Biomedical subjects

G Dawes

Publications and source records attributed to G Dawes.

6 recordsLinked to original sources

PubMed2000

Molecular breeding of viruses.

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

3T3 Cells↗

PubMed1997

Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening.

An efficient beta-fucosidase was evolved by DNA shuffling from the Escherichia coli lacZ beta-galactosidase. Seven rounds of DNA shuffling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native beta-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for o-nitrophenyl fucopyranoside versus o-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galactopyranoside. The evolved cell line showed a 66-fold increase in p-nitrophenyl fucosidase specific activity. The evolved fucosidase has a 10- to 20-fold increased kcat/Km for the fucose substrates compared with the native enzyme. The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme. This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuffling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology. Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.

Escherichia coli↗

PubMed1997

PDZ domain of neuronal nitric oxide synthase recognizes novel C-terminal peptide sequences.

PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.

Amino Acid Sequence↗

PubMed1997

Molecular evolution of an arsenate detoxification pathway by DNA shuffling.

Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo. Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determined arsenate resistance from plasmid pl258 encoded by genes arsR, arsB, and arsC was evolved in Escherichia coli. Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance. Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome. In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal. The integrated ars operon had 13 mutations. Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance. While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12-fold. These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation. These mechanisms may be difficult to explain and are likely to be overlooked by rational design.

Adenosine Triphosphatases↗

PubMed1994

Computerized evaluation of fetal heart-rate patterns.

Antepartum Cardiotocography (CTG) is one of the few techniques available today to assess fetal conditions in high risk pregnancies. Visual interpretation of CTG traces has been shown to be unreliable. In order to eliminate observer variability and to increase the accuracy of CTG, numerical on-line analysis of fetal heart rate (FHR) patterns was introduced. The experience with computerized systems has shown that the best indicator of fetal conditions is variation, long- or short-term. Mean range (Long-term variation) values of less than 20 msec are associated with an increased rate of fetal acidaemia and perinatal mortality. The availability of numerical measurements enables the detection of small changes in FHR occurring in time, so when initial deterioration signs arise, the single fetus can be followed up longitudinally. Moreover, a more accurate correlation with other biophysical and biochemical parameters of the fetus can be done, as well as with perinatal outcome. In conclusion, computerized CTG improves accuracy and allows to distinguish fetuses that are truly jeopardized because of hypoxemia, from those who are not.

Cardiotocography↗

PubMed1991

Umbilical artery resistance index as a screening test for fetal well-being. I: Prospective revealed evaluation.

Abnormalities of the human fetoplacental circulation, a common association with fetal morbidity and mortality, can be detected by Doppler ultrasound examination of the umbilical arterial blood flow. The value of this procedure as a screening test was assessed in 369 women whose fetuses were evaluated on 1354 occasions using both computerized antenatal fetal heart rate (FHR) analysis and continuous-wave Doppler ultrasound. The FHR analyses were used to guide management, but the results of the Doppler measurements were not made available to the clinical staff. The mean duration of each nonstress test (NST) was 27 minutes, compared with 6 minutes for the Doppler examination. Increased resistance indices in the umbilical artery identified those fetuses with an abnormal NST or a clinical diagnosis of antenatal distress with a high sensitivity and negative predictive value. The high rate of false-positive results with respect to these two end points was reduced when fetal distress in labor was included as a third end point. The value of the Doppler examination in predicting fetal distress was applicable to appropriately grown as well as small for gestational age fetuses.

Female↗