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PubMed2004

Genetics in reverse.

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Fragile X Messenger Ribonucleoprotein 1↗
PubMed2000

Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters.

A major question in plant physiology is how the large amount of sucrose made in leaves is transported to the rest of the plant. Although physiological, biochemical, and anatomical investigations have been performed in this field, to date there have been very few genetic studies. Using a reverse genetic screen, we have identified mutant Arabidopsis plants containing transferred DNA insertions in the gene encoding a phloem-specific sucrose transporter, SUC2. SUC2 is thought to function in loading sugar from the apoplast into the conducting sieve tubes. In the homozygous state, these mutations resulted in stunted growth, retarded development, and sterility. The source leaves of mutant plants contained a great excess of starch, and radiolabeled sugar failed to be transported efficiently to roots and inflorescences. These data provide genetic proof that apoplastic phloem loading is critical for growth, development, and reproduction in Arabidopsis and that SUC2 is at least partially responsible for this step.

Arabidopsis↗
PubMed2005

Reverse engineering genetic networks using evolutionary computation.

This paper proposes an improved evolutionary method for constructing the underlying network structure and inferring effective kinetic parameters from the time series data of gene expression using decoupled S-system formalism. We employed Trigonometric Differential Evolution (TDE) as the optimization engine of our algorithm for capturing the dynamics in gene expression data. A more effective fitness function for attaining the sparse structure, which is the hallmark of biological networks, has been applied. Experiments on artificial genetic network show the power of the algorithm in constructing the network structure and predicting the regulatory parameters. The method is used to evaluate interactions between genes in the SOS signaling pathway in Escherichia coli using gene expression data.

Algorithms↗
PubMed1986

Autosomal modifiers of the bobbed phenotype are a major component of the rDNA magnification paradox in Drosophila melanogaster.

rDNA magnification in Drosophila melanogaster is defined experimentally as the ability of bb/Ybb- males to produce exceptional progeny that are wild type with respect to rDNA associated phenotypes. Here, we show that some of these bobbed-plus progeny result not from genetic reversion at the bb locus but rather from variants at two or more autosomal loci that ameliorate the bobbed phenotype of rDNA deficient males in Drosophila. In doing so we resolve several aspects of a long-standing paradox concerning the phenomenon of rDNA magnification. This problem arose from the use of two genetic assays, which were presumed to be identical, but paradoxically, produced conflicting data on both the kinetics of reversion and the stability of magnified bb+ chromosomes. We resolve this problem by demonstrating that in one assay bobbed-plus progeny arise primarily by genetic reversion at the bobbed locus, whereas in the other assay bobbed-plus progeny arise both by reversion and by an epistatic effect of autosomal modifiers on the bobbed phenotype. We further show that such modifiers can facilitate the appearance of phenotypically bobbed-plus progeny even under conditions where genetic reversion is blocked by magnification defective mutants. Finally, we present a speculative model relating the action of these modifiers to the large increases in rDNA content observed in males undergoing magnification.

Animals↗
PubMed1995

Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies.

Reverse transcription of retroviral genomic RNA in a target cell is influenced by cellular factors, including the concentration of deoxyribonucleoside triphosphates (dNTPs). In addition, recent data have demonstrated that reverse transcription can be driven within human immunodeficiency virus type 1 virions, prior to infection of a cell, by increasing extracellular concentrations of dNTPs. In attempts to increase the transduction efficiency of recombinant murine leukemia virus vectors, endogenous reverse transcription was initiated within cell-free, recombinant murine leukemia virus virions in the presence of relatively high concentrations of dNTPs. As a result, the expression of transduced genes via these retroviral vectors was increased approximately 10-fold by treatment of virions with dNTPs. Combined with our previous data, these observations suggest that virion-associated DNA synthesis can occur in diverse groups of retroviruses and positively alter retroviral infectivity. As such, these manipulations may be useful for increasing the efficiency of retrovirus-mediated gene delivery.

Animals↗
PubMed1969

Antipolarity in the ilv operon of Escherichia coli K-12.

The genes governing three of the enzymes of the isoleucine-valine biosynthetic pathway form the operon: operator-ilvA-ilvD-ilvE. The enzymes are: ilvA, l-threonine deaminase; ilvD, dihydroxy acid dehydrase; and ilvE, transaminase B. A nonsense mutation in the ilvD gene (D-ochre) and a nonsense mutation in the ilvE gene (E-amber) affect the properties of the proximal gene product, l-threonine deaminase (TD), in addition to inactivating the enzymes produced by the genes in which the mutations have occurred. The D-ochre mutation causes TD to move in diffusion and gel filtration experiments as though it were 30% smaller than the wild-type enzyme. The E-amber mutation causes TD to move in similar experiments as though it were much larger than the wild-type enzyme. Both mutations completely abolish the sensitivity of TD to l-isoleucine, the normal feedback inhibitor of the wild-type enzyme. The effects of the nonsense mutations on TD can be reversed in three ways: by genetic reversion of the D-ochre mutation; by treatment of the altered enzymes with 3.0 m urea; and by forming a heterozygous diploid, containing the wild-type allele as well as the mutant allele of ilvD or ilvE. The results suggest that the subunits of TD undergo abnormal aggregation in the presence of the partial polypeptides produced by the mutant alleles of ilvD or ilvE; multi-enzyme aggregates in extracts of wild type, however, could not be detected.

Chromosome Mapping↗
PubMed1999

Sexual behavior mutants revisited: molecular and cellular basis of Drosophila mating.

The study of Drosophila melanogaster by a combination of forward genetics with specific mutants, and reverse genetics, in which a given gene is expressed in an appropriate brain area to test its effect on behavior, provides a unique opportunity to explore the causal relationship between a particular gene, its function in the cell and the behavioral outcome at the organismic level. Enhanced male-to-male courtship has been shown to occur as a result of mutations in several different genes. For example, the Voila mutant exhibits intense GAL4 reporter expression in the tarsal gustatory sensilla, suggesting the importance of tapping by a male on the female abdomen with his forelegs. Feminization of parts of the antennal lobe and mushroom body by targeted expression of a female-determining gene transformer+ (tra+) drives the male to court other males. Mutations in the tra target gene fruitless (fru), which is expressed in the antennal lobe as well as the suboesophageal ganglion (the gustatory inputs are processed here), also induce homosexual courtship in males. These results suggest that sensory inputs mediated and/or processed by the tarsal receptors, suboesophageal ganglion, antennal lobe and mushroom body contribute to the regulation of male-female courtship. Mosaic analysis localized the neural center for male courtship behavior to the posterior dorsal brain, in which the sensory information processed by the aforementioned neural structures may be integrated. Another mosaic study mapped the neural center for female sexual behavior, as measured by her receptiveness to copulation, to the anterior dorsal brain. The issue as to how the mutations that reduce female sexual receptiveness, e.g. dissatisfaction (dsf), spinster (spin) and chaste (cht), affect the structure and/or function of this neural center deserves to be addressed urgently.

Animals↗
PubMed2006

Components of the protein quality control system are expressed in a strain-dependent manner in the mouse hippocampus.

Inbred mouse strains are used in forward-genetic experiments, designed to uncover genes contributing to their highly distinct neurophenotypes and multiple reports of variations in mutant phenotypes due to genetic background differences in reverse-genetic approaches have been published. Information on strain-specific protein expression-phenotypes however, is limited and a comprehensive screen of an effect of strain on brain protein levels has not yet been carried out. Herein a proteomic approach, based upon two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) was used to show significant genetic variation in hippocampal protein levels between five mouse strains. Considering recent evidence for the importance of the intracellular protein quality control system for synaptic plasticity-related mechanism we decided to focus on the analysis of molecular chaperones and components of the ubiquitin-proteasome system. Sixty-six spots, depicting 36 proteins have been unambiguously identified by mass spectrometry. Quantification revealed strain-dependent levels of 18 spots, representing 12 individual gene products. We thus present proteome analysis of hippocampal tissues of several mouse strains as suitable tool to address fundamental questions about genetic control of protein levels and to demonstrate molecular networks of protein metabolism and chaperoning. The findings are useful for designing future studies on these cascades and interpretation of results show that data on brain protein levels cannot be simply extrapolated among different mouse strains.

Animals↗
PubMed2004

Taming plastids for a green future.

Plant genetic engineering will probably contribute to the required continued increase in agricultural productivity during the coming decades, and moreover, plants can potentially provide inexpensive production platforms for pharmaceuticals and nutraceuticals. With the advent of technologies for altering the genetic information inside chloroplasts, a new attractive target for genetic engineering has become available to biotechnologists. Potential advantages over conventional nuclear transformation include high transgene expression levels and increased biosafety because of maternal organelle inheritance in most crops. This review summarizes the state of the art in chloroplast genetic engineering and describes how reverse genetics approaches enhance our understanding of photosynthesis and other important chloroplast functions. Furthermore, promising strategies by which chloroplast genetic engineering might contribute to the successful modification of plant metabolism are discussed.

Chloroplasts↗
PubMed2026

A Novel Approach to Engineering Tomato Spotted Wilt Virus Infectious Clones by Disarming Key Nodes in Antiviral Defenses.

Tomato spotted wilt virus (TSWV) is an economically devastating pathogen that rapidly overcomes genetic resistance in major crops. Reverse genetic systems are crucial for investigating plant-virus interactions and resistance-breaking mechanisms, and developing these tools for segmented ambisense RNA viruses remains a crucial challenge. Current TSWV clones rely on extensively modified Asian isolates requiring co-delivery of multiple replication helpers and viral silencing suppressors. Streamlining these systems for regionally significant strains with minimal genetic alterations is essential. Here, we developed the first infectious clone of a U.S. TSWV isolate (PA01). Three binary plasmids contain cDNAs for the antigenomic L and S segments, as well as the genomic M segment, with enhanced GFP replacing NSs on the S segment. Co-delivery of the cucumovirus 2b alone or in combination with tombusvirus P19 or begomovirus AL2 achieved a high proportion of systemically infected Nicotiana benthamiana and Capsicum annuum plants. In N. tabacum, co-delivering the Caenorhabditis elegans cell death suppressor CED-9 or using NahG transgenic plants produced 30 to 33% systemically infected plants. Co-delivery of 2b boosted infection levels in NahG plants to 62%. These data indicate that in addition to the antiviral RNA-silencing machinery, additional host defense pathways influence TSWV rescue and systemic infection from cDNA. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tospovirus↗
PubMed1992

The molecular and genetic analysis of mouse development.

This review describes some recent advances in the molecular-genetic analysis of mouse development. Reversed genetics and gene assignment have been used to isolate genes affected in developmental mutations. The establishment of a high-density molecular-genetic map promises to facilitate cloning of additional genes with developmental functions. Based on molecular, biochemical or other biological criteria many mouse genes that code for transcriptional regulators, growth-factor-like molecules and their receptors have been isolated. The role of these genes during development can be analysed in vivo after producing targeted mutations. Mutations can be generated by homologous recombination in the genome of embryonic stem cells and can then be introduced into the mouse germ line by means of germ-line chimaeras. Additional approaches employing stem cells to identify and mutate putative developmental genes are coming into use.

Animals↗
PubMed2005

Identification of Drosophila genes modulating Janus kinase/signal transducer and activator of transcription signal transduction.

The JAK/STAT pathway was first identified in mammals as a signaling mechanism central to hematopoiesis and has since been shown to exert a wide range of pleiotropic effects on multiple developmental processes. Its inappropriate activation is also implicated in the development of numerous human malignancies, especially those derived from hematopoietic lineages. The JAK/STAT signaling cascade has been conserved through evolution and although the pathway identified in Drosophila has been closely examined, the full complement of genes required to correctly transduce signaling in vivo remains to be identified. We have used a dosage-sensitive dominant eye overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway to screen 2267 independent, newly generated mutagenic P-element insertions. After multiple rounds of retesting, 23 interacting loci that represent genes not previously known to interact with JAK/STAT signaling have been identified. Analysis of these genes has identified three signal transduction pathways, seven potential components of the pathway itself, and six putative downstream pathway target genes. The use of forward genetics to identify loci and reverse genetic approaches to characterize them has allowed us to assemble a collection of genes whose products represent novel components and regulators of this important signal transduction cascade.

Animals↗
PubMed2025

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Schizosaccharomyces↗
PubMed2003

Forward genetics and map-based cloning approaches.

Whereas reverse genetics strategies seek to identify and select mutations in a known sequence, forward genetics requires the cloning of sequences underlying a particular mutant phenotype. Map-based cloning is tedious, hampering the quick identification of candidate genes. With the unprecedented progress in the sequencing of whole genomes, and perhaps even more with the development of saturating marker technologies, map-based cloning can now be performed so efficiently that, at least for some plant model systems, it has become feasible to identify some candidate genes within a few months. This, in turn, will boost the use of forward genetics approaches, as applied (for example) to isolating genes involved in natural variation and genes causing phenotypic mutations as derived from (second-site) mutagenesis screens.

Arabidopsis↗