Screening for oxidative resistance.
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Culture Media↗
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Results for “directed evolution”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
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Out of the warm little pond: prerequisites for an evolvable system.
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Directed Molecular Evolution↗
Spectroscopic and functional studies of a novel quadruple myoglobin variant with increased peroxidase activity.
A quadruple variant of horse heart myoglobin (Thr39Ile/Lys45Asp/Phe46Leu/Ile107Phe) that exhibits significantly (approximately 25-fold) greater peroxidase activity than the wild-type protein has been studied to determine its midpoint reduction potential (24(2) mV vs. SHE; pH 6.0, mu = 0.1 M, 25 degrees C) and to characterize the kinetics of its reaction with hydrogen peroxide. In addition, Fourier transform infrared (FTIR) spectra of the carbonyl and azide adducts of the protein have been obtained to gain initial insight into the effects of these substitutions on the ligand binding properties of the reduced and oxidized variant. All of the results obtained in this work are consistent with a variant heme binding pocket with increased hydrophilic character.
Animals↗
Protein engineering of subtilisin.
The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.
Calcium↗
Biotechnology in the 21st century.
Although the future is unpredictable, it is highly likely that biotechnology will play a much more visible and significant role in the 21st century than it did in the 20th century. The number and kinds of drugs provided by biotechnology will expand markedly and biotechnology will stand at the center of the oncoming revolution in bioinformatics.
Biotechnology↗
Reinventing the wheel.
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Directed Molecular Evolution↗
Microbial technologies for the discovery of novel bioactive metabolites.
Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development and application of molecular methods for the detection of uncommon genera of actinomycetes in soil DNA and for the rapid dereplication of actinomycete isolates. The results indicate a substantial presence in many soils of the uncommon genera and a large diversity of isolated actinomycetes. However, while uncommon actinomycete strains may provide an increased chance of yielding novel structures, their genetics and physiology are poorly understood. To speed up their manipulation, we have developed vectors capable of stably maintaining large segments of actinomycete DNA in Escherichia coli and of integrating site specifically in the Streptomyces genome. These vectors are suitable for the reconstruction of gene clusters from smaller segment of cloned DNA, the preparation of large-insert libraries from unusual actinomycete strains and the construction of environmental libraries.
Actinobacteria↗
An evolutionary basis for scrapie disease: identification of a fish prion mRNA.
Infectious prion proteins cause neurodegenerative disease in mammals owing to the acquisition of an aberrant conformation. We cloned a Fugu rubripes gene that encodes a structurally conserved prion protein, and found rapid rates of molecular divergence among prions from different vertebrate classes, along with molecular stasis within each class. We propose that a directional trend in the evolution of prion sequence motifs associated with pathogenesis and infectivity could account for the origin of scrapie in mammals.
Amino Acid Sequence↗
Maternal effects, paternal effects and sexual selection.
Maternal and paternal effects can lead to complicated evolutionary dynamics, including evolution in the opposite direction to selection. Recent studies demonstrate that parental effects on sexually selected traits, as well as preferences for those traits, might be large. Although these findings are likely to have consequences for both the evolutionary dynamics and equilibria of sexual selection, theory is lacking. Because parents are expected to maximize their own fitness, rather than that of a specific offspring, the magnitude (and even direction) of parental effects are context dependent. By extension, this dynamic nature of parental effects might help to explain the maintenance of variation in many sexually selected traits.
Journal Article↗
The modern synthesis, Ronald Fisher and creationism.
The 'modern evolutionary synthesis' convinced most biologists that natural selection was the only directive influence on adaptive evolution. Today, however, dissatisfaction with the synthesis is widespread, and creationists and antidarwinians are multiplying. The central problem with the synthesis is its failure to show (or to provide distinct signs) that natural selection of random mutations could account for observed levels of adaptation.
Journal Article↗
[Triple association: extramembranous glomerulonephritis, renal adenocarcinoma and splenic hamartoma. Apropos of a case].
We report the case of a 36 year old woman who presented a renal cell carcinoma, associated to a membranous nephropathy as a paraneoplastic syndrome. The concomitant association with a splenic hamartoma was probably fortuitous. Five years after nephrectomy, the patient was asymptomatic and her proteinuria was very low. We studied in the literature 93 cases which reported a such association between cancer, nephrotic syndrome and membranous nephropathy. Carcinoma of the lung and adenocarcinoma of the gastrointestinal tract are the most frequently implicated. This association can occur at every age and more often in men (75%) than in women. The survival is directly linked to the evolution of the cancer. Proteinuria and membranous nephropathy can totally disappear after surgical resection of the carcinoma. The glomerular injury is mediated by immune complexes composed at least in part of tumour associated antigens. The development of several types of glomerular injury in patients with carcinoma have been described but membranous glomerulonephritis is the most commonly observed.
Adenocarcinoma↗
Genetics of diabetes. Trans-racial gene mapping studies.
A major component of inherited susceptibility to IDDM is associated with one or more loci in the MHC. Identification of the primary susceptibility genes has been complicated by the low frequency of recombination, i.e. linkage disequilibrium, within the MHC. It is difficult to distinguish whether a detected genetic association with the disease is primary, or secondary due to linkage disequilibrium with an allele at another locus which is directly predisposing. During the evolution of different races, however, recombination within the MHC has occurred and population-specific MHC haplotypes exist. Primary susceptibility allels should be associated with disease in all racial groups, regardless of genetic background. It is unlikely that disease associations secondary to linkage disequilibrium will be consistent in these groups. This chapter reviews the known associations of candidate class II susceptibility alleles with IDDM in the five largest racial groups; white Caucasians, Asian Indians, Negroids, Japanese and Chinese. These trans-racial studies suggest that the DQ molecule has a primary role in predisposition to IDDM. There are consistent findings of a positive association with the DQA1*0301 allele and negative associations with the DQB1*0602 and DQB1*0603 alleles. These two alleles differ by a single codon and so the encoded DQ beta chains are likely to have similar functions. DR4-associated susceptibility is associated with the DQA1*0301 allele in all races tested so far but this allele cannot be the only susceptibility factor on this haplotype. The identity of the DR3-associated susceptibility factor remains unclear but the DQB1*0201 allele is a candidate. If DQB1*0201 is involved, the existence of a protective factor on the neutral DR7-DQB1*0201 haplotypes is indicated. Analysis of DR9 associated susceptibility implicates a non-DR/DQ predisposing factor.
Asian People↗
Novel concepts for selection of catalytic activity.
The selection of mutant enzymes with novel properties from libraries is emerging as a very powerful strategy for enzyme engineering. The past year has witnessed significant progress on several fronts: new and improved methods have been developed for the creation of libraries and advances have been made in screening and selection techniques. The results achieved demonstrate the enormous potential of the methods and leave questions open for further studies.
Catalysis↗
In vitro display technologies: novel developments and applications.
In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.
Binding Sites↗
Towards novel processes for the fine-chemical and pharmaceutical industries.
In response to the need in the pharmaceutical industry for more complex, chiral molecules, fine-chemical companies are embracing new manufacturing technologies to produce compounds of these specifications. In particular, recent developments in biocatalysis combined with novel process engineering are providing improved methods for the production of valuable chemical intermediates.
Catalysis↗
Expression of ribozymes in gene transfer systems to modulate target RNA levels.
The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.
Animals↗
Biotechnology match making: screening orphan ligands and receptors.
To date there has been a considerable amount of interest and success in the pharmaceutical industry in the discovery of drug targets and diagnostics via genomic technologies, namely DNA sequencing, mutation/polymorphism detection and expression monitoring of mRNA. As the ultimate targets for the majority of these methods are actually proteins, more and more emphasis has been placed upon protein-based methods in an effort to define the function of proteins discovered by genomic technologies. One of the most challenging areas of drug target discovery facing researchers today is the search for novel receptor-ligand pairs. Database mining techniques in conjunction with other computational methods are able to identify many novel sequences of putative receptors, but the ability to similarly identify the receptor's natural ligand is not possible by these methods. The past few years have seen an increase in methodology and instrumentation focused on the ability to discover and characterize protein-protein interactions, as well as receptor-ligand pairs. Significant advances have been made in the areas of instrumentation (biosensors and fluorescent plate readers) as well as methodologies relating to phage/ribosome display and library construction.
Animals↗
Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages.
The cave bear, Ursus spelaeus, represents one of the most frequently found paleontological remains from the Pleistocene in Europe. The species has always been confined to Europe and was contemporary with the brown bear, Ursus arctos. Relationships between the cave bear and the two lineages of brown bears defined in Europe, as well as the origins of the two species, remain controversial, mainly due to the wide morphological diversity of the fossil remains, which makes interpretation difficult [1, 2]. Sequence analysis of ancient DNA is a useful tool for resolving such problems because it provides an independent source of data [3]. We previously amplified a short DNA fragment of the mitochondrial DNA control region (mt control region) of a 40,000-year-old Ursus spelaeus sample [4]. In this paper, we describe the DNA analysis of two mtDNA regions, the control region and the cytochrome b gene. Control region sequences were obtained from ten samples of cave bears ranging from 130,000 to 20,000 years BP, and one particularly well-conserved sample gave a complete cyt b sequence. Our data demonstrate that cave bears split largely before the lineages of brown bears around 1.2 million years ago. Given its abundance, its wide distribution in space and time, and its large morphological diversity, the cave bear is a promising model for direct observation of the evolution of sequences throughout time, extinction periods, and the differentiation of populations shaped by climatic fluctuations during the Pleistocene.
Animals↗