Systematic genome-wide screens of gene function.
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Animals↗
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Results for “Genomic screening”
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Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray.
Although there have been studies on the toxicity of the pesticide thiuram, the present study is the first one to attempt to integrate a whole genomic response using microarray technology. From the DNA microarray experiment it was found that exposure to thiuram led to alterations of gene expression in yeast cells and that many genes involved in detoxification and stress response were highly induced. The induced genes were classified according to the MIPS yeast database. The induction of genes concerned with folding and proteolysis reflects the protein denaturing and degradation effects of the thiuram treatment The induction of genes involved in redox and defense against reaction oxygen species also suggests that thiuram has other effects, such as oxidative stress. Genes classified for carbohydrate metabolism and energy were also highly induced, and these gene products may play the role of providing the energy for the detoxification mechanism. In addition, in view of the induction of some genes involved in DNA repair, thiuram potentially causes DNA damage. Therefore, as stated in previous reports, thiuram is a potential positive toxic chemical. On the other hand, YKL071W, YCR102C, YLR303W, and YLL057C were selected based on the result of a DNA microarray experiment and used for the promoter activity assay. Thiuram treatment affected the promoter of these genes, indicating that this technique could be used for the selection of biomarker candidates.
Biomarkers↗
Transcendent elements: whole-genome transposon screens and open evolutionary questions.
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Animals↗
A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes.
DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.
Amino Acid Sequence↗
Computational identification of noncoding RNAs in E. coli by comparative genomics.
Some genes produce noncoding transcripts that function directly as structural, regulatory, or even catalytic RNAs [1, 2]. Unlike protein-coding genes, which can be detected as open reading frames with distinctive statistical biases, noncoding RNA (ncRNA) gene sequences have no obvious inherent statistical biases [3]. Thus, genome sequence analyses reveal novel protein-coding genes, but any novel ncRNA genes remain invisible. Here, we describe a computational comparative genomic screen for ncRNA genes. The key idea is to distinguish conserved RNA secondary structures from a background of other conserved sequences using probabilistic models of expected mutational patterns in pairwise sequence alignments. We report the first whole-genome screen for ncRNA genes done with this method, in which we applied it to the "intergenic" spacers of Escherichia coli using comparative sequence data from four related bacteria. Starting from >23,000 conserved interspecies pairwise alignments, the screen predicted 275 candidate structural RNA loci. A sample of 49 candidate loci was assayed experimentally. At least 11 loci expressed small, apparently noncoding RNA transcripts of unknown function. Our computational approach may be used to discover structural ncRNA genes in any genome for which appropriate comparative genome sequence data are available.
Animals↗
Genetics of atopy and asthma: the rationale behind promoter-based candidate gene studies (IL-4 and IL-10).
The genetics of atopy and asthma has become a very interesting area for research. Potential candidate genes identified either by the immunopathogenesis of asthma or bronchial hyperresponsiveness, or uncovered by the whole-genome screen, will lead to new and better ways of diagnosing asthma and, more importantly, the potential for drug discovery related to the products of the candidate genes identified in the various genome screening efforts. The candidate gene approach has been applied to the promoter region of a number of cytokine genes, both within and outside of the human 5q33 cytokine gene cluster. As a prototype for both cytokines, work relating to an interleukin (IL)-4 promoter polymorphism and an IL-10 promoter polymorphism will be reviewed as providing a potential molecular mechanism for dysregulation of these cytokine genes in asthma.
Asthma↗
[Genome-wide screening of transcriptional factors regulating limb development].
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Animals↗
Genome-wide screening for genetic changes in a matched pair of benign and prostate cancer cell lines using array CGH.
Copy number alterations in a matched pair of benign epithelial and prostate cancer cell lines derived from the same patient were assessed using array-based comparative genomic hybridisation (aCGH). The cancer cell line showed a gain of chromosome 7, deletion of chromosome 8, gains (including high level) and losses on chromosome 11, loss of 18p and gain of 20q. Deletions on chromosome 8 were confirmed with microsatellite markers. The aCGH results were compared to gene expression data obtained using DNA microarrays and suggested the involvement of caspases and ICEBERG on 11q and E2F1 on chromosome 20q.
Cell Line, Tumor↗
Genome-wide screen for allelic imbalance in a mouse model for neuroblastoma.
We have used the rat tyrosine hydroxylase promotor to overexpress MYCN in the neural crest of transgenic mice, resulting in a mouse model for neuroblastoma. Using PCR analysis of microsatellite markers, we conducted a genome-wide analysis in tumors from these animals. Regions of chromosomes 1, 3, 10, 11, 14, and 18 were affected in 20-50% of tumors. Analysis of a subset of these tumors by comparative genomic hybridization was consistent with the microsatellite data. The changes on mouse chromosomes 1, 11, 14, and 18 were syntenic with corresponding regions of loss of heterozygosity in human neuroblastoma, suggesting that genes implicated in the mouse tumors may also play a role in the pathogenesis of the human disease. One-third of the mouse tumors shared abnormalities on chromosomes 1, 3, and 10, whereas the remainder of tumors did not show this combination. These data suggest that genetic mutations on chromosomes 1, 3, and 10 cooperate in the pathogenesis of neuroblastoma and that neuroblastoma in the mouse arises from at least two distinct genetic pathways, one of which is dependent on lesions in chromosomes 1, 3, and 10, the other of which is not.
Animals↗
Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast.
BACKGROUND: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. RESULTS: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. CONCLUSION: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.
Adaptation, Biological↗
Multiple susceptibility loci for multiple sclerosis.
Multiple sclerosis (MS) is a common and frequently disabling autoimmune disorder mediated by autoaggressive T cells and autoantibodies that target central nervous system myelin. While numerous studies have demonstrated a strong genetic component to MS, it has been difficult to identify the specific genes involved. Several genomic screens have been undertaken to locate such genes, but have not provided consistent gene localization, except for the MHC on chromosome 6p21 and a locus on chromosome 19q13. To determine which of the original genomic locations presented in the US genome screen could be replicated, a more detailed analysis of additional families was performed. The results, derived from a population of 266 affected individuals belonging to 98 multiplex families, continue to support linkage to chromosomes 6p21, 6q27, and 19q13 with LOD scores>3.0, and suggest that regions on chromosomes 12q23-24 and 16p13 may also harbor susceptibility loci for MS. Analysis taking into account the known HLA-DR2 association identified two additional potential linkage regions on chromosomes 7q21-22 and 13q33-34. These regions can now be targeted for detailed study to identify the underlying MS susceptibility genes.
Chromosomes, Human, Pair 13↗
Vector-based RNAi approaches for stable, inducible and genome-wide screens.
RNA interference (RNAi) has revolutionized the study of biology and offers numerous applications in basic biology as well as in drug discovery research. Since the discovery of RNAi, several tools have been developed to enable loss-of-function studies in mammalian systems. The efficacy of RNAi is dependent on specific and versatile RNAi triggers that have evolved to enable transient, stable and in-vivo applications. Recently developed genome-wide short hairpin RNA (shRNA) and microRNA-adapted short hairpin RNA (shRNAmir) libraries incorporate advances in shRNA design and molecular 'barcodes' to enable more complex RNAi screens and the opportunity to progress to more complex genetics in whole animals.
Animals↗
Genetic susceptibility to MS: a second stage analysis in Canadian MS families.
Four published genome screens have identified a number of markers with increased sharing in multiple sclerosis (MS) families, although none has reached statistical significance. One hundred and five markers previously identified as showing increased sharing in Canadian, British, Finnish, and American genome screens were genotyped in 219 sibling pairs ascertained from the database of the Canadian Collaborative Project on Genetic Susceptibility to MS (CCPGSMS). No markers examined met criteria for significant linkage. Markers located at 5p14 and 17q22 were analyzed in a total of 333 sibling pairs and attained mlod scores of 2.27 and 1.14, respectively. The known HLA Class II DRB1 association with MS was confirmed (P<0.0001). Significant transmission disequilibrium was also observed for D17S789 at 17q22 (P=0.0015). This study highlights the difficulty of searching for genes with only mild-to-moderate effects on susceptibility, although large effects of specific loci may still be present in individual families. Future progress in the genetics of this complex trait may be helped by (1) focussing on more ethnically homogeneous samples, (2) using an increased number of MS families, and (3) using transmission disequilibrium analysis in candidate regions rather than the affected relative pair linkage analysis.
Canada↗
Screening of genomic DNA to identify mutations in the gene for Bruton's tyrosine kinase.
Mutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen PCR products from 30 unrelated families presumed to carry a Btk mutation. It was possible to amplify DNA in every reaction from every patient, indicating that large deletions in Btk are uncommon. Twenty three different mutations were found in 25 unrelated families, including one family in whom DNA was available from a carrier but not an affected patient. Seven mutations were single base pair substitutions resulting in premature stop codons scattered throughout the gene. Small insertions or deletions causing frameshifts and secondary premature stop codons constituted an additional seven mutations. One patient had a point mutation in the start codon and one patient had a mutation in a splice donor site. Point mutations resulting in amino acid substitutions were seen in nine patients. Northern blot analysis of RNA from three patients with premature stop codons showed an absence of Btk transcript whereas four patients with amino acid substitutions had normal amounts of transcript of normal size. These studies document the considerable variability in the Btk mutations causing XLA and they demonstrate an approach that will be useful for carrier detection as well as mutation identification.
Agammaglobulinaemia Tyrosine Kinase↗
No evidence for association with Parkinson disease for 13 single-nucleotide polymorphisms identified by whole-genome association screening.
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Adult↗
Scan statistics to scan markers for susceptibility genes.
Scan statistics are applied to combine information on multiple contiguous genetic markers used in a genome screen for susceptibility loci. This information may be, for example, allele sharing proportions for sib pairs or logarithm of odds (lod) scores in general small families. We focus on a dichotomous outcome variable, for example, case and control individuals or affected-affected versus affected-unaffected siblings, and suitable single-marker statistics. A significant scan statistic based on the single-marker statistics represents evidence of the presence of a susceptibility gene. For a given length of the scan statistic, we assess its significance by Monte Carlo permutation tests. Comparing P values for varying lengths of scan statistics, we treat the smallest observed P value as our statistic of interest and determine its overall significance level. We applied this method to a genome screen with autism families. The result was informative and surprising: A susceptibility region was found (genome-wide significance level, P = 0.038), which is missed with conventional approaches.
Alleles↗
Mapping susceptibility genes for asthma and allergy.
Allergy and asthma are related conditions caused by a complex interaction of genetic factors and environmental influences. With family data from several different populations, linkage analysis has been performed and used to identify regions of the genome that contain susceptibility genes for these conditions. To date, 4 genome screens have been completed and have successfully identified several chromosomal locations that are likely to contain asthma and allergy genes. Many of these regions contain potential biologic candidate genes that modulate immunologic responses or airways inflammation. By focusing on the common regions that have been replicated in these 4 genome screens, the major susceptibility genes for asthma and allergy should be identified. This will lead to an improved understanding of pathogenic factors that lead to development or progression of asthma and allergic diseases.
Asthma↗
New locus for autosomal dominant high myopia maps to the long arm of chromosome 17.
PURPOSE: To map the gene(s) associated with autosomal dominant (AD) high-grade myopia. METHODS: A multigeneration English/Canadian family with AD severe myopia was ascertained. Myopes were healthy, with no clinical evidence of syndromic disease, anterior segment abnormalities, or glaucoma. The family contained 22 participating members (12 affected). The average age of diagnosis of myopia was 8.9 years (range, birth to 11 years). The average refractive error for affected adults was -13.925 D (range, -5.50 to -50.00). Microsatellite markers for genotyping were used to assess linkage to several candidate loci, including three previously identified AD high-myopia loci on 18p11.31, 12q22-q23, and 7q36. Syndromic myopia linkage was excluded by using intragenic or flanking markers for Stickler syndrome types 1, 2, and 2B; Marfan syndrome; Ehlers-Danlos syndrome type 4; and juvenile glaucoma. A full genome screening was performed, with 327 microsatellite markers spaced by 5 to 10 cM. Two-point linkage was analyzed using the FASTLINK program run at 90% penetrance and a myopia gene frequency of 0.0133. RESULTS: Linkage to all candidate loci was excluded. The genome screening yielded a maximum two-point lod score of 3.17 at theta = 0 with microsatellite marker D17S1604. Fine mapping and haplotype analysis defined the critical interval of 7.71 cM at 17q21-22. CONCLUSIONS: A novel putative disease locus for AD high-grade myopia has been identified and provides additional support for genetic heterogeneity for this disorder.
Child↗