Biomedical subjects
Publications and source records attributed to A Coulson.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.
Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.
OBJECTIVES: To evaluate the potential effect of computer support on general practitioners' management of familial breast and ovarian cancer, and to compare the effectiveness of two different types of computer program. DESIGN: Crossover experiment with balanced block design. PARTICIPANTS: Of a random sample of 100 general practitioners from Buckinghamshire who were invited, 41 agreed to participate. From these, 36 were selected for a fully balanced study. INTERVENTIONS: Doctors managed 18 simulated cases: 6 with computerised decision support system Risk Assessment in Genetics (RAGs), 6 with Cyrillic (an established pedigree drawing program designed for clinical geneticists), and 6 with pen and paper. MAIN OUTCOME MEASURES: Number of appropriate management decisions made (maximum 6), mean time taken to reach a decision, number of pedigrees accurately drawn (maximum 6). Secondary measures were method of support preferred for particular aspects of managing family histories of cancer; importance of specific information on cancer genetics that might be provided by an "ideal computer program." RESULTS: RAGs resulted in significantly more appropriate management decisions (median 6) than either Cyrillic (median 3) or pen and paper (median 3); median difference between RAGs and Cyrillic 2.5 (95% confidence interval 2.0 to 3.0; P<0.0001). RAGs also resulted in significantly more accurate pedigrees (median 5) than both Cyrillic (median 3.5) and pen and paper (median 2); median difference between RAGs and Cyrillic 1.5 (1.0 to 2.0; P<0.0001). The time taken to use RAGs (median 178 seconds) was 51 seconds longer per case (95% confidence interval 36 to 65; P<0.0001) than pen and paper (median 124 seconds) but was less than Cyrillic (median 203 seconds; difference 23. (5 to 43; P=0.02)). 33 doctors (92% (78% to 98%)) preferred using RAGs overall. The most important elements of an "ideal computer program" for genetic advice in primary care were referral advice, the capacity to create pedigrees, and provision of evidence and explanations to support advice. CONCLUSIONS: RAGs could enable general practitioners to be more effective gatekeepers to genetics services, empowering them to reassure the majority of patients with a family history of breast and ovarian cancer who are not at increased genetic risk.
Gene discovery programs centred around expressed sequence tag (EST) and genome sequencing projects have predictably led to an exponential surge in the number of parasite gene sequences deposited in public databases. To take advantage of this wealth of sequence information, it is essential to develop rapid methods for elucidating the biological function or mode of action of individual genes. Here, Patricia Kuwabara and Alan Coulson discuss the virtues of a powerful epigenetic gene disruption technique, RNA-mediated interference (RNAi), which was originally developed for the nematode Caenorhabditis elegans. It is anticipated that this technique will not only provide insights into gene function, but also help investigators to mine the genome for candidate drug intervention or vaccine development targets, some of which may not be readily apparent on the basis of sequence information alone.
OBJECTIVES: To explore general practitioners' attitudes towards and use of a computer program for assessing genetic risk of cancer in primary care. DESIGN: Qualitative analysis of semistructured interviews and video recordings of simulated consultations. PARTICIPANTS: Purposive sample of 15 general practitioners covering a range of computer literacy, interest in genetics, age, and sex. INTERVENTIONS: Each doctor used the program in two consultations in which an actor played a woman concerned about her family history of cancer. Consultations were videotaped and followed by interviews with the video as a prompt to questioning. MAIN OUTCOME MESURESs: Use of computer program in the Consultation. RESULTS: The program was viewed as an appropriate application of information technology because of the complexity of cancer genetics and a sense of "guideline chaos" in primary care. Doctors found the program easy to use, but it often affected their control of the consultation. They needed to balance their desire to share the computer screen with the patient, driven by their concerns about the effect of the computer on doctor-patient communication, against the risk of premature disclosure of bad news. CONCLUSIONS: This computer program could provide the necessary support to assist assessment of genetic risk of cancer in primary care. The potential impact of computer software on the consultation should not be underestimated. This study highlights the need for careful evaluation when developing medical information systems.
BACKGROUND: The value of antibiotic prophylaxis before insertion of an intrauterine device (IUD) remains uncertain. We undertook a triple-masked, randomised, placebo-controlled trial to find out whether such prophylaxis reduces the rate of IUD removal within 90 days. METHODS: 11 clinic sites in southern California enrolled women who requested IUD insertion and were at low risk of sexually transmitted infection according to self-reported medical history. We randomly assigned 1985 participants either 500 mg azithromycin or placebo capsules of identical appearance taken about 1 h before insertion of a Copper T 380A IUD. 118 women did not have an IUD inserted. We followed up 1833 of the remaining 1867 (98%) participants for at least 90 days after insertion. FINDINGS: The rate of IUD removal for any reason other than partial expulsion was 3.8% (35/918) in the antibiotic group and 3.4% (31/915) in the placebo group (relative risk 1.1 [95% CI 0.7-1.8]). The two treatment groups sought medical attention with equal frequency (mean 38 visits per 100 women). During the 90 days after IUD insertion, only one woman from each assignment group had salpingitis, as defined by established criteria. INTERPRETATION: Prophylaxis with azithromycin did not affect the likelihood that a woman would retain her IUD at 90 days or the frequency of postinsertion medical attention. In appropriately screened women, the risk of upper-genital-tract infection is negligible after IUD insertion, with or without the administration of prophylactic antibiotics.
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STUDY OBJECTIVE: The purpose of this study was to investigate the use of an intracoronary cannula during minimally invasive direct coronary artery bypass (MIDCAB) surgery. DESIGN: Evaluation of the coronary perfusion catheter was conducted during MIDCAB surgery to determine if there was any trauma to the coronary artery. The study was retrospective with 45% follow-up arteriograms. SETTING: A community hospital provided the setting for the study. Twenty patients undergoing MIDCAB surgery were included. INTERVENTIONS: Insertion of an intracoronary cannula was performed during MIDCAB surgery. MEASUREMENTS AND RESULTS: Measurements were determined on 45% follow-up arteriograms, and clinical follow-up was done on patient status. CONCLUSION: The use of an intracoronary cannula during MIDCAB surgery did not result in any trauma to the cannulated coronary artery. The cannula prevented ischemia of the service area of the target coronary artery. ECG changes were avoided as were hypotension and arrhythmias. The cannula also helped stabilize the target coronary area during suturing.
Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 A resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 A resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 A resolution. The electron density map showed no indication of substrate binding.
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Pfs230 is a surface protein of the gametes of Plasmodium falciparum and has been demonstrated to be a target of malaria transmission-blocking antibodies; it is an important candidate antigen for a transmission-blocking vaccine. The target epitopes of transmission-blocking antibodies against Pfs230 are almost all reduction sensitive suggesting that disulfide bonds are critical for folding the native molecule. Following the cloning of the Pfs230 gene attempts are now underway to express subunits of the protein for use in vaccine trials. It will be important to understand the disulfide-bond structure of the Pfs230 to achieve this goal. In this paper we present a model for this structure based on the observation that the Pfs230 molecule contains a series of regularly repeated cysteine-containing motifs. Four such motifs have been identified, together with a fifth cysteineless motif, which occur in the same relative order, with regular alternating omission of specific motifs, 14 times throughout the length of the protein. Each of the 14 sets of motifs contains an even number of cysteine residues (2, 4 or 6). We postulate that each set folds into a separate disulfide-bonded domain in which corresponding pairs of cysteines form an equivalent disulfide bond in every such domain. The postulated bonding arrangements in the different domains are mutually confirmatory throughout the sequence of Pfs230. We have identified two other malaria proteins, Pfs48/45 and Pf12, which share the same arrangements of motifs and conform to the same disulfide-bond structure proposed for Pfs230; no other proteins in the sequence data base share these characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)
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The genetic map of each Caenorhabditis elegans chromosome has a central gene cluster (less pronounced on the X chromosome) that contains most of the mutationally defined genes. Many linkage group termini also have clusters, though involving fewer loci. We examine the factors shaping the genetic map by analyzing the rate of recombination and gene density across the genome using the positions of cloned genes and random cDNA clones from the physical map. Each chromosome has a central gene-dense region (more diffuse on the X) with discrete boundaries, flanked by gene-poor regions. Only autosomes have reduced rates of recombination in these gene-dense regions. Cluster boundaries appear discrete also by recombination rate, and the boundaries defined by recombination rate and gene density mostly, but not always, coincide. Terminal clusters have greater gene densities than the adjoining arm but similar recombination rates. Thus, unlike in other species, most exchange in C. elegans occurs in gene-poor regions. The recombination rate across each cluster is constant and similar; and cluster size and gene number per chromosome are independent of the physical size of chromosomes. We propose a model of how this genome organization arose.
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