Bi-Directional SIFT Predicts a Subset of Activating Mutations

Advancements in sequencing technologies have empowered recent efforts to identify polymorphisms and mutations on a global scale. The large number of variations and mutations found in these projects requires high-throughput tools to identify those that are most likely to have an impact on function. Numerous computational tools exist for predicting which mutations are likely to be functional, but none that specifically attempt to identify mutations that result in hyperactivation or gain-of-function. Here we present a modified version of the SIFT (Sorting Intolerant from Tolerant) algorithm that utilizes protein sequence alignments with homologous sequences to identify functional mutations based on evolutionary fitness. We show that this bi-directional SIFT (B-SIFT) is capable of identifying experimentally verified activating mutants from multiple datasets. B-SIFT analysis of large-scale cancer genotyping data identified potential activating mutations, some of which we have provided detailed structural evidence to support. B-SIFT could prove to be a valuable tool for efforts in protein engineering as well as in identification of functional mutations in cancer.     

Simple Quantitative PCR Approach to Reveal Naturally Occurring and Mutation-Induced Repetitive Sequence Variation on the Drosophila Y Chromosome

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.     

Screens, maps & networks: from genome sequences to personalized medicine

Genome sequencing of tumors provides a wealth of information on mutations and structural variations, instilling hope that this data can be used to predict individual tumor progression and response to treatment. Yet currently, our ability to predict the functional consequences of these aberrations remains poor. How do cancer-associated mutations give rise to the hallmark phenotypes of cancer? Recently, information about the genetic makeup of cancer cells has been combined with novel functional genomics approaches to identify novel targets, exploit synthetic lethality and explore the rewiring of cellular pathways. Here, we highlight recent developments revealing the hidden landscape of genetic interactions in model organisms and cancer cells, a key step toward personalized cancer diagnostics and therapy.     

High-resolution mutation mapping reveals parallel experimental evolution in yeast

Understanding the genetic basis of evolutionary adaptation is limited by our ability to efficiently identify the genomic locations of adaptive mutations. Here we describe a method that can quickly and precisely map the genetic basis of naturally and experimentally evolved complex traits using linkage analysis. A yeast strain that expresses the evolved trait is crossed to a distinct strain background and DNA from a large pool of progeny that express the trait of interest is hybridized to oligonucleotide microarrays that detect thousands of polymorphisms between the two strains. Adaptive mutations are detected by linkage to the polymorphisms from the evolved parent. We successfully tested our method by mapping five known genes to a precision of 0.2–24 kb (0.1–10 cM), and developed computer simulations to test the effect of different factors on mapping precision. We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose–galactose environment. All four strains had acquired one or more missense mutations in GAL80, the repressor of the galactose utilization pathway. When transferred into the ancestral strain, the gal80 mutations conferred the fitness advantage that the evolved strains show in the transition from glucose to galactose. Our results show an example of parallel adaptation caused by mutations in the same gene.     

Two-dimensional DNA displays for comparisons of bacterial genomes

We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms.First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of >800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason asecond method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA. Published: June 15, 2003     

Use of model organisms for the study of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses are a group of fatal progressive neurodegenerative diseases predominantly affecting children. Identification of mutations that cause neuronal ceroid lipofuscinosis, and subsequent functional and pathological studies of the affected genes, underpins efforts to investigate disease mechanisms and identify and test potential therapeutic strategies. These functional studies and pre-clinical trials necessitate the use of model organisms in addition to cell and tissue culture models as they enable the study of protein function within a complex organ such as the brain and the testing of therapies on a whole organism. To this end, a large number of disease models and genetic tools have been identified or created in a variety of model organisms. In this review, we will discuss the ethical issues associated with experiments using model organisms, the factors underlying the choice of model organism, the disease models and genetic tools available, and the contributions of those disease models and tools to neuronal ceroid lipofuscinosis research. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.     

Diversification of DNA sequences in the symbiotic genome of Rhizobium etli

Bacteria of the genus Rhizobium and related genera establish nitrogen-fixing symbioses with the roots of leguminous plants. The genetic elements that participate in the symbiotic process are usually compartmentalized in the genome, either as independent replicons (symbiotic plasmids) or as symbiotic regions or islands in the chromosome. The complete nucleotide sequence of the symbiotic plasmid of Rhizobium etli model strain CFN42, symbiont of the common bean plant, has been reported. To better understand the basis of DNA sequence diversification of this symbiotic compartment, we analyzed the distribution of single-nucleotide polymorphisms in homologous regions from different Rhizobium etli strains. The distribution of polymorphisms is highly asymmetric in each of the different strains, alternating regions containing very few changes with regions harboring an elevated number of substitutions. The regions showing high polymorphism do not correspond with discrete genetic elements and are not the same in the different strains, indicating that they are not hypervariable regions of functional genes. Most interesting, some highly polymorphic regions share exactly the same nucleotide substitutions in more than one strain. Furthermore, in different regions of the symbiotic compartment, different sets of strains share the same substitutions. The data indicate that the majority of nucleotide substitutions are spread in the population by recombination and that the contribution of new mutations to polymorphism is relatively low. We propose that the horizontal transfer of homologous DNA segments among closely related organisms is a major source of genomic diversification.     

A catalog of neutral and deleterious polymorphism in yeast

The abundance and identity of functional variation segregating in natural populations is paramount to dissecting the molecular basis of quantitative traits as well as human genetic diseases. Genome sequencing of multiple organisms of the same species provides an efficient means of cataloging rearrangements, insertion, or deletion polymorphisms (InDels) and single-nucleotide polymorphisms (SNPs). While inbreeding depression and heterosis imply that a substantial amount of polymorphism is deleterious, distinguishing deleterious from neutral polymorphism remains a significant challenge. To identify deleterious and neutral DNA sequence variation within Saccharomyces cerevisiae, we sequenced the genome of a vineyard and oak tree strain and compared them to a reference genome. Among these three strains, 6% of the genome is variable, mostly attributable to variation in genome content that results from large InDels. Out of the 88,000 polymorphisms identified, 93% are SNPs and a small but significant fraction can be attributed to recent interspecific introgression and ectopic gene conversion. In comparison to the reference genome, there is substantial evidence for functional variation in gene content and structure that results from large InDels, frame-shifts, and polymorphic start and stop codons. Comparison of polymorphism to divergence reveals scant evidence for positive selection but an abundance of evidence for deleterious SNPs. We estimate that 12% of coding and 7% of noncoding SNPs are deleterious. Based on divergence among 11 yeast species, we identified 1,666 nonsynonymous SNPs that disrupt conserved amino acids and 1,863 noncoding SNPs that disrupt conserved noncoding motifs. The deleterious coding SNPs include those known to affect quantitative traits, and a subset of the deleterious noncoding SNPs occurs in the promoters of genes that show allele-specific expression, implying that some cis-regulatory SNPs are deleterious. Our results show that the genome sequences of both closely and distantly related species provide a means of identifying deleterious polymorphisms that disrupt functionally conserved coding and noncoding sequences.     

Computational analysis of human genome polymorphism

While genome-era technologies focused on complete genome sequencing in various organisms, post-genome technologies aim at the understanding of the mechanisms of genetic information processing and elucidation of within-species variation. Single nucleotide polymorphisms (SNPs) are the most common source of genome variation in the human population. Nonsynonymous SNPs that occur in coding gene regions and result in amino acid substitutions are of particular interest. It is thought that such SNPs are responsible for phenotypic variation, quantitative traits, and the etiology of common diseases. PolyPhen is a computational tool for the prediction of putatively functional nonsynonymous SNPs by combining information of various types. The application areas of PolyPhen and similar methods include the genetics of complex diseases and congenital defects, the identification of functional mutations in model organisms, and evolutionary genetics.     

High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies

Whole-genome sequencing is a powerful technique for obtaining the reference sequence information of multiple organisms. Its use can be dramatically expanded to rapidly identify genomic variations, which can be linked with phenotypes to obtain biological insights. We explored these potential applications using the emerging next-generation sequencing platform Solexa Genome Analyzer, and the well-characterized model bacterium Bacillus subtilis. Combining sequencing with experimental verification, we first improved the accuracy of the published sequence of the B. subtilis reference strain 168, then obtained sequences of multiple related laboratory strains and different isolates of each strain. This provides a framework for comparing the divergence between different laboratory strains and between their individual isolates. We also demonstrated the power of Solexa sequencing by using its results to predict a defect in the citrate signal transduction pathway of a common laboratory strain, which we verified experimentally. Finally, we examined the molecular nature of spontaneously generated mutations that suppress the growth defect caused by deletion of the stringent response mediator relA. Using whole-genome sequencing, we rapidly mapped these suppressor mutations to two small homologs of relA. Interestingly, stable suppressor strains had mutations in both genes, with each mutation alone partially relieving the relA growth defect. This supports an intriguing three-locus interaction module that is not easily identifiable through traditional suppressor mapping. We conclude that whole-genome sequencing can drastically accelerate the identification of suppressor mutations and complex genetic interactions, and it can be applied as a standard tool to investigate the genetic traits of model organisms.     

Genome sequence of Bacillus subtilis subsp. spizizenii gtP20b, isolated from the Indian ocean

Bacillus subtilis is an aerobic spore-forming Gram-positive bacterium that is a model organism and of great industrial significance as the source of diverse novel functional molecules. Here we present, to our knowledge, the first genome sequence of Bacillus subtilis strain gtP20b isolated from the marine environment. A subset of candidate genes and gene clusters were identified, which are potentially involved in production of diverse functional molecules, like novel ribosomal and nonribosomal antimicrobial peptides. The genome sequence described in this paper is due to its high strain specificity of great importance for basic as well as applied researches on marine organisms.     

Molecular hyperdiversity and evolution in very large populations

The genomic density of sequence polymorphisms critically affects the sensitivity of inferences about ongoing sequence evolution, function and demographic history. Most animal and plant genomes have relatively low densities of polymorphisms, but some species are hyperdiverse with neutral nucleotide heterozygosity exceeding 5%. Eukaryotes with extremely large populations, mimicking bacterial and viral populations, present novel opportunities for studying molecular evolution in sexually reproducing taxa with complex development. In particular, hyperdiverse species can help answer controversial questions about the evolution of genome complexity, the limits of natural selection, modes of adaptation and subtleties of the mutation process. However, such systems have some inherent complications and here we identify topics in need of theoretical developments. Close relatives of the model organisms Caenorhabditis elegans and Drosophila melanogaster provide known examples of hyperdiverse eukaryotes, encouraging functional dissection of resulting molecular evolutionary patterns. We recommend how best to exploit hyperdiverse populations for analysis, for example, in quantifying the impact of noncrossover recombination in genomes and for determining the identity and micro‐evolutionary selective pressures on noncoding regulatory elements.     

Reverse PCA,a Systematic Approach for Identifying Genes Important for the Physical Interaction between Protein Pairs

Protein-protein interactions (PPIs) are of central importance for many areas of biological research. Several complementary high-throughput technologies have been developed to study PPIs. The wealth of information that emerged from these technologies led to the first maps of the protein interactomes of several model organisms. Many changes can occur in protein complexes as a result of genetic and biochemical perturbations. In the absence of a suitable assay, such changes are difficult to identify, and thus have been poorly characterized. In this study, we present a novel genetic approach (termed “reverse PCA”) that allows the identification of genes whose products are required for the physical interaction between two given proteins. Our assay starts with a yeast strain in which the interaction between two proteins of interest can be detected by resistance to the drug, methotrexate, in the context of the protein-fragment complementation assay (PCA). Using synthetic genetic array (SGA) technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify those mutations that disrupt the physical interaction of interest. We were able to successfully validate this novel approach by identifying mutants that dissociate the conserved interaction between Cia2 and Mms19, two proteins involved in Iron-Sulfur protein biogenesis and genome stability. This method will facilitate the study of protein structure-function relationships, and may help in elucidating the mechanisms that regulate PPIs.     

SNP discovery in nonmodel organisms: strand bias and base‐substitution errors reduce conversion rates

Single nucleotide polymorphisms (SNPs) have become the marker of choice for genetic studies in organisms of conservation, commercial or biological interest. Most SNP discovery projects in nonmodel organisms apply a strategy for identifying putative SNPs based on filtering rules that account for random sequencing errors. Here, we analyse data used to develop 4723 novel SNPs for the commercially important deep‐sea fish, orange roughy (Hoplostethus atlanticus), to assess the impact of not accounting for systematic sequencing errors when filtering identified polymorphisms when discovering SNPs. We used SAMtools to identify polymorphisms in a velvet assembly of genomic DNA sequence data from seven individuals. The resulting set of polymorphisms were filtered to minimize ‘bycatch’—polymorphisms caused by sequencing or assembly error. An Illumina Infinium SNP chip was used to genotype a final set of 7714 polymorphisms across 1734 individuals. Five predictors were examined for their effect on the probability of obtaining an assayable SNP: depth of coverage, number of reads that support a variant, polymorphism type (e.g. A/C), strand‐bias and Illumina SNP probe design score. Our results indicate that filtering out systematic sequencing errors could substantially improve the efficiency of SNP discovery. We show that BLASTX can be used as an efficient tool to identify single‐copy genomic regions in the absence of a reference genome. The results have implications for research aiming to identify assayable SNPs and build SNP genotyping assays for nonmodel organisms.     

TILLING在水稻育种中的应用前景

TILLING(Targeting Induced Local Lesions in Genomes)是功能基因组研究中应用的一种反向遗传学技术。它能高通量低成本地在EMS诱变群体中鉴定出发生在特定基因上的点突变。在其基础上发展出的EcoTILLING技术则可发现种质资源中的SNP位点及小插入或缺失多态性位点。水稻是非常重要的粮食作物, 也是已经完成了全基因组序列测定,有丰富的生物信息学资源可以利用的基因组研究模式植物。水稻的分子标记辅助育种将在育种中扮演越来越重要的角色。在这样的背景下,本文从基于特定基因的种质资源鉴定、EMS诱变育种、及水稻功能标记开发等方面论述了其在水稻育种中的应用前景。     

From mouse to man: generating megabase chromosome rearrangements

Experimental approaches for deciphering the function of human genes rely heavily on our ability to generate mutations in model organisms such as the mouse. However, because recessive mutations are masked by the wild-type allele in the diploid context, conventional mutagenesis and screening is often laborious and costly. Chromosome engineering combines the power of gene targeting in embryonic stem (ES) cells with Cre--loxP technology to create mice that are functionally haploid in discrete portions of the genome. Chromosome deletions, duplications and inversions can be tagged with visible markers, facilitating strain maintenance. These approaches allow for more refined mutagenesis screens that will greatly accelerate functional mouse genomics and generate mammalian models for developmental processes and cancer.