galactose renders the centromere nonfunctional and the plasmids are segregated in an asymmetric fashion. We identified "lines" of cells containing increased numbers of plasmids after transformation. Cell lines containing as many as five to ten active centromeres are stably maintained in the absence of genetic selection. Thus haploid yeast cells can tolerate a 50% increase in their centromere number without affecting progression through the cell cycle. This system provides the opportunity to address issues of specific cellular controls on centromere copy number.

Centromere↗
PubMed1987

UV-induced damage and repair in centromere DNA of yeast.

The centromere is the region within a chromosome that is required for proper segregation during mitosis and meiosis. Lesions in this sequence represent a unique type of damage, as loss of function could result in catastrophic loss of the genetic material of an entire chromosome. We have measured the induction by ultraviolet (UV) light of pyrimidine dimers in a 2550-bp restriction fragment that includes the centromere region of chromosome III in Saccharomyces cerevisiae. Yeast cells were exposed to ultraviolet light, cellular DNA was gently extracted, and subsequently treated with a UV-specific endonuclease to cleave all pyrimidine dimers. The sites of UV-specific nuclease scission within the centromere were determined by separating the DNA according to molecular weight, transferring the fragments to nitrocellulose, and hybridizing to a radiolabeled 624-bp fragment homologous to the centromere DNA from chromosome III. Several hotspots were identified in chromatin DNA from cells, as well as in irradiated deproteinized DNA. Double strand damage due to closely opposed pyrimidine dimers was also observed. At biological doses (35% survival) there are approximately 0.1 to 0.2 pyrimidine dimers per centromere. These dimers are efficiently repaired in the centromere and surrounding region.

Base Sequence↗