The use of the maharanobis and modified distances for the improvement of simulation of glutamic acid production

A modified simulation procedure based on a statistical approach was investigated. The procedure predicts the time course of fed-batch culture for glutamic acid production by a temperature-sensitive strain of Brevibacterium flavum. The statistical approach requires only a data base of state points obtained in experiments, and not perfect identification of fermentation models. The simulation procedure is based on regression analysis to estimate specific rate parameters of system equations using the data points selected with reference to the Euclid distance. It was modified in that the data selection procedure included the use of the Maharanobis distance as well as a modified distance defined in this study. Simulation results using the modified procedure allow reasonable prediction of the time course of fed-batch culture for glutamic acid compared to that involving the Euclid distance alone.     

A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants

We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.     

Automated metaphase finding: an assessment of the efficiency of the METAFER2 system in a routine mutagenicity assay

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparation from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure.METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-staines second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.     

High-throughput fluorescence-based isolation of live C. elegans larvae

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.     

Empirical Bayes procedure for estimating genetic distance between populations and effective population size

We developed an empirical Bayes procedure to estimate genetic distances between populations using allele frequencies. This procedure makes it possible to describe the skewness of the genetic distance while taking full account of the uncertainty of the sample allele frequencies. Dirichlet priors of the allele frequencies are specified, and the posterior distributions of the various composite parameters are obtained by Monte Carlo simulation. To avoid overdependence on subjective priors, we adopt a hierarchical model and estimate hyperparameters by maximizing the joint marginal-likelihood function. Taking advantage of the empirical Bayesian procedure, we extend the method to estimate the effective population size using temporal changes in allele frequencies. The method is applied to data sets on red sea bream, herring, northern pike, and ayu broodstock. It is shown that overdispersion overestimates the genetic distance and underestimates the effective population size, if it is not taken into account during the analysis. The joint marginal-likelihood function also estimates the rate of gene flow into island populations.     

On the distribution of temporal variations in allele frequency: consequences for the estimation of effective population size and the detection of loci undergoing selection

The effective population size (Ne) is frequently estimated using temporal changes in allele frequencies at neutral markers. Such temporal changes in allele frequencies are usually estimated from the standardized variance in allele frequencies (Fc). We simulate Wright-Fisher populations to generate expected distributions of Fc and of Fc (Fc averaged over several loci). We explore the adjustment of these simulated Fc distributions to a chi-square distribution and evaluate the resulting precision on the estimation of Ne for various scenarios. Next, we outline a procedure to test for the homogeneity of the individual Fc across loci and identify markers exhibiting extreme Fc-values compared to the rest of the genome. Such loci are likely to be in genomic areas undergoing selection, driving Fc to values greater (or smaller) than expected under drift alone. Our procedure assigns a P-value to each locus under the null hypothesis (drift is homogeneous throughout the genome) and simultaneously controls the rate of false positive among loci declared as departing significantly from the null. The procedure is illustrated using two published data sets: (i) an experimental wheat population subject to natural selection and (ii) a maize population undergoing recurrent selection.     

General method for cloning Neurospora crassa nuclear genes by complementation of mutants.

We have developed a sib selection procedure for cloning Neurospora crassa nuclear genes by complementation of mutants. This procedure takes advantage of a modified N. crassa transformation procedure that gives as many as 10,000 to 50,000 stable transformants per microgram of DNA with recombinant plasmids containing the N. crassa qa-2+ gene. Here, we describe the use of the sib selection procedure to clone genes corresponding to auxotrophic mutants, nic-1 and inl. The identities of the putative clones were confirmed by mapping their chromosomal locations in standard genetic crosses and using restriction site polymorphisms as genetic markers. Because we can obtain very high N. crassa transformation frequencies, cloning can be accomplished with as few as five subdivisions of an N. crassa genomic library. The sib selection procedure should, for the first time, permit the cloning of any gene corresponding to an N. crassa mutant for which an appropriate selection can be devised. Analogous procedures may be applicable to other filamentous fungi before the development of operational shuttle vectors.     

Palmar pattern frequencies as indicators of relationships among Sardinian linguistic groups of males

Palmar pattern frequencies were used to calculate distance coefficients between Sardinian linguistic groups of males for the purpose of verifying, by means of correlation matrix analyses, whether or not the dermatoglyphic traits considered lead to a reliable identification of the biological relationships on the basis of the linguistic backgrounds of these groups. With Sanghvi's as the distance measure and by using palmar pattern frequencies in the Hy area, Th/I, II, III, and IV interdigital areas and all traits together for palms combined or separated were calculated dermatoglyphic distance measures. Mantel tests of matrix correspondence showed that, by using palmar pattern frequencies in the Th/I interdigital area (palms combined), in the II, III, and IV interdigital areas, or all traits together for palms combined and separated, statistical significance between dermatoglyphic and linguistic distances can be obtained, even when the effect of geography is removed; there is no statistically significant correspondence between geographic and dermatoglyphic distance matrices, even when the effect of language is removed. The results obtained in this study by means of the Mantel test procedure demonstrate that the dermatoglyphic traits analyzed, with the exception of palmar pattern frequencies in the Hy area and in the Th/I interdigital area for plams separated when these areas are used singly, can be considered as a good set of variables to use in finding biological relationships between Sardinian linguistic groups of males examined on the basis of their linguistic backgrounds.     

Increasing specificity in high-throughput yeast two-hybrid experiments

Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions. However, Y2H screens are sometimes criticized for generating high rates of false-positives. Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H. Here, we summarize various approaches that reduce false-positives in HT-Y2H projects. We evaluated the potential of examining putative positives after removing the prey encoding plasmid by negative selection. We found that this method reliably identifies false-positives caused by spontaneous conversion of baits into auto-activators and provides significant time-savings in HT screens. In addition, we present a method to eliminate an important source of false-positives: contaminating prey plasmids. Y2H interactors can be wrongly identified due to the presence of two or more different plasmids in the cells of a single yeast colony. Of these independent plasmids, only one encodes a genuine interactor. Contaminating plasmids are eliminated by extended culture of yeast cells under positive selection for the interaction, allowing the identification of the true interaction partner.     

Genetics of P-element transposition into Drosophila melanogaster centric heterochromatin

Heterochromatin is a major component of higher eukaryotic genomes, but progress in understanding the molecular structure and composition of heterochromatin has lagged behind the production of relatively complete euchromatic genome sequences. The introduction of single-copy molecular-genetic entry points can greatly facilitate structure and sequence analysis of heterochromatic regions that are rich in repeated DNA. In this study, we report the isolation of 502 new P-element insertions into Drosophila melanogaster centric heterochromatin, generated in nine different genetic screens that relied on mosaic silencing (position-effect variegation, or PEV) of the yellow gene present in the transposon. The highest frequencies of recovery of variegating insertions were observed when centric insertions were used as the source for mobilization. We propose that the increased recovery of variegating insertions from heterochromatic starting sites may result from the physical proximity of different heterochromatic regions in germline nuclei or from the association of mobilizing elements with heterochromatin proteins. High frequencies of variegating insertions were also recovered when a potent suppressor of PEV (an extra Y chromosome) was present in both the mobilization and selection generations, presumably due to the effects of chromatin structure on P-element mobilization, insertion, and phenotypic selection. Finally, fewer variegating insertions were recovered after mobilization in females, in comparison to males, which may reflect differences in heterochromatin structure in the female and male germlines. FISH localization of a subset of the insertions confirmed that 98% of the variegating lines contain heterochromatic insertions and that these schemes produce a broader distribution of insertion sites. The results of these schemes have identified the most efficient methods for generating centric heterochromatin P insertions. In addition, the large collection of insertions produced by these screens provides molecular-genetic entry points for mapping, sequencing, and functional analysis of Drosophila heterochromatin.     

The carcinoGENOMICS project: critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.     

The complete automation of cell culture: improvements for high-throughput and high-content screening

Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. High-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other "omic" approaches into networks and can be used for the identification of therapeutic targets. Current methods used for HT-HC screens are laborious, time-consuming, and prone to human error. The authors thus developed an automated high-throughput system with an integrated fluorescent imager for HC screens called the AI.CELLHOST. The implementation of user-defined culturing and assay plate setup parameters allows parallel operation of multiple screens in diverse mammalian cell types. The authors demonstrate that such a system is able to successfully maintain different cell lines in culture for extended periods of time as well as significantly increasing throughput, accuracy, and reproducibility of HT and HC screens.     

Evolutionary distances for protein-coding sequences: modeling site- specific residue frequencies

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon- level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.      

Computational identification of insertional mutagenesis targets for cancer gene discovery

Insertional mutagenesis is a potent forward genetic screening technique used to identify candidate cancer genes in mouse model systems. An important, yet unresolved issue in the analysis of these screens, is the identification of the genes affected by the insertions. To address this, we developed Kernel Convolved Rule Based Mapping (KC-RBM). KC-RBM exploits distance, orientation and insertion density across tumors to automatically map integration sites to target genes. We perform the first genome-wide evaluation of the association of insertion occurrences with aberrant gene expression of the predicted targets in both retroviral and transposon data sets. We demonstrate the efficiency of KC-RBM by showing its superior performance over existing approaches in recovering true positives from a list of independently, manually curated cancer genes. The results of this work will significantly enhance the accuracy and speed of cancer gene discovery in forward genetic screens. KC-RBM is available as R-package.     

Development and application of a DNA microarray-based yeast two-hybrid system

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.     

Identifying signatures of sexual selection using genomewide selection components analysis

Sexual selection must affect the genome for it to have an evolutionary impact, yet signatures of selection remain elusive. Here we use an individual‐based model to investigate the utility of genome‐wide selection components analysis, which compares allele frequencies of individuals at different life history stages within a single population to detect selection without requiring a priori knowledge of traits under selection. We modeled a diploid, sexually reproducing population and introduced strong mate choice on a quantitative trait to simulate sexual selection. Genome‐wide allele frequencies in adults and offspring were compared using weighted F_ST values. The average number of outlier peaks (i.e., those with significantly large F_ST values) with a quantitative trait locus in close proximity (“real” peaks) represented correct diagnoses of loci under selection, whereas peaks above the F_ST significance threshold without a quantitative trait locus reflected spurious peaks. We found that, even with moderate sample sizes, signatures of strong sexual selection were detectable, but larger sample sizes improved detection rates. The model was better able to detect selection with more neutral markers, and when quantitative trait loci and neutral markers were distributed across multiple chromosomes. Although environmental variation decreased detection rates, the identification of real peaks nevertheless remained feasible. We also found that detection rates can be improved by sampling multiple populations experiencing similar selection regimes. In short, genome‐wide selection components analysis is a challenging but feasible approach for the identification of regions of the genome under selection.     

Fluorescence-based phenotypic selection allows forward genetic screens in haploid human cells

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.     

Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends

Recent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53 000 clones tested using naïve screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high‐throughput expression for subsequent production and characterization. The pre‐screening of clone libraries by naïve screens followed by the pyrosequencing of the inserts allowed for a 106‐fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time‐consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine‐tuned naïve‐ and sequence‐based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market.     

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions.