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1.
Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability. 相似文献
2.
Discovery of induced point mutations in maize genes by TILLING 总被引:4,自引:0,他引:4
Bradley J Till Steven H Reynolds Clifford Weil Nathan Springer Chris Burtner Kim Young Elisabeth Bowers Christine A Codomo Linda C Enns Anthony R Odden Elizabeth A Greene Luca Comai Steven Henikoff 《BMC plant biology》2004,4(1):1-8
Background
Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.Results
We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.Conclusions
Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project. 相似文献3.
Functional genomics tools for the analysis of zebrafish pigment 总被引:3,自引:0,他引:3
Pickart MA Sivasubbu S Nielsen AL Shriram S King RA Ekker SC 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2004,17(5):461-470
Genetic model organisms are increasingly valuable in the post-genomics era to provide a basis for comparative analysis of the human genome. For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice. A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g. neural crest development. The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease. Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research. An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model. Morpholino phosphorodiamidate oligonucleotide (MO) 'knockdown' of pigment gene expression provides a non-conventional antisense tool for the analysis of genes involved in pigment cell biology and disease. In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology. Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come. 相似文献
4.
We have developed a new community resource, called the WiscDsLox collection, for performing reverse-genetic analysis in arabidopsis.
This resource is composed of 10,459 T-DNA lines generated using the Arabidopsis thaliana ecotype Columbia. The flanking sequence tag for each T-DNA insertion has been deposited in public databases, and seed for
each line is currently available from the Arabidopsis Biological Resource Center. The pDsLox vector used to create this new population contains a Ds transposon and Cre/Lox recombination sites. Each WiscDsLox line therefore has the potential to serve as a launch-pad for performing local saturation
mutagenesis by mobilization of the Ds element. In addition, Cre-Lox recombination between the T-DNA and a transposed Ds element should enable targeted deletion of specific genomic regions. We generated the WiscDsLox collection using an improved
high-throughput pipeline that streamlines analysis of large numbers of independent Arabidopsis thaliana (L.) Hyenh. lines. In this paper we describe the details of this novel method and also provide potential users of WiscDsLox
T-DNA lines with useful background information about this collection. Experiments to characterize the utility of the Ds transposon and Cre/Lox elements present in the WiscDsLox lines are in progress and will be reported in the future. 相似文献
5.
S Wray M Self;NINDS Parkinson's Disease iPSC Consortium;NINDS Huntington's Disease iPSC Consortium;NINDS ALS iPSC Consortium PA Lewis JW Taanman NS Ryan CJ Mahoney Y Liang MJ Devine UM Sheerin H Houlden HR Morris D Healy JF Marti-Masso E Preza S Barker M Sutherland RA Corriveau M D'Andrea AH Schapira RJ Uitti M Guttman G Opala B Jasinska-Myga A Puschmann C Nilsson AJ Espay J Slawek L Gutmann BF Boeve K Boylan AJ Stoessl OA Ross NJ Maragakis J Van Gerpen M Gerstenhaber K Gwinn TM Dawson 《PloS one》2012,7(8):e43099
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community. 相似文献
6.
Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis 总被引:29,自引:0,他引:29
Greene EA Codomo CA Taylor NE Henikoff JG Till BJ Reynolds SH Enns LC Burtner C Johnson JE Odden AR Comai L Henikoff S 《Genetics》2003,164(2):731-740
Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of approximately 1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, >99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage. 相似文献
7.
The Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster 总被引:1,自引:0,他引:1
The majority of genes of multicellular organisms encode proteins with functions that are not required for viability but contribute to important physiological functions such as behavior and reproduction. It is estimated that 75% of the genes of Drosophila melanogaster are nonessential. Here we report on a strategy used to establish a large collection of stocks that is suitable for the recovery of mutations in such genes. From approximately 72,000 F(3) cultures segregating for autosomes heavily treated with ethyl methanesulfonate (EMS), approximately 12,000 lines in which the treated second or third chromosome survived in homozygous condition were selected. The dose of EMS induced an estimated rate of 1.2-1.5 x 10(-3) mutations/gene and predicts five to six nonessential gene mutations per chromosome and seven to nine alleles per locus in the samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic mutations induced in the initial exposure to the mutagen, many of the lines are segregating or are now fixed for lethal mutations on the mutagenized chromosome. The features of this collection, known as the Zuker collection, make it a valuable resource for forward and reverse genetic screens for mutations affecting a wide array of biological functions. 相似文献
8.
Coupled mutagenesis screens and genetic mapping in zebrafish 总被引:4,自引:0,他引:4
Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. 相似文献
9.
David Wilson‐Sánchez Silvia Rubio‐Díaz Rafael Muñoz‐Viana José Manuel Pérez‐Pérez Sara Jover‐Gil María Rosa Ponce José Luis Micol 《The Plant journal : for cell and molecular biology》2014,79(5):878-891
The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T‐DNA is responsible for the phenotype. To quickly map non‐annotated T‐DNA insertions, we developed a reliable, cost‐effective and easy method based on whole‐genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf ( http://genetics.umh.es/phenoleaf ) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal–distal cell cycle‐driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ. 相似文献
10.
One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional
study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim
is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a
number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the
search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations
from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained
from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function,
with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally
efficient route to generating clinically relevant and functionally important mouse models. 相似文献
11.
Ordered collections of Arabidopsis thaliana lines containing mapped T-DNA insertions have become an important resource for plant scientists performing genetic studies. Previous reports have indicated that T-DNA insertion lines can have chromosomal translocations associated with the T-DNA insertion site, but the prevalence of these rearrangements has not been well documented. To determine the frequency with which translocations are present in a widely-used collection of T-DNA insertion lines, we analyzed 64 independent lines from the Salk T-DNA mutant collection. Chromosomal translocations were detected in 12 of the 64 lines surveyed (19%). Two assays were used to screen the T-DNA lines for translocations: pollen viability and genome-wide genetic mapping. Although the measurement of pollen viability is an indirect screen for the presence of a translocation, all 11 of the T-DNA lines showing an abnormal pollen phenotype were found to contain a translocation when analyzed using genetic mapping. A normal pollen phenotype does not, however, guarantee the absence of a translocation. We observed one T-DNA line with normal pollen that nevertheless had a translocation based on genetic mapping results. One additional phenomenon that we observed through our genetic mapping experiments was that the T-DNA junctions on the 5'- and 3'-sides of a targeted gene can genetically separate from each other in some cases. Two of the lines in our survey displayed this 'T-DNA borders separate' phenomenon. Experimental procedures for efficiently screening T-DNA lines for the presence of chromosomal abnormalities are presented and discussed. 相似文献
12.
In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines. 相似文献
13.
Druka A Franckowiak J Lundqvist U Bonar N Alexander J Houston K Radovic S Shahinnia F Vendramin V Morgante M Stein N Waugh R 《Plant physiology》2011,155(2):617-627
Since the early 20th century, barley (Hordeum vulgare) has been a model for investigating the effects of physical and chemical mutagens and for exploring the potential of mutation breeding in crop improvement. As a consequence, extensive and well-characterized collections of morphological and developmental mutants have been assembled that represent a valuable resource for exploring a wide range of complex and fundamental biological processes. We constructed a collection of 881 backcrossed lines containing mutant alleles that induce a majority of the morphological and developmental variation described in this species. After genotyping these lines with up to 3,072 single nucleotide polymorphisms, comparison to their recurrent parent defined the genetic location of 426 mutant alleles to chromosomal segments, each representing on average <3% of the barley genetic map. We show how the gene content in these segments can be predicted through conservation of synteny with model cereal genomes, providing a route to rapid gene identification. 相似文献
14.
While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 μM, we identified that ~55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells. 相似文献
15.
Christopher Schuetze Michelle Peters Jia-Jen Duong Matthieu Cavey Ruth D?rig Paul Lasko Beat Suter 《Génome》2004,47(5):832-838
Chromosomal mutations induced by ethyl methanesulfonate (EMS) treatment can cause female sterility or maternal-effect lethality in Drosophila. EMS is particularly useful to researchers because it creates mutations independent of position effects. However, because researchers have little control over the chromosomal site of mutation, post-mutagenic genetic mapping is required to determine the cytological location of the mutation. To make a valuable set of mutants more useful to the research community, we have mapped the uncharacterized part of the female-sterile - maternal-effect lethal Tubingen collection. We mapped 49 female-sterile - maternal-effect lethal alleles and 72 lethal alleles to individual deficiency intervals on the third chromosome. In addition, we analyzed the phenotype of ovaries resulting from female sterile mutations. The observed phenotypes range from tumorous ovaries and early blocks in oogenesis, to later blocks, slow growth, blocks in stage 10, to apparently full development of the ovary. The mapping and phenotypic characterization of these 121 mutations provide the necessary information for the researcher to consider a specific mutant as a candidate for their gene of interest. 相似文献
16.
TILLING (Targeting induced local lesions in genomes) is a general reverse-genetic strategy that is used to locate an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and cost-effective detection of induced point mutations in populations of chemically mutagenized individuals. The technique can be applied not only to model organisms but also to economically important organisms in plants. Owing to its full of advantages such as simple procedure, high sensitivity, and high efficiency, TILLING provides a powerful approach for gene discovery, DNA polymorphism assessment, and plant improvement. Coupled with other genomic resources, TILLING and EcoTILLING can be used immediately as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants. 相似文献
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Drosophila melanogaster is a widely used model organism for genetic dissection of developmental processes. To exploit its full potential for studying the genetic basis of male fertility, we performed a large-scale screen for male-sterile (ms) mutations. From a collection of 12,326 strains carrying ethyl-methanesulfonate-treated, homozygous viable second or third chromosomes, 2216 ms lines were identified, constituting the largest collection of ms mutations described to date for any organism. Over 2000 lines were cytologically characterized and, of these, 81% failed during spermatogenesis while 19% manifested postspermatogenic processes. Of the phenotypic categories used to classify the mutants, the largest groups were those that showed visible defects in meiotic chromosome segregation or cytokinesis and those that failed in sperm individualization. We also identified 62 fertile or subfertile lines that showed high levels of chromosome loss due to abnormal mitotic or meiotic chromosome transmission in the male germ line or due to paternal chromosome loss in the early embryo. We argue that the majority of autosomal genes that function in male fertility in Drosophila are represented by one or more alleles in the ms collection. Given the conservation of molecular mechanisms underlying important cellular processes, analysis of these mutations should provide insight into the genetic networks that control male fertility in Drosophila and other organisms, including humans. 相似文献
20.
Isolation of Mutations in the Drosophila Homologues of the Human Neurofibromatosis 2 and Yeast Cdc42 Genes Using a Simple and Efficient Reverse-Genetic Method 总被引:2,自引:0,他引:2
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Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci. However, because the screens performed to date primarily have generated autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes. We have designed a reverse genetic screen that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene that serves as an autosomally linked duplication of a small region of the X chromosome. The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologues of two well-studied genes, the human Neurofibromatosis 2 tumor suppressor and the yeast CDC42 gene. The method we describe should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new alleles of existing X-linked or autosomal mutations in Drosophila. 相似文献