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 415 records · Page 23Linked to original sources

Recent advances in understanding of the DNA double-strand break repair machinery of plants.

Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA double-strand break (DSB) is both particularly threatening and common. Double-strand breaks arise from exposure to DNA damaging agents, but also from cell metabolism-in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D)J gene rearrangement. Cells possess several overlapping repair pathways to deal with these breaks, generally designated as genetic recombination. Genetic and biochemical studies have provided considerable amounts of data about the proteins involved in recombination processes and their functions within these processes. Although they have long played a key role in building understanding of genetics, relatively little is known at the molecular level of the genetic recombination processes in plants. The use of reverse genetic approaches and the public availability of sequence tagged mutants in Arabidopsis thaliana have led to increasingly rapid progress in this field over recent years. The rapid progress of studies of recombination in plants is obviously not limited to the DSB repair machinery as such and we ask readers to understand that in order to maintain the focus and to rest within a reasonable length, we present only limited discussion of the exciting advances in the of plant meiosis field, which require a full review in their own right . We thus present here an update on recent advances in understanding of the DSB repair machinery of plants, focussing on Arabidopsis and making a particular effort to place these in the context of more general of understanding of these processes.

Adenosine Triphosphatases↗

Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains.

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 41 nucleotide changes, 12 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.

5' Untranslated Regions↗

Protein sorting in yeast: the role of the vacuolar proton-translocating ATPase.

We are investigating the physiological roles of organelle acidification in yeast by two different approaches. First, we have identified two mutants which are defective in acidification of the yeast lysosome-like vacuole from among a collection of mutants which mis-sort soluble vacuolar proteins to the cell surface. These mutants have been helpful in identifying other cellular functions linked to acidification, such as the activation of vacuolar zymogens. We have complemented this classical genetic approach to acidification with direct biochemical and reverse genetic studies on the yeast vacuolar proton-translocating ATPase (H+-ATPase), the enzyme responsible for vacuolar network acidification. Our biochemical characterization of this enzyme indicates that it is a multisubunit complex with many structural similarities to other vacuolar H+-ATPases. Like the other vacuolar H+-ATPases characterized, it also shares some structural features with the F1F0-type ATPases of mitochondria, chloroplasts, and Escherichia coli. We are currently cloning the genes for the subunits of the yeast vacuolar H+-ATPase. Mutagenesis of the cloned genes will allow us to determine the phenotype of yeast cells expressing a vacuolar H+-ATPase altered in well controlled ways. We are also beginning to investigate how the subunits of the vacuolar H+-ATPase are assembled into the enzyme complex and targeted to their proper cellular location.

Biological Transport↗

Analysis of the floral transcriptome uncovers new regulators of organ determination and gene families related to flower organ differentiation in Gerbera hybrida (Asteraceae).

Development of composite inflorescences in the plant family Asteraceae has features that cannot be studied in the traditional model plants for flower development. In Gerbera hybrida, inflorescences are composed of morphologically different types of flowers tightly packed into a flower head (capitulum). Individual floral organs such as pappus bristles (sepals) are developmentally specialized, stamens are aborted in marginal flowers, petals and anthers are fused structures, and ovaries are located inferior to other floral organs. These specific features have made gerbera a rewarding target of comparative studies. Here we report the analysis of a gerbera EST database containing 16,994 cDNA sequences. Comparison of the sequences with all plant peptide sequences revealed 1656 unique sequences for gerbera not identified elsewhere within the plant kingdom. Based on the EST database, we constructed a cDNA microarray containing 9000 probes and have utilized it in identification of flower-specific genes and abundantly expressed marker genes for flower scape, pappus, stamen, and petal development. Our analysis revealed several regulatory genes with putative functions in flower-organ development. We were also able to associate a number of abundantly and specifically expressed genes with flower-organ differentiation. Gerbera is an outcrossing species, for which genetic approaches to gene discovery are not readily amenable. However, reverse genetics with the help of gene transfer has been very informative. We demonstrate here the usability of the gerbera microarray as a reliable new tool for identifying novel genes related to specific biological questions and for large-scale gene expression analysis.

Asteraceae↗

[From positional cloning to gene identification and its function in genetic diseases].

The huge development of genetic tools, together with the availability of powerful computers have enabled the mapping of several loci responsible for genetic diseases by the positional cloning technique, also called reverse genetics. The step consisting of identifying the gene itself remains a difficult step and the function of the gene identified may remain difficult to establish. In this chapter, we will examine the various steps allowing the identification of the gene, techniques allowing the creation of animal models of human diseases and the recent progresses of these techniques.

Animals↗

Marker-assisted study of genetic background and gene-targeted locus modifiers in lymphopoietic phenotypes.

Forward and reverse genetic approaches facilitate the molecular dissection of individual gene functions and the integration of individual gene functions into multi-gene processes in the context of the whole organism. Variations in mutant phenotypes due to genetic background differences have been well documented through the analysis of mouse mutants. Nevertheless, recommendations concerning the assessment of genetic background as it impacts on phenotype, and utilization of genetic background differences to identify and integrate gene functions have been largely overlooked. Genetic background as it relates to immunological mutants will be discussed utilizing an Ets1-targeted allele to exemplify phenotypic variation due to background. Marker-assisted strategies for the identification of genetic modifiers, especially those linked to the targeted locus, will also be considered.

Animals↗

A modified viral satellite DNA-based gene silencing vector is effective in association with heterologous begomoviruses.

We have previously reported effective gene silencing of a transgene and endogenous plant genes in tobacco and tomato plants using a modified viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV). In this study, we constructed a similar gene silencing vector (DNADeltaC12beta) based on the satellite DNAbeta associated with Tobacco curly shoot virus (TbCSV) by replacing its betaC1 gene with a multiple cloning site. Strong and stable silencing of cognate genes was achieved when this vector, carrying a fragment of the green fluorescent protein (GFP) transgene or a sulfur (Su) endogenous gene encoding one unit of the chloroplast enzyme magnesium chelatase required for chlorophyll II production, was co-agroinoculated with TbCSV used as a helper virus. GFP silenced transgenic Nicotiana benthamiana plants appear red under UV illumination due to loss of green fluorescence, while the Su silenced plants appear white as a result of failure to synthesize chlorophyll. Our results show that the efficiency of Su silencing is independent of the insert orientation in both N. benthamiana and N. glutinosa plants. Most significant however, is the observation that in association with heterologous begomoviruses, such as TYLCCNV or Malvastrum yellow vein virus, the DNADeltaC12beta vector could still effectively induce transgene and endogenous gene silencing in tobacco plants. These observations suggest that the modified viral satellite DNA vector can be applied as a reverse genetics tool for the study, analysis and discovery of gene function in more plants.

Blotting, Southern↗

Genetic exchange in Trypanosoma brucei: selection of hybrid trypanosomes by introduction of genes conferring drug resistance.

Genetic exchange in Trypanosoma brucei ssp. can occur when 2 different strains are cotransmitted through the tsetse fly vector. We have introduced heterologous genes for drug resistance (neo or hph) into parental trypanosome lines by electroporation. Drug resistant lines were then crossed in vivo in the fly or in vitro. Hybrids were subsequently selected by double drug resistance. Analysis of trypanosomes from both fly midguts and salivary glands showed the latter to be the probable site of genetic exchange. This is one of the first applications of reverse genetics to a longstanding problem in parasite biology.

Animals↗

The soybean cyst nematode, Heterodera glycines: a genetic model system for the study of plant-parasitic nematodes.

Despite advances in understanding plant responses to nematode infection, little information exists regarding parasitic mechanisms. Recently, it has become possible to perform genetic analysis of soybean cyst nematode. Integration of classic and reverse genetics and genomic approaches for the parasite, with host genetics and genomics will expand our knowledge of nematode parasitism.

Animals↗

A point mutation within the replicase gene differentially affects coronavirus genome versus minigenome replication.

During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

Animals↗

Inhibition of virus replication by RNA interference.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.

Animals↗

Recombinant respiratory syncytial viruses lacking the C-terminal third of the attachment (G) protein are immunogenic and attenuated in vivo and in vitro.

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpDeltaG118, rA2cpDeltaG174, rA2cpDeltaG193, and rA2cpDeltaG213. All rA2cpDeltaG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37 degrees C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpDeltaG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpDeltaG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpDeltaG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

Animals↗

The development of live attenuated cold-adapted influenza virus vaccine for humans.

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.

Adaptation, Physiological↗

Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus.

Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA.

Animals↗

No association between alpha-adducin 460 polymorphism and essential hypertension in a Japanese population.

Many unknown genetic factors are involved in the pathogenesis of hypertension. Recently, the reverse genetic approach revealed that some genetic variants, such as angiotensinogen, lipoprotein lipase, and alpha-adducin gene polymorphisms, increase the risk for hypertension. Both in rat and human, the genetic predisposition to hypertension was confirmed only for angiotensinogen and alpha-adducin genes. Adducin is a membrane cytoskeletal protein, which is thought to regulate sodium transport. Abnormalities of membrane sodium transport in the kidney play an important role in hypertension. A recent report by Cusi et al showed that the Trp allele of alpha-adducin polymorphism (Gly 460 Trp) is associated with an increased risk of hypertension in whites, which led us to carry out a case-control study to examine whether the same association is observed in the Japanese population. We recruited 170 hypertensive and 194 normotensive Japanese subjects and compared the genotype distribution of alpha-adducin 460 polymorphism between cases and controls and between whites and Japanese. Trp allele frequency of controls in the Japanese subjects was twice as high as in the whites. However, no association was observed between alpha-adducin polymorphism and hypertension. Furthermore, alpha-adducin 460 polymorphism was not associated with any clinical characteristics. Accordingly, we concluded that alpha-adducin 460 polymorphism is not a major genetic risk for hypertension in Japanese people.

Alleles↗

Target-selected inactivation of the zebrafish rag1 gene.

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 F1 fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 F1 fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.

Amino Acid Substitution↗

The molecular genetics of complex inherited diseases.

The majority of inherited diseases are due to unknown biochemical defects, or are either polygenetic or multifactorial. Due to advances in molecular genetics, it is now possible to study these conditions, either by reverse genetics (using cloning techniques to move from linkage to gene, and then determining the protein involved) or by dissecting a complex disease into a set of monogenic paradigms.

Chromosome Mapping↗

XY sex-reversal syndrome in the domestic horse.

The XY sex-reversal syndrome occurs when a phenotypic mare is born that has the karyotype of a stallion. The syndrome is manifested by both genotypic and phenotypic heterogeneity. The sex-reversed genetic condition occurs frequently within certain pedigrees where XY females have been found and can be readily detected by chromosome karyotyping. The phenotypic spectrum ranges from the feminine mare with a reproductive tract that is within normal limits to the greatly masculinized mare. Pedigree analysis suggests that there are two modes of inheritance: (1) an X-linked recessive or autosomal sex-limited dominant transmitted through the female and (2) an autosomal sex-limited dominant or a Y chromosomal mutation with variable expression transmitted through the male. The proportion of female to male progeny of the 69 top-producing Arabian stallions in the world is 53 to 47, indicating that the occurrence of genetic defects affecting the sex ratio such as the XY sex-reversal syndrome is relatively infrequent. In nine cases where the sex ratios of stallions deviated significantly from the expected sex ratio, the incidence of female infertility also increased. Cytogenetic screening of young animals would provide for early detection and avoid the expense and disappointment of continuous breeding attempts. Just as importantly, it would aid in the reduction and the possible elimination of this genetic condition from breeding herds.

Animals↗