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Results for “Forward genetics”

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PubMed2004

Genetic and genomic approaches to reward and addiction.

Drug addiction is recognized as a mental disease affecting the brain's natural reward system. Drugs of abuse strongly activate reward structures in the brain and induce lasting changes in behavior that reflect changes in neuron physiology and biochemistry. With the ultimate goal of developing therapeutic interventions, it is of interest to determine the molecular and cellular components of motivation and reward, and identify those gene products that contribute to the process of drug addiction. Our laboratory has chosen three general genetic approaches to examine reward and addiction: reverse genetics to assess the role of candidate genes in drug responsiveness, forward genetics to discover novel regulators of dopamine transmission, and gene expression profiling to define gene sets in different brain structures that contribute to the molecular and neurobiological basis of reward.

Animals↗
PubMed2001

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice.

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.

Animals↗
PubMed2002

Reverse genetics by fast neutron mutagenesis in higher plants.

Fast neutron mutagenesis has long been used for forward genetic studies in plants. Only recently has it been used for reverse genetics. By combining fast neutron mutagenesis and high throughput PCR screening, a new knockout methodology, Deleteagene (Delete-a-gene), was developed in Arabidopsis and rice to obtain deletion mutants for target genes. Since fast neutron is a highly efficient mutagen that produces deletion mutations easily detectable by PCR, this method has the potential to enable reverse genetic screens in most plant species. In this review, we will discuss the use of fast neutron as a mutagen for creating deletion mutations, and strategies for successful application of Deleteagene in other plant species.

Arabidopsis↗
PubMed2005

Genetic dissection of the zebrafish retinal stem-cell compartment.

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.

Animals↗
PubMed2005

Use of the zebrafish system to study primitive and definitive hematopoiesis.

The zebrafish (Danio rerio) has emerged as an ideal organism for the study of hematopoiesis, the process by which all the cellular elements of the blood are formed. These elements, including erythrocytes, granulocytes, monocytes, lymphocytes, and thrombocytes, are formed through complex genetic signaling pathways that are highly conserved throughout phylogeny. Large-scale forward genetic screens have identified numerous blood mutants in zebrafish, helping to elucidate specific signaling pathways important for hematopoietic stem cells (HSCs) and the various committed blood cell lineages. Here we review both primitive and definitive hematopoiesis in zebrafish, discuss various genetic methods available in the zebrafish model for studying hematopoiesis, and describe some of the zebrafish blood mutants identified to date, many of which have known human disease counterparts.

Animals↗
PubMed2006

Harvesting the potential of induced biological diversity.

For most of the past century, chemical and physical mutagens have been used in plant genetic research to introduce novel genetic variation. In crop improvement, more than 2000 plant varieties that contain induced mutations have been released for cultivation having faced none of the regulatory restrictions imposed on genetically modified material. In plant science, mutational approaches have found extensive use in forward genetics and for enhancer and suppressor screens - particularly in model organisms where positional cloning is easily achieved. However, new approaches that combine mutagenesis with novel and sensitive methods to detect induced DNA sequence variation are establishing a new niche for mutagenesis in the expanding area of (crop) plant functional genomics and providing a bridge that links discovery in models to application in crops.

Gene Frequency↗
PubMed2005

Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.

Animals↗
PubMed2002

The art and design of genetic screens: Drosophila melanogaster.

The success of Drosophila melanogaster as a model organism is largely due to the power of forward genetic screens to identify the genes that are involved in a biological process. Traditional screens, such as the Nobel-prize-winning screen for embryonic-patterning mutants, can only identify the earliest phenotype of a mutation. This review describes the ingenious approaches that have been devised to circumvent this problem: modifier screens, for example, have been invaluable for elucidating signal-transduction pathways, whereas clonal screens now make it possible to screen for almost any phenotype in any cell at any stage of development.

Animals↗
PubMed1999

Plant tagnology.

Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence-function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species.

Journal Article↗
PubMed2001

Fishing for lymphoid genes.

Thymic organogenesis and T-cell lymphopoiesis are crucial interdependent processes that establish a functional vertebrate immune system. The current understanding of vertebrate thymic development during embryogenesis remains incomplete and would benefit from novel approaches. The zebrafish Danio rerio is a powerful developmental and genetic system for the dissection of early events in the ontogeny of the immune system. Forward genetic screens have uncovered genes involved in hematopoiesis, and specific screens are being designed to examine the genes that regulate T-cell development and the origin of the thymus. Studies of the zebrafish should improve our understanding of lymphoid development in vertebrates.

Animals↗
PubMed2005

The art and design of genetic screens: mouse.

Humans are mammals, not bacteria or plants, yeast or nematodes, insects or fish. Mice are also mammals, but unlike gorilla and goat, fox and ferret, giraffe and jackal, they are suited perfectly to the laboratory environment and genetic experimentation. In this review, we will summarize the tools, tricks and techniques for executing forward genetic screens in the mouse and argue that this approach is now accessible to most biologists, rather than being the sole domain of large national facilities and specialized genetics laboratories.

Animals↗
PubMed2003

New insights into the structure and function of glutamate receptors: the orphan receptor delta2 reveals its family's secrets.

The ionotropic glutamate receptors (iGluRs) form ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the mammalian brain. These receptors play central roles not only in normal neurodevelopmental and neurophysiological processes but also in certain neuropathological processes. Molecular cloning of genes for iGluRs in the past decade has advanced our understanding of the basic properties of iGluRs, such as ion selectivity, ligand binding, and anchoring at synapses. Although the gene for the delta2 glutamate receptor (GluRdelta2) was cloned on the basis of homology screening, GluRdelta2 has been referred to as an "orphan" receptor because it does not form functional glutamate-gated ion channels. However, ataxia in many types of mice is caused by spontaneous mutation of GluRdelta2. Analysis of two such mutants, lurcher and hotfoot, has provided key insights into the GluRdelta2 signaling in neurons. Furthermore, characterization of mutant GluRdelta2 has revealed unexpected clues to two fundamental features regarding the structure and function of iGluRs--gating and assembly. Studies have recently shown that the transmembrane region where the lurcher mutation is located probably plays a crucial role in channel gating. The mechanism that controls iGluR subunit assembly seems to involve the extracellular N-terminal domain where the hotfoot mutation is located. An understanding of mechanisms responsible for gating and assembly is essential for the comprehension of neuronal function and dysfunction. Although reverse genetics is useful in deciphering glutamate signaling, these findings demonstrate the power of classic approaches to forward genetics on mutant mice.

Amino Acid Sequence↗
PubMed2005

Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics.

IR64, the most widely grown indica rice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M4 generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M4 mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.

DNA, Plant↗
PubMed1999

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Amino Acid Sequence↗
PubMed2005

Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii.

Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at approximately 300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of approximately 592 cM and an average map unit of approximately 104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies.

Animals↗
PubMed2000

The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Amino Acid Sequence↗
PubMed2026

Advances in genomics-driven genetic decoding and genomic design breeding in tomato.

Tomatoes are highly nutritious and represent one of the important vegetable fruits worldwide. Both historically and moving forward, genetic decoding and precision breeding remain fundamental to tomato improvement. Here, we summarize pivotal advances in decoding tomato genomes across domestication, improvement and evolution processes and provide a perspective on future breeding through precision design. In-depth population genetic studies have revealed how artificial selection systematically prioritized yield-related alleles at the cost of narrowing genetic diversity, especially at flavor-related loci-highlighting the urgent need to reconcile these trade-offs. Comparative genomics across species, viewed through an evolutionary lens, has uncovered critical insights into functional genes, deepening our understanding of the genetic architecture and regulatory mechanisms underlying key traits. Collectively, these advances have enabled precise identification and functional characterization of key genetic elements, paving the way for systematic redomestication of tomato through precision genomic design. Looking ahead, more efficient and precise breeding strategies will be required to accelerate genetic gains in tomato in the coming decades. The integration of recent genomic advances, coupled with genomic selection and artificial intelligence, into genomic design breeding offers a transformative framework, unlocking unprecedented opportunities for developing highly flavorful and consumer-customized tomato varieties.

Journal Article↗
PubMed2000

Dissection of behavior and psychiatric disorders using the mouse as a model.

Mouse genetic models have played an important role in the elucidation of molecular pathways underlying human disease. This approach is becoming an increasingly popular way to study the genetic underpinning of psychiatric disorders. Genes within candidate regions for susceptibility to psychiatric illness can be evaluated through the phenotypic assessment of mutants mapped to the corresponding regions in the mouse genome. Alternatively, one can search for mouse mutants displaying characteristics that might correspond to physiological and behavioral markers of a psychiatric disorder, sometimes referred to as endophenotypes. Mice with anomalies in these traits can be generated by targeted mutagenesis in known genes (gene-based mutagenesis or reverse genetics), or can be identified among progeny of mice in a random mutagenesis screen (phenotype-based mutagenesis or forward genetics). In this review, we discuss recently generated behavioral mutants in the mouse. We also give an overview of several robust and commonly used behavioral phenotypes, their relevance to human disease and lessons learned from recent successes in mouse behavioral genetics.

Animals↗