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 307 records · Page 17Linked to original sources
PubMed1993

Leptomonas seymouri as a model system for the analysis of gene expression in trypanosomatids.

Leptomonas seymouri, a monogenetic trypanosomatid originally isolated from Dysdercus suturellus (Hemiptera), was used to develop a reverse genetic system for trypanosomatid flagellates. In many eukaryotic cell types, reverse genetics has proven to be a powerful tool for defining structure/function relationships within genes. The mini-exon genes of trypanosomatids encode key components of all cellular mRNAs. This component is a 5' "leader" RNA that is spliced onto all mRNA precursors during mRNA formation within the cell nucleus. The data presented here indicate that structure/function relationships within the mini-exon gene can be probed using the molecular genetic system developed and characterized for L. seymouri.

Animals↗
PubMed2004

Genetic approaches to studying mouse models of human seizure disorders.

In conclusion, we have discussed a reverse genetics approach to studying seizure disorders in mice (Fig. 1), employing a targeted mutagenesis method to exploit the genetic defects identified in human epilepsy families. After detailed characterization of the nature of the human mutation and the mouse counterpart gene, a targeting vector containing the human disease allele is created. The endogenous mouse gene is replaced by the human disease allele through homologous recombination in ES cells, leading to the generation of chimeric animals. Mice carrying one copy or both copies of the human mutation can be bred to study the phenotypic effect of heterozygous and homozygous mutations. At this stage, one may want to split the newly created mice into two groups. One group will go through seizure phenotyping tests, while the other group will be used to generate disease allele-carrying mice on a different genetic background. Phenotypic characterization of mice on different inbred strains includes behavioral monitoring and EEG analysis looking for the occurrence of spontaneous seizures, as well as routine cage examination looking for handling-provoked seizure and ECT- and PTZ- induced seizure paradigms looking for sensitivity to these stimuli. A complete evaluation of the seizure phenotype at the whole-animal level establishes the relevance of the mouse model to the human condition. Further investigation including imaging, electrophysiology and AED response in these mouse models will shed light on the mechanistic basis of the convulsive disorder. Current epilepsy research in mouse genetics offers promise for understanding the molecular mechanisms that underlie epileptogenesis in humans. A large-scale forward genetic effort to create novel mouse mutants with seizure phenotypes by in vivo chemical mutagenesis with ethyl-nitroso urea (ENU) is underway at the Jackson Laboratory (http://www.jax.org/nmf/). Genetic mapping and isolation of the affected genes in these seizure-prone models will provide additional molecular pathways involved in seizures. The mutant mice generated through both forward and reverse genetic approaches will be a valuable resource for the biomedical community to study epilepsy at the molecular level and to characterize the pathological consequences of seizures in the whole organism.

Animals↗
PubMed1989

Long-range mapping in the research and diagnosis of genetic disease.

This paper reviews current genetic and molecular biological methods that may be used in the so-called "reverse genetics" approach. These methods are the mapping, isolation, and study of the chromosomal DNA containing a previously unidentified gene responsible for a genetic disease, beginning with its chromosomal localization. In principle, the reverse genetics methodology follows the same path for different diseases studied. An overall outline of the steps to be undertaken is given and discussed. Several stages are illustrated with reference to current research in the fields of Duchenne muscular dystrophy, Huntington's disease, and polycystic kidney disease.

Chromosome Mapping↗
PubMed2006

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Animal Diseases↗
PubMed2006

Mutagenesis strategies in zebrafish for identifying genes involved in development and disease.

It has been nearly a decade since the completion of two large-scale chemical mutagenesis screens in zebrafish, and two years since the completion of a large-scale insertional mutagenesis. In this article, we use the accumulated data from these screens to compare the efficiency of each mutagen to isolate mutants and to identify mutated genes, and argue that the two mutagens target the same set of genes. We then review how both forward genetic screens and reverse genetic techniques, such as morpholinos and TILLING, and transgenics are being used to develop models of human disease.

Animals↗
PubMed2003

Molecular regulation of leaf senescence.

Leaf senescence is a process of programmed cell death, which is induced in an age-dependent manner and by various environmental cues. The mechanisms that regulate the induction and progression of leaf senescence remain unclear because of their complexity. However, recent genetic and reverse-genetic approaches have identified key components of the regulation of leaf senescence and have revealed glimpses of the underlying molecular mechanisms.

Apoptosis↗
PubMed1999

Forward genetics in mammalian cells: functional approaches to gene discovery.

Definitive proof of function in biological systems requires genetic analysis. Only when the loss of a particular protein corresponds to the loss of a specific function can one be sure that the protein truly affects the function. Changing the pattern of gene expression through random mutagenesis or by introducing expression libraries, followed by selection of mutant or variant cells and identification of a missing or overexpressed protein, has the power to reveal or confirm the roles of specific components of signaling pathways and to provide mutant cell lines and cDNA reagents to be used in defining detailed mechanisms through structure-function analyses. These examples of forward genetics contrast with reverse genetic approaches, where the function of a known gene product is explored by knockout or replacement. Here we review a broad range of techniques that have been used to alter gene expression randomly in mammalian cells, with examples of specific discoveries that have resulted from these applications of forward genetics.

Animals↗
PubMed2000

Establishment of a rescue system for canine distemper virus.

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

Animals↗
PubMed2000

Transdominant genetics, peptide inhibitors and drug targets.

The future of medical therapy is tied to the discovery of high-quality drug targets and drugs. Transdominant genetics provides a function-based route to peptide inhibitors that can be used as probes to identify protein targets or as reagents for drug development. Both forward-genetic and reverse-genetic applications have been implemented. The move is now underway to expand upon advances made in model systems and exploit screens of real therapeutic value.

Animals↗
PubMedDate not supplied

Cis-acting signals and trans-acting factors involved in influenza virus RNA synthesis.

Influenza A virus RNA replication and expression is directed from cis-acting sequences present on the viral RNAs with the help of trans-acting factors encoded by the virus. Ribonucleoprotein (RNP) complexes reconstituted from synthetic cDNA-derived RNA and purified viral proteins have facilitated the dissection of these cis-acting signals and trans-acting factors. Prior to these studies influenza viruses and other negative-strand RNA viruses were refractory to molecular genetic manipulations. These reverse genetic studies have helped in defining the promoter and polyadenylation signals required for viral RNA synthesis. Studies involving the use of reconstituted RNP complexes have revealed that the viral proteins PB1, PB2, PA, and the nucleoprotein (NP) are necessary for replication and expression of influenza virus RNA. Inroads have also been made in determining the cellular proteins that participate in influenza virus gene expression and replication. The yeast interactive trap system has been used to identify and clone a gene (NPI-1), which encodes a protein that interacts with the influenza virus NP suggesting that this cellular protein is a trans-acting factor functioning in viral RNA synthesis.

Base Sequence↗
PubMed2003

The devil in the details of life-history evolution: instability and reversal of genetic correlations during selection on Drosophila development.

The evolutionary relationships between three major components of Darwinian fitness, development rate, growth rate and preadult survival, were estimated using a comparison of 55 distinct populations of Drosophila melanogaster variously selected for age-specific fertility, environmental-stress tolerance and accelerated development. Development rate displayed a strong net negative evolutionary correlation with weight at eclosion across all selection treatments, consistent with the existence of a size-versus-time tradeoff between these characters. However, within the data set, the magnitude of the evolutionary correlation depended upon the particular selection treatments contrasted. A previously proposed tradeoff between preadult viability and growth rate was apparent only under weak selection for juvenile fitness components. Direct selection for rapid development led to sharp reductions in both growth rates and viability. These data add to the mounting results from experimental evolution that illustrate the sensitivity of evolutionary correlations to (i) genotype-by-environment (G x E) interaction, (ii) complex functional-trait interactions, and (iii) character definition. Instability, disappearance and reversal of patterns of genetic covariation often occur over short evolutionary time frames and as the direct product of selection, rather than some stochastic process. We suggest that the functional architecture of fitness is a rapidly evolving matrix with reticulate properties, a matrix that we understand only poorly.

Animals↗
PubMed2001

T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics.

T-DNA insertion mutants have become a valuable resource for studies of gene function in Arabidopsis. In the course of both forward and reverse genetic projects, we have identified novel interchromosomal rearrangements in two Arabidopsis T-DNA insertion lines. Both rearrangements were unilateral translocations associated with the left borders of T-DNA inserts that exhibited normal Mendelian segregation. In one study, we characterized the embryo-defective88 mutation. Although emb88 had been mapped to chromosome I, molecular analysis of DNA adjacent to the T-DNA left border revealed sequence from chromosome V. Simple sequence length polymorphism mapping of the T-DNA insertion demonstrated that a >40-kbp region of chromosome V had inserted with the T-DNA into the emb88 locus on chromosome I. A similar scenario was observed with a prospective T-DNA knockout allele of the LIGHT-REGULATED RECEPTOR PROTEIN KINASE (LRRPK) gene. Whereas wild-type LRRPK is on lower chromosome IV, mapping of the T-DNA localized the disrupted LRRPK allele to chromosome V. In both these cases, the sequence of a single T-DNA-flanking region did not provide an accurate picture of DNA disruption because flanking sequences had duplicated and inserted, with the T-DNA, into other chromosomal locations. Our results indicate that T-DNA insertion lines--even those that exhibit straightforward genetic behavior--may contain an unexpectedly high frequency of rearrangements. Such duplication/translocations can interfere with reverse genetic analyses and provide misleading information about the molecular basis of mutant phenotypes. Simple mapping and polymerase chain reaction methods for detecting such rearrangements should be included as a standard step in T-DNA mutant analysis.

Arabidopsis↗
PubMed2005

Gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication.

The avian coronavirus Infectious bronchitis virus (IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins.

Animals↗
PubMed2001

Vaccinia virus-free recovery of vesicular stomatitis virus.

The advent of reverse-genetics represents a powerful new approach to elucidate aspects of negative-sense RNA virus replication. The reverse-genetics system established previously for vesicular stomatitis virus (VSV) required four plasmids encoding the nucleoprotein (N), phosphoprotein (P), polymerase (L), and the full-length, anti-genomic RNA. Transcription to yield the antigenomic RNA as well as the N, P, and L, mRNAs was initiated by bacteriophage T7 polymerase expressed from a recombinant Vaccinia virus. In this report, we describe the successful recovery of infectious VSV in the absence of Vaccinia virus. The N, P, and L genes of VSV were inserted downstream of both the T7 promoter and an internal ribosomal entry site (IRES element). T7 polymerase was expressed constitutively from BSR-T7/5 cells. RTPCR was used to confirm that the recovered VSV was derived from transfected DNA. Virion protein profile, CPE in tissue culture, and virus titer of the recombinant VSV were indistinguishable from those of parental VSV. Thus, the need for Vaccinia virus is eliminated with this system, making it an attractive, alternative approach for the recovery of infectious VSV from DNA.

Animals↗
PubMed1999

[Molecular-genetic approach to congenital malformation syndromes].

The causes of congenital malformation syndromes had long remained obscure, because their biochemical bases were unknown. However, with recently developed reverse-genetic and/or Human Genome Project-derived technology, we have been able to approach such disorders. These new methods include positional cloning techniques, candidate gene approach and positional candidate strategies. Among them, positional cloning has become most reliable nowadays, and three ways are mainly available to obtain position information of disease genes, i.e., data from linkage analysis of disease families; comparative maps between different species; breakpoints of disease-related de novo chromosomal translocations. I present here some examples for each strategy: a large kindred with mesomelic dysplasia Kantaputra type for linkage data, and its seemingly allelic bone dysplasia associated with t (2; 8) for positional cloning that starts from a chromosomal translocation; Waardenburg syndrome for candidate gene approach; and the p57KIP2 gene for positional candidate approach to Wiedemann-Beckwith syndrome. I finally emphasize that a human link composed of general physicians, molecular geneticists and clinical researchers is essential to reach a goal of reverse genetics. Without such a goal, we can neither understand the genesis of diseases nor develop their therapy.

Beckwith-Wiedemann Syndrome↗
PubMedDate not supplied

[Protease and reverse transcriptase genetic polymorphism in HIV type 1 subtype A variants predominating in cis countries].

To define frequencies of drug resistance mutations among HIV-1 variants circulating within the territory of Russia, subtype A HIV-1 nucleotide sequences encoding protease and reverse transcriptase were analyzed. The analysis was carried out in 141 antiretroviral-naive individuals. Low frequency (less than 1%) of primary drug resistance mutations was shown. However, high frequencies of secondary mutations V77I in protease and A62V in RT (67% H 63%, respectively) linked to each other in most cases were observed. The HIV-1 isolates bearing both substitutions (MutV77I/A62V) were also characterized by the presence of several synonymous mutations, suggesting common origin for these viruses. HIV Biochip Hybridization microarray and/or Restriction fragment-length polymorphism analyses were performed to characterize gene pol polymorphism in additional 178 subtype A HIV-1 isolates. Among total 319 samples studied, Mutv77IA62V variant accounted for 56%, and was found to predominate in Russia in terms of both its geographical distribution and number of cases caused. Moreover, these viruses were prevalent in the regions known to have highest incidence of HIV-1 infection (Irkutsk, Samara, and Moscow regions). In addition, three other variants were found: viruses not containing the substitutions V77I or A62V, and variants bearing only one of them. Evolutional relationships between all four HIV-1 variants, as well as potential impact of the gene pol polymorphism on HIV-1 replicative fitness and drug resistance development are discussed.

Amino Acid Substitution↗
PubMed2004

Downregulation of gene expression with negatively charged peptide nucleic acids (PNAs) in zebrafish embryos.

We found that negatively charged, highly soluble PNA analogs with alternating phosphonates (HypNA-pPNAs) are effective and specific antisense agents in zebrafish embryos, showing comparable potency and greater specificity against chordin, ntl and uroD. In addition, we successfully phenocopied a dharma mutant that had not been found susceptible to MO knockdown. Both MO and HypNA-pPNAs against a tumor suppressor gene induced comparable upregulation of p53, illustrating similar effects on transcription profiles. HypNA-pPNAs are therefore a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes in zebrafish or validating newly identified orthologs, and perhaps for reverse genetic studies in other organisms.

Animals↗
PubMedDate not supplied

Molecular biology in arteriosclerosis research.

The topics discussed in this article illustrate how molecular biology will have a dramatic impact on arteriosclerosis research. DNA clones for a small number of relevant proteins have been isolated, and studies are underway in numerous laboratories to extend these initial studies. The techniques of molecular biology will provide major advances in our understanding of numerous proteins directly or indirectly involved in the atherogenic process. Cloning technology will solve the primary structures of many proteins that can not be purified in quantities sufficient for classical methods of analysis. Studies of regulation will benefit from the availability of DNA probes, the ability to generate site-directed antibodies, and the use of reverse genetics to identify nucleic acid sequences involved in the regulation of gene expression. Studies of gene structure and genetic polymorphisms will unravel the genetic basis for defects in lipid and lipoprotein metabolism and should provide valuable reagents for clinical screening and diagnosis. The reverse genetics approach will permit the systematic analysis of structure-function relationships at the protein level in a manner not previously possible. Each of these will contribute to our understanding of the atherogenic process and should provide insight into ways of preventing and treating arteriosclerosis.

Amino Acid Sequence↗