Restriction site mapping for three or more enzymes

Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined. Received on July 30, 1989; accepted on April 4, 1990     

Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.     

Cloning and Expression of a 5-Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase

Abstract: In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [¹⁴C]glucose or the [β-³⁵S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

Development of predictive models of laboratory animal growth using artificial neural networks

Traditional regression analysis of body weight growth curvesencounters problems .when the data are extremely variable. Whiletransformations are often employed to meet the criteria of theanalysis, some transformations are inadequate for normalizingthe data. Regression analysis also requires presuppositionsregarding the model to be fit and the techniques to be usedin the analysis. An alternative approach using artificial neuralnetworks is presented which may be suitable for developing predictivemodels of growth. Neural networks are simulators of the processesthat occur in the biological brain during the learning process.They are trained on the data, developing the necessary algorithmswithin their internal architecture, and produce a predictivemodel based on the learned facts. A dataset of Sprague–Dawleyrat (Rattus norvegicus) weights is analyzed by both traditionalregression analysis and neural network training. Predictionsof body weight are made from both models. While both methodsproduce models that adequately predict the body weights, theneural network model is superior in that it combines accuracyand precision, being less influenced by longitudinal variabilityin the data. Thus, the neural network provides another toolfor researchers to analyze growth curve data.     

One hundred and one new simple sequence repeat-based markers for the canine genome

One hundred and one new dinucleotide repeat polymorphisms specific for the canine genome have been identified and characterized. Screening of both primary libraries and marker selected libraries enriched for simple sequence repeats led to the isolation of large numbers of genomic clones that contained (CA)_n repeats. Over 200 of these clones were sequenced, and PCR primers that bracket the repeat were developed for those that contained ten or more continuous (CA)_n units. This effort led to the production of 101 polymorphic markers, which were assigned to one of four categories depending on their degree of polymorphism. Fiftyfour markers were found to be highly or very highly polymorphic as they had four or more alleles when tested on a panel of unrelated dogs. This group of markers will be useful for following inheritance of traits in crosses between dogs.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers indicated in Table 1.