Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis

In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease. The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene. The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding. Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3%. From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified. The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene.     

Primary sequences of proteins from complete genomes display a singular periodicity: Alignment-free N-gram analysis

A method is proposed to represent and to analyze complete genome sequences (52 species from procaryotes and eukaryotes), based upon n-gram sequence's frequencies of amino acid pairs (bigrams), separated by a given number of other residues. For each of the species analyzed, it allows us to construct over-abundant and over-deficient occurrence profiles, summarizing amino acid bigram frequencies over the entire genome. The method deals efficiently with a sparseness of statistical representations of individual sequences, and describes every gene sequence in the same way, independently of its length and of the genome sizes. The frequency of over-abundant and over-deficient occurrences of bigrams presents a singular periodicity around 3.5 peptide bonds, suggesting a relation with the alpha helical secondary structure.     

Drift or Selection: A Statistical Test of Gene Frequency Variation over Generations

The method used by Fisher and Ford (1947) to study the spread of a gene in a natural population has been modified to analyze the variation in allele frequencies from generation to generation in a common experimental procedure. A further analysis has been developed that is more sensitive to directional trends in the allele frequency over generations, and its use in detecting the action of directional selection on gene frequency at a locus is discussed. The power of each of these statistical tests is calculated for a number of cases, and the tests are applied to sets of isozyme data from Drosophila pseudoobscura and Zea mays.     

Analysis of deletion mutations in the PARK2 gene in idiopathic Parkinson's disease

A method for analysis of deletions and duplications of individual exons and groups of exons in the parkin gene (PARK2) in both homozygous and heterozygous states has been developed. The method is based on semiquantitative polymerase chain reaction (PCR). The method has been used for analysis of the frequency of deletions in gene PARK2 in patients with idiopathic Parkinson's disease from Bashkortostan. Two unrelated patients have been found to carry a deletion of the 12th (last) exon of gene PARK2. Possibly, this deletion has caused the disease in the given patients.     

Experimental designs for reliable detection of linkage disequilibrium in unstructured random population association studies

A method is given for design of experiments to detect associations (linkage disequilibrium) in a random population between a marker and a quantitative trait locus (QTL), or gene, with a given strength of evidence, as defined by the Bayes factor. Using a version of the Bayes factor that can be linked to the value of an F-statistic with an existing deterministic power calculation makes it possible to rapidly evaluate a comprehensive range of scenarios, demonstrating the feasibility, or otherwise, of detecting genes of small effect. The Bayes factor is advocated for use in determining optimal strategies for selecting candidate genes for further testing or applications. The prospects for fine-scale mapping of QTL are reevaluated in this framework. We show that large sample sizes are needed to detect small-effect genes with a respectable-sized Bayes factor, and to have good power to detect a QTL allele at low frequency it is necessary to have a marker with similar allele frequency near the gene.     

Systematic Detection of Large-Scale Multigene Horizontal Transfer in Prokaryotes

Horizontal gene transfer (HGT) is central to prokaryotic evolution. However, little is known about the “scale” of individual HGT events. In this work, we introduce the first computational framework to help answer the following fundamental question: How often does more than one gene get horizontally transferred in a single HGT event? Our method, called HoMer, uses phylogenetic reconciliation to infer single-gene HGT events across a given set of species/strains, employs several techniques to account for inference error and uncertainty, combines that information with gene order information from extant genomes, and uses statistical analysis to identify candidate horizontal multigene transfers (HMGTs) in both extant and ancestral species/strains. HoMer is highly scalable and can be easily used to infer HMGTs across hundreds of genomes. We apply HoMer to a genome-scale data set of over 22,000 gene families from 103 Aeromonas genomes and identify a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Analysis of these HMGTs reveals interesting relationships between gene function, phylogenetic distance, and frequency of multigene transfer. Among other insights, we find that 1) the observed relative frequency of HMGT increases as divergence between genomes increases, 2) HMGTs often have conserved gene functions, and 3) rare genes are frequently acquired through HMGT. We also analyze in detail HMGTs involving the zonula occludens toxin and type III secretion systems. By enabling the systematic inference of HMGTs on a large scale, HoMer will facilitate a more accurate and more complete understanding of HGT and microbial evolution.     

Analysis of the angiotensinogen gene T174M polymorphism in populations of the Volga-Ural region

The method of polymerase chain reaction was used to analyze T174M polymorphism at the angiotensinogen (AGT) gene in a number of populations of the Volga-Ural region, belonging to Finno-Ugric (Komi-Permyaks, Maris, Mordovians, and Udmurts), Turkic (Chuvashes, Tatars, and Bashkirs), and Eastern-Slavic (Russians) ethnic groups. Population-specific patterns of the polymorphic alleles and genotypes frequency distribution were established. Comparison of the results with the literature data on the AGT gene polymorphism in different world populations provided identification of specific trends in the changes of genotype frequency of the AGT gene depending on the ethnicity of the populations.     

A model of population fitness and the maintenance of genetic polymorphism when two ecological patches are available

A model is presented for population fitness and the maintenance of genetic polymorphism. when the environment is subdivided into two ecological patches. Movement of individuals within each geneation is governed by a two-state continuous-time-parameter Markov chain. The outcome of this basic formulation is used to specify the equilibrium gene frequency for two alleles at one locus. The change in gene frequency giving the equilibrium condition is a weighted average of the gene frequency changes appropriate to a coarse-grained and a fine-grained environment. A numerical example is given to illustrate the model.     

Analysis of Deletion Mutations in the PARK2 Gene in Idiopathic Parkinson's Disease

A method for analysis of deletions and duplications of individual exons and groups of exons in the parkin gene (PARK2) in both homozygous and heterozygous states has been developed. The method is based on semiquantitative polymerase chain reaction (PCR). The method has been used for analysis of the frequency of deletions in gene PARK2 in patients with idiopathic Parkinson's disease from Bashkortostan. Two unrelated patients have been found to carry a deletion of the 12th (last) exon of gene PARK2. Possibly, this deletion has caused the disease in the given patients.     

Progeny testing for recessive genes: procedures and interpretations

Summary Methods for calculating the probability of detecting a carrier of a recessive gene by utilizing matings among related individuals are presented for single and litter bearing species. The confidence level for detection of heterozygosity depends upon: (1) the genetic relationship between mates, (2) the number of mates per male and the number of offspring per mate, (3) whether an estimate of recessive gene frequency before selection is available and (4) the magnitude of that frequency. Methods of computing probability of heterozygosity vs homozygosity utilizing Bayes theorem also are presented. In the conventional progeny test method, a sire initially is assumed heterozygous before calculations are made, but no prior information concerning his probable genotype is utilized. In the method using Bayes theorem, prior sources of information from relatives or from estimates of population allele frequency are utilized. This method gives the exact probability that a sire is not a carrier, given prior information and that he produces all normal offspring. These methods could be used in any sexually reproducing species to identify not only detrimental genes but beneficial genes as well.     

Gene CATCHR--gene cloning and tagging for Caenorhabditis elegans using yeast homologous recombination: a novel approach for the analysis of gene expression

Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression.     

Analysis of the Angiotensinogen Gene T174M Polymorphism in Populations of the Volga–Ural Region

The method of polymerase chain reaction was used to analyze T174M polymorphism at the angiotensinogen (AGT) gene in a number of populations of the Volga–Ural region, belonging to Finno–Ugric (Komi-Permyaks, Maris, Mordovians, and Udmurts), Turkic (Chuvashes, Tatars, and Bashkirs), and Eastern-Slavic (Russians) ethnic groups. Population-specific patterns of the polymorphic alleles and genotypes frequency distribution were established. Comparison of the results with the literature data on the AGT gene polymorphism in different world populations provided identification of specific trends in the changes of genotype frequency of the AGT gene depending on the ethnicity of the populations.     

On an extremum principle in the genetical theory of natural selection

Natural selection causes gene frequency changes in a large population leading to genetic evolution over evolutionary time scales. Such gene frequency changes, however, involve an optimizing principle. According to Kimura, such changes, over a short interval of time, occur in a manner such that the increase in population fitness is maximum for a given distance between parent and daughter generation gene frequencies. But according to Ewens, of all gene frequency changes, including those that lead to the same partial increase in mean fitness as the natural selection gene frequency changes, the natural selection values minimize the generalized distance measure between parent and daughter generation gene frequency values. These two optimality principles happen to be mirror images of each other. However, the optimality principles are restricted to the case where the increase in mean fitness is to thefirst order in natural selection gene frequency changes. I show in this paper that, instead of linear approximation to the increase in mean fitness, the treatment can be fairly general, and the exact increase in mean fitness can be considered so as to include the dominance effects of the genes.     

Resonating vector strength: what happens when we vary the “probing” frequency while keeping the spike times fixed

The synchrony vector, whose length stands for the vector strength (VS), is a means to quantify the amount of periodicity in a neuronal response to a given periodic signal, say, the stimulus. One usually chooses the input angular frequency and evaluates the synchrony vector as a weighted sum of exponentials taken at given experimental spike times of the neuronal response in combination with the driving input frequency. Given the experimental spike times, we replace the stimulus frequency by a variable probing frequency, study the synchrony vector in dependence upon this probing frequency, i.e., as a function of the frequency as a real variable, and exhibit both mathematically and experimentally a resonance behavior once the variable frequency is in the neighborhood of the stimulus frequency. Furthermore, a “resonating” VS is shown to be quite useful since one need not know the external frequency but can simply stick to the given spike times and analyze the ensuing resonance as the frequency varies, for example, to determine at the same time a “best” frequency and the corresponding VS. Finally, it is straightforward to determine the corresponding phase originating from, say, a delay as well.     

An evolutionary approach for gene selection and classification of microarray data based on SVM error-bound theories

Microarrays have thousands to tens-of-thousands of gene features, but only a few hundred patient samples are available. The fundamental problem in microarray data analysis is identifying genes whose disruption causes congenital or acquired disease in humans. In this paper, we propose a new evolutionary method that can efficiently select a subset of potentially informative genes for support vector machine (SVM) classifiers. The proposed evolutionary method uses SVM with a given subset of gene features to evaluate the fitness function, and new subsets of features are selected based on the estimates of generalization error of SVMs and frequency of occurrence of the features in the evolutionary approach. Thus, in theory, selected genes reflect to some extent the generalization performance of SVM classifiers. We compare our proposed method with several existing methods and find that the proposed method can obtain better classification accuracy with a smaller number of selected genes than the existing methods.     

Variation of gene conversion and intragenic recombination frequencies in the genome of Ascobolus immersus.

Summary Eighty mutants in 17 ascospore character genes were studied for their conversion patterns. The correlation between conversion pattern and mutagenic origin, previously found in genes b1 and b2 was extended to all the genes studied. Aberrant 4:4 asci were found in most genes irrespective of their conversion frequency. From gene to gene, the conversion frequency showed an almost 100 times variation. The frequency of intragenic recombination also showed sharp variation from gene to gene. The mean conversion frequency and the maximal intragenic recombination frequency were shown to be highly correlated in 5 genes for which these 2 values are known. This correlation was extended to 12 other genes in other Ascomycetes: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora, and Sordaria. From this study it is concluded that, 1) the probability of hybrid DNA formation undergoes considerable changes according to the region of the genome; 2) the intragenic recombination frequency primarily reflects the frequency of hybrid DNA formation rather than the physical length of the gene; 3) for a given physical distance on the DNA, a similar fraction of the gene conversion events lead to recombination in the 5 Ascomycetes.