Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “reverse genetics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed2004

Conservation of the global sex determination gene tra-1 in distantly related nematodes.

Sex determination has long intrigued evolutionists, geneticists, and developmental biologists in a similar way. Substantial evidence indicates that sex determination evolves rapidly and, therefore, can be used to study how molecular patterning processes evolve. In Caenorhabditis elegans, sex determination relies on a signaling pathway that involves a cascade of negatively acting factors, finally triggering the GLI-family zinc-finger transcription factor TRA-1. We have started to investigate sex determination in the nematode satellite species Pristionchus pacificus that is separated from C. elegans for 200-300 million years. In P. pacificus, animals with two X chromosomes develop as hermaphrodites, whereas XO animals develop as males. We used an unbiased forward genetic approach and isolated several mutants with a hermaphrodite to male transformation of the XX karyotype. We identified one complementation group as representing the P. pacificus ortholog of tra-1, providing the first evidence for the conservation of a global sex determination gene over a time period of at least 200 million years. A Ppa-tra-1 morpholino phenocopies Ppa-tra-1 mutants and establishes the morpholino technology as a reverse genetic approach in P. pacificus.

Alleles↗
PubMed2005

Genetic analysis of innate immunity: identification and function of the TIR adapter proteins.

The innate immune system senses pathogens largely through signals initiated by a collection of phylogenetically related proteins known as "Toll-like receptors" (TLRs), of which ten representatives are encoded in the human genome. The sensing role of the TLRs first came to light when one member of this family, TLR4, was shown to serve the detection of endotoxin (lipopolysaccharide; LPS) in mice. This discovery was based upon positional cloning of a spontaneous mutation affecting a locus known as Lps. The recognition specificities of other TLRs have since been established by reverse genetic methods. The understanding of the biochemical circuitry that maintains the innate capacity for immune recognition and response has loomed as the next hurdle in the field. A total of five adapter proteins with cytoplasmic domain homology to the TLRs are known to exist in mammals. These proteins are not entirely promiscuous in their interaction with TLRs, but rather, show preferential association with individual family members, giving a particular character to the signals that distinct micro-organisms initiate. The adaptive immune response is dependent upon upregulation of costimulatory molecules (UCM) such as CD80 and CD86. Very recently, it has been shown that this upregulation is dependent upon an adapter encoded by a locus known as Lps2, known as Trif or Ticam-1, and upon type I interferon receptor signaling. LPS and dsRNA both signal via Trif, but dsRNA has an accessory pathway for UCM, that is independent of both Trif/Ticam-1 and the known dsRNA receptor, TLR3. Other key innate immunity genes have also been disclosed by germline mutagenesis, and are discussed in the present review.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Genetic manipulation of negative-strand RNA virus genomes.

Negative-strand RNA viruses have been refractory to genetic manipulation using recombinant DNA techniques. Recently, new techniques were developed that allowed the rescue of synthetic RNA molecules into influenza A viruses and, subsequently, into other negative-strand RNA viruses. These techniques are presently being used to study the molecular biology of these viruses. Questions concerning cis- and trans-acting elements that are involved in transcription and replication of negative-sense RNA viral genomes can now be addressed with reverse genetic approaches. Further development of this methodology has enabled the construction--by recombinant DNA techniques--of influenza A viruses that contain altered genomes. The phenotypic characteristics and possible applications of these novel transfectant viruses are also discussed.

Base Sequence↗
PubMed2005

Tall tales from sly dwarves: novel functions of gibberellins in plant development.

Gibberellins (GAs) are endogenous hormones controlling numerous aspects of plant growth and development. Our present understanding of GA physiology is based largely on genetic analysis in model plants such as Arabidopsis. In spite of the success of this approach, the discovery of additional physiological roles for GAs in seed development, pollen tube growth and meristem development indicates that the existing collection of GA-related mutants (identified partially or entirely on the basis of vegetative phenotypes) has failed to uncover all aspects of plant development that are controlled by GAs. The continued use of ever improving forward and reverse genetic techniques is expected to lead to the discovery of further novel roles for GAs in plant development.

Arabidopsis↗
PubMed2002

Infectious bursal disease virus capsid protein VP3 interacts both with VP1, the RNA-dependent RNA polymerase, and with viral double-stranded RNA.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus of the Birnaviridae family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we identified the domains responsible for the interaction between VP3 and VP1. Using the yeast two-hybrid system we found that VP1 binds to VP3 through an internal domain, while VP3 interacts with VP1 solely by its carboxy-terminal 10 amino acids. These results were confirmed by using a reverse-genetics system that allowed us to analyze the interaction of carboxy-terminally truncated VP3 molecules with VP1 in infected cells. Coimmunoprecipitations with VP1- and VP3-specific antibodies revealed that the interaction is extremely sensitive to truncation of VP3. The mere deletion of the C-terminal residue reduced coprecipitation almost completely and also fully abolished production of infectious virions. Surprisingly, these experiments additionally revealed that VP3 also binds to RNA. RNase treatments and reverse transcription-PCR analyses of the immunoprecipitates demonstrated that VP3 interacts with dsRNA of both viral genome segments. This interaction is not mediated by the carboxy-terminal domain of VP3 since C-terminal truncations of 1, 5, or 10 residues did not prevent formation of the VP3-dsRNA complexes. VP3 seems to be the key organizer of birnavirus structure, as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1, and the two genomic dsRNAs.

Animals↗
PubMed2006

Venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation.

RNA viruses are notorious for their genetic plasticity and propensity to exploit new host-range opportunities, which can lead to the emergence of human disease epidemics such as severe acute respiratory syndrome, AIDS, dengue, and influenza. However, the mechanisms of host-range change involved in most of these viral emergences, particularly the genetic mechanisms of adaptation to new hosts, remain poorly understood. We studied the emergence of Venezuelan equine encephalitis virus (VEEV), an alphavirus pathogen of people and equines that has had severe health and economic effects in the Americas since the early 20th century. Between epidemics, VEE disappears for periods up to decades, and the viral source of outbreaks has remained enigmatic. Combined with phylogenetic analyses to predict mutations associated with a 1992-1993 epidemic, we used reverse genetic studies to identify an envelope glycoprotein gene mutation that mediated emergence. This mutation allowed an enzootic, equine-avirulent VEEV strain, which circulates among rodents in nearby forests to adapt for equine amplification. RNA viruses including alphaviruses exhibit high mutation frequencies. Therefore, ecological and epidemiological factors probably constrain the frequency of VEE epidemics more than the generation, via mutation, of amplification-competent (high equine viremia) virus strains. These results underscore the ability of RNA viruses to alter their host range, virulence, and epidemic potential via minor genetic changes. VEE also demonstrates the unpredictable risks to human health of anthropogenic changes such as the introduction of equines and humans into habitats that harbor zoonotic RNA viruses.

Amino Acids↗
PubMed2001

MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat.

We have undertaken a systematic reverse genetic approach to understand R2R3-MYB gene function in Arabidopsis. Here, we report the functional characterization of MYB61 based on the phenotype of three independent insertion alleles. Wide-ranging phenotype screens indicated that MYB61 mutants were deficient in seed mucilage extrusion upon imbibition. This phenotype was expressed in the sporophytic tissues of the seed. Deposition and extrusion of the principal component of the mucilage, a relatively unbranched rhamnogalacturonan, were reduced in the MYB61 mutant seed coats. Additional defects in the maturation of the testa epidermal cells suggested a potential deficiency in extracellular secretion in myb61 lines. Consistent with a proposed role in testa development, reverse transcription-polymerase chain reaction analysis showed the highest MYB61 expression in siliques, which was localized to the seed coat by a beta-glucuronidase (GUS) reporter gene fusion. Lower levels of GUS expression were detected in developing vascular tissue. Parallel analysis of the ttg1-1 mutant phenotype indicated that this mutant showed more severe developmental defects than myb61 and suggested that MYB61 may function in a genetic pathway distinct from that of TTG1. The transient nature of seed epidermal characteristics in the ttg1-1 mutant suggested that TTG1 was required for maintenance rather than initiation of testa epidermal differentiation. Germination and seedling establishment were compromised in the myb61 and ttg1-1 mutants under conditions of reduced water potential, suggesting a function for Arabidopsis seed mucilage during germination in dry conditions.

Arabidopsis↗
PubMed2005

Trypanosoma brucei DMC1 does not act in DNA recombination, repair or antigenic variation in bloodstream stage cells.

Homologous recombination acts in the repair of cellular DNA damage and can generate genetic variation. Some of this variation provides a discrete purpose in the cell, although it can also be genome-wide and contribute to longer-term natural selection. In Trypanosoma brucei, a eukaryotic parasite responsible for sleeping sickness disease in sub-Saharan Africa, homologous recombination acts to catalyse antigenic variation, an immune evasion strategy involving switches in variant surface glycoprotein. In addition, T. brucei can undergo genetic exchange by homologous recombination in the tsetse vector, and some evidence suggests that this occurs by meiosis. Here, we show that T. brucei, Trypanosoma cruzi and Leishmania major each contain a single copy gene whose product is highly related to the eukaryotic meiosis-specific protein Dmc1, which is structurally and functionally related to Rad51. We show that T. brucei DMC1 is transcribed in the bloodstream stage of the parasite, where the gene can be mutated by reverse genetic disruption. DMC1 mutation does not, however, result in detectable alterations in DNA repair, recombination or antigenic variation efficiency in this life cycle stage.

Amino Acid Sequence↗
PubMed1994

Cytosine deaminations catalyzed by DNA cytosine methyltransferases are unlikely to be the major cause of mutational hot spots at sites of cytosine methylation in Escherichia coli.

Sites of cytosine methylation are hot spots for C to T mutations in Escherichia coli DNA. We have developed a genetic reversion assay that allows direct selection of C to T mutations at a site of methylation. Because the mutant gene is on a plasmid, this system can be used to study mutational effects of biochemical agents in vitro as well as in vivo. Using this system we show that in vitro an E. coli methyltransferase can cause C to U deaminations at a site of methylation. Reaction conditions that are known to inhibit a side reaction of the methyltransferase also suppress reversion frequency, suggesting that this side reaction is required for deamination. Furthermore, a mutation in the enzyme that eliminates its catalytic activity but not its ability to bind DNA eliminates the ability of the enzyme to cause C to U deaminations. Despite this, in vivo experiments strongly suggest that enzyme-catalyzed deaminations of cytosine do not play a major role in making methylation sites in E. coli hot spots for mutations. For example, although uracil-DNA glycosylase (Ung) suppresses the occurrence of mutations due to C to U deaminations, the frequency of C to T mutations at a methylation site remains high in ung+ cells. Furthermore, the reversion frequencies in ung+ and ung- cells are quite similar.

Base Sequence↗
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↗
PubMed2006

Genes involved in Dictyostelium discoideum sexual reproduction.

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.

Animals↗
PubMedDate not supplied

Participation of nuclear genes in chloroplast gene expression.

The expression of the plastid genome is dependent on a large number of nucleus-encoded factors. Some of these factors have been identified through biochemical assays, and many others by genetic screens in Arabidopsis, Chlamydomonas and maize. Nucleus-encoded factors function in each step in plastid gene expression, including transcription, RNA editing, RNA splicing, RNA processing, RNA degradation, and translation. Many of the factors discovered via biochemical approaches play general roles as components of the basic gene expression machinery, whereas the majority of those identified by genetic approaches are specifically required for the expression of small subsets of chloroplast genes and are involved in post-transcriptional steps. Some of the nucleus-encoded factors may play regulatory roles and modulate chloroplast gene expression in response to developmental or environmental cues. They may also serve to couple chloroplast gene expression with the assembly of the protein products into the large complexes of the photosynthetic apparatus. The convergence of biochemical approaches with those of classical and reverse genetics, and the contributions from large scale genomic sequencing should result in rapid advances in our understanding of the regulatory interactions that govern plastid gene expression.

Cell Nucleus↗
PubMed2002

Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression.

Nitrate is an essential element for plant growth, both as a primary nutrient in the nitrogen assimilation pathway and as an important signal for plant development. The uptake of nitrate from the soil and its translocation throughout the plant has been the subject of intensive physiological and molecular studies. Using a reverse genetic approach, the AtNRT2.1 gene has been shown to be involved in the inducible component of the high-affinity nitrate transport system in Arabidopsis. The Arabidopsis Genome Initiative has released nearly the whole genome sequence of Arabidopsis, allowing the identification of a small NRT2 multigene family in this species. Thus, we investigated the phylogenetic relationship between NRT2 proteins belonging to several kingdoms and compared the structure of the different members of the Arabidopsis family. We analyzed, by semiquantitative reverse transcriptase-polymerase chain reaction, the expression pattern of each gene depending on plant organ and development or nutritional status, and compared the relative level of each gene by real-time polymerase chain reaction. We also evaluated the significance of each paralog on the basis of the relative levels of gene expression. The results are discussed in relation with distinct roles for the individual members of the AtNRT2 family.

Anion Transport Proteins↗
PubMed2004

Reversion of molecularly engineered, partially attenuated, very virulent infectious bursal disease virus during infection of commercial chickens.

A molecularly cloned, tissue culture-adapted infectious bursal disease virus (BD-3tc) was generated from a very virulent strain by the reverse genetics approach following site-directed mutagenesis (Q253H and A284T in VP2). The pathogenicity of BD-3tc was tested in commercial chickens. The wild-type strain (BD-3wt) and the molecularly cloned parental strain (BD-3mc) were included for comparison. The subclinical course of the disease, with delayed and milder pathological lesions followed by quick follicular regeneration in the bursa of Fabricius in BD-3tc-inoculated birds, suggested that these amino acid substitutions made BD-3tc partially attenuated. However, severe bursa atrophy was observed at 14 days after inoculation. Reverse transcription-polymerase chain reaction coupled with restriction enzyme analysis revealed that both point mutations in BD-3tc had reverted 14 days after inoculation. Further investigations demonstrated that the codon for amino acid at position 284 had already reverted to the wild-type phenotype (T284A) 3 days after inoculation.

Animals↗
PubMed2006

Viral-based modelling and correction of neurodegenerative diseases by RNA interference.

Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.

Animals↗
PubMed2004

RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence.

Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.

Genes, Reporter↗
PubMed2003

Lentivirus-delivered stable gene silencing by RNAi in primary cells.

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Dendritic Cells↗
PubMed1988

Physiological processes revealed through an analysis of inborn errors.

To develop the significance of human inborn errors of membrane function in terms of their potential for the elucidation of physiological processes, an overview of the genetics of inherited metabolic disorders is first presented, concisely covering classical Mendelian inheritance and progressing to molecular applications currently being used in the isolation of a number of disease-causing genes. Second, approaches to the isolation of several genes implicated in inherited disorders of membrane function are discussed. Examples are drawn from studies of the Shaker/IA K+ channel mutants of Drosophila and human inborn errors in the low-density lipoprotein receptor and Kidd antigen/urea transporter. These examples illustrate the techniques of "reverse" genetics, restriction fragment-length polymorphism analysis, chromosomal walking, and cDNA library screening with oligonucleotides and antibodies. Finally, the need to develop a physiological functional analysis of the protein structures derived from isolated human disease-causing genes is presented as a necessary direction for future research.

Animals↗