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At least 145 records · Page 8Linked to original sources
PubMed2004

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Arabidopsis↗
PubMed2002

Toward understanding the pathogenicity of wild-type measles virus by reverse genetics.

The Edmonston (Ed) strain of measles virus (MV) isolated in primary human kidney cells in 1954 has long been thought of as a representative MV strain. But this view has been challenged by wild-type MV strains isolated in marmoset B-lymphoblastoid B95a cells. Although the Ed strain is not pathogenic in monkey models, wild-type MV isolated in B95a cells from measles patients induces clinical signs typical of human measles, indicating that wild-type MV retains its pathogenicity. In addition, wild-type MV has restricted cell tropism and replicates only in B95a and some lymphocyte cell lines. This is in sharp contrast to the ability of the Ed strain to replicate in a variety of human cell lines. To understand the molecular basis for the pathogenicity and the cell tropism of wild-type MV, we have established a reverse genetics system based on a highly pathogenic wild-type MV strain (IC-B) isolated in B95a cells. By using this system, we have constructed recombinant wild-type and Ed strains of MV bearing heterologous envelope hemagglutinin (H) proteins, and we have examined roles of the H protein in determining the cell tropism. Our results clearly indicate that the MV cell tropism is determined by not only the H protein, but also other viral proteins. We thus propose the presence of another unidentified MV receptor on the surface of Vero cells.

Base Sequence↗
PubMed2006

Reverse genetic analysis of neurogenesis in the zebrafish retina.

To gain an understanding of molecular events that underlie pattern formation in the retina, we evaluated the expression profiles of over 8000 transcripts randomly selected from an embryonic zebrafish library. Detailed analysis of cDNAs that display restricted expression patterns revealed factors that are specifically expressed in single cell classes and are potential regulators of neurogenesis. These cDNAs belong to numerous molecular categories and include cell surface receptors, cytoplasmic enzymes, and transcription factors. To test whether expression patterns that we have uncovered using this approach are indicative of function in neurogenesis, we used morpholino-mediated knockdown approach. The knockdown of soxp, a transcript expressed in the vicinity of the inner plexiform layer, revealed its role in cell type composition of amacrine and ganglion cell layers. Blocking the function of cxcr4b, a chemokine receptor specifically expressed in ganglion cells, suggests a role in ganglion cell survival. These experiments demonstrate that in situ hybridization-based reverse genetic screens can be applied to isolate genetic regulators of neurogenesis. This approach very well complements forward genetic mutagenesis studies previously used to study retinal neurogenesis in zebrafish.

Animals↗
PubMed2001

Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40.

DT40 is an avian leucosis virus-transformed chicken B-lymphocyte line which exhibits high ratios of targeted to random integration of transfected DNA constructs. This efficient targeted integration may be related to the ongoing diversification of the variable segment of the immunoglobulin gene through homologous DNA recombination-controlled gene conversion. DT40s are a convenient model system for making gene-targeted mutants. Another advantage is the relative tractability of these cells, which makes it possible to disrupt multiple genes in a single cell and to generate conditionally gene-targeted mutants including temperature-sensitive mutants. There are strong phenotypic similarities between murine and DT40 mutants of various genes involved in DNA recombination. These similarities confirm that the DT40 cell line is a reasonable model for the analysis of vertebrate DNA recombination, despite obvious concerns associated with the use of a transformed cell line, which may have certain cell-line-specific characteristics. Here we describe our studies of homologous DNA recombination in vertebrate somatic cells using reverse genetics in DT40 cells.

Animals↗
PubMed2006

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as 'Hit and Run' and 'vector recycling' which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.

Animals↗
PubMed1999

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis.

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.

Arabidopsis↗
PubMed2005

[Generation of attenuated H5N1 and H5N2 subtypes of influenza virus recombinants by reverse genetics system].

The HA connecting peptide at cleavage site, PQRERRKKR / GL, of an H5N1 subtype avian influenza virus (AIV) was replaced with PQRESR / GL, and then the modified HA gene was cloned into the transcription/expression vector, pHW2000, constructing a plasmid named pHW524-HA. The NA (N1) gene from the H5N1 virus and the NA (N2) gene from an H9N2 AIV were also cloned into pHW2000 separately, resulting in plasmids pHW506-NA and pHW206-NA. With the organization of pHW524-HA, pHW506-NA or pHW206-NA, and six plasmids containing internal genes from A/WSN/33 backbone virus, two transfectants, H5N1/WSN and H5N2/WSN, were subsequently generated by eight-plasmid system. After 15 consecutive passages in embryonated eggs, the two recombinants grew up to high titers of 1:2(9) in hemagglutination test with no changes in nucleotide sequences of the surface genes detected. Both the recombinant viruses belonged to mildly pathogen when evaluated by the pathogenicity test in six-week-old SPF chickens. H5N2/WSN recombinant virus was obviously less pathogenic than H5N1/WSN virus for embryonated chicken eggs. This presentation showed that the reverse genetics system is a very useful tool for studying the construction and function of individual genes and for the generation of virus as vaccine candidate.

Animals↗
PubMed2006

Identification and reverse genetic analysis of mitochondrial processing peptidase and the core protein of the cytochrome bc1 complex of Caenorhabditis elegans, a model parasitic nematode.

Mitochondria could be a good target for anti-parasitic drugs. The alpha and beta subunits of mitochondrial processing peptidase (MPP) and the core subunits of the cytochrome bc1 complex, UCR-1 and UCR-2, are homologous to one another and are important for mitochondrial functions. However, our knowledge of these proteins in nematodes is very limited. Caenorhabditis elegans, a free-living nematode, has six genes coding for proteins homologous to these subunits. On primary structure comparison, and immunochemical and enzymological analyses, the gene products were assigned as follows: Y71G12B.24, alpha-MPP; ZC410.2, beta-MPP; F56D2.1, UCR-1; VW06B3R.1, T10B10.2; and T24C4.1, UCR-2. The primary structures of beta-MPP and UCR-1 from Brugia malayi, a parasitic nematode causing human filariasis, were deduced from their cDNA structures. Phylogenetic analysis showed that the UCR-1s from both C. elegans and B. malayi were less related to mammalian UCR-1s than to MPPs from various organisms. MPP and the bc1 complex are essential for the life cycle of C. elegans, because their reverse genetic inhibition is lethal. This suggests the possibility that these proteins are also essential for the viability of B. malayi and other parasitic nematodes, and are potential targets for anti-parasitic agents.

Amino Acid Motifs↗
PubMed2005

Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.

A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after transfection of RNA transcripts was dependent on the presence of bile acids, consistent with our recent identification of a bile acid-mediated signaling pathway required for PEC replication (Chang et al., Proc. Natl. Acad. Sci. USA 101:8733-8788, 2004). Recovery of virus was verified by detection of PEC antigen in transfected cells by immunofluorescence and enzyme-linked immunosorbent assays, direct observation of recovered viral particles by electron microscopy, and partial sequence analysis of their genomes (first 1,070 nucleotides) to differentiate them from tissue culture-adapted parental virus. The recovered virus retained its ability to infect piglets when administered by the oral route and showed an attenuated phenotype similar to that of the tissue culture-adapted parental virus. This reverse genetics system for PEC provides a new tool to study the molecular basis of replication and pathogenesis for caliciviruses associated with diarrheal disease.

Animals↗
PubMed2003

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice.

A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.

Blotting, Southern↗
PubMed2001

Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses.

BACKGROUND: Post-transcriptional gene silencing (PTGS) by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs) for their ability to silence cytoplasmic RNA genomes. RESULTS: Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. CONCLUSIONS: Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Gene Silencing↗
PubMed2001

Population admixture may appear to mask, change or reverse genetic effects of genes underlying complex traits.

Association studies using random population samples are increasingly being applied in the identification and inference of genetic effects of genes underlying complex traits. It is well recognized that population admixture may yield false-positive identification of genetic effects for complex traits. However, it is less well appreciated that population admixture can appear to mask, change, or reverse true genetic effects for genes underlying complex traits. By employing a simple population genetics model, we explore the effects and the conditions of population admixture in masking, changing, or even reversing true genetic effects of genes underlying complex traits.

Genetics, Population↗
PubMed1997

Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance.

The human mind was engaged with fundamental questions on the nature of heredity long before the study of genetics became a scientific discipline. Many traits, such as height, eye color, blood pressure, or cancer susceptibility, have been known to run in families, although the genes or combination of genes that underlie these observable characteristics remain unknown in most cases. Differences in susceptibility to environmental agents in humans are likewise determined by variations in genetic background--genetic polymorphisms. In this article, we review the current status of studies on human polymorphisms in drug-metabolizing enzymes and discuss various approaches to the analysis of genetic polymorphisms. We expect that in the near future, novel methods in genetic analysis of human populations will be likely to play a key role in the identification of genes of toxicological relevance.

Cytochrome P-450 Enzyme System↗
PubMedDate not supplied

[Determinant of resistance to kanamycin in Streptomycin rimosus: amplification in the chromosome and reversible genetic instability].

The study on the kanamycin resistance determinant (Kanr) in an oxytetracycline--producing strain of S. rimosus showed that it was capable of amplifying in the chromosome during selection for increasing the antibiotic resistance level. The amplification of the DNA fragment with a molecular weight of 10.3 MDa containing Kanr amounted to 300 copies per genome, which resulted in a more than 1000-fold increase in kanamycin resistance level. Cloning of the Kanr determinant on plasmid SLP1.2 in S. lividans strain 66 was performed. In Streptomyces lividans strain 66 the Kanr determinant preserved the capacity for amplification in the hybrid plasmid pSU10 integrated into the chromosome. The Kanr determinant in the strains of S. rimosus and S. lividans was characterized by transfers Kanr in equilibrium Kans with a frequency of 1 X 10(-3). It was shown that the mutation in S. lividans strain 66 resulting in phenotype Kans was not connected with the structural Kanr gene on plasmid pSU10 but was localized on the chromosome. Phenotype Kans was promoted by a decrease in the number of the copies of the regulatory genetic element designated RES1. The reverse to phenotype Kanr might be due to one of the following events: amplification to the initial level of RES1 and amplification up to 200 copies per the genome of the hybrid plasmid pSU10 containing the Kanr determinant. Amplification of the Kanr determinant with preserved initial level of RES1 element resulted in a more than 1000 times increase in the resistance level.

Chromosome Mapping↗
PubMed2005

Reverse genetic analyses of gamete-enriched genes revealed a novel regulator of the cAMP signaling pathway in Dictyostelium discoideum.

Sexual development in Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodiesterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.

Amino Acid Sequence↗
PubMed1993

High-frequency genetic reversion mediated by a DNA duplication: the mouse pink-eyed unstable mutation.

The mouse pink-eyed unstable (p(un)) mutation, affecting coat color, exhibits one of the highest reported reversion frequencies of any mammalian mutation and is associated with a duplication of genomic DNA at the p locus. In this study, genomic clones containing the boundaries of the p(un) duplication were isolated and characterized. The structure of these sequences and their wild-type and revertant counterparts were analyzed by restriction mapping, PCR product analysis, DNA sequence analysis, and pulsed-field gel electrophoresis. DNA from p(un) was distinguished from wild-type and revertant DNA by a head-to-tail tandem duplication of approximately 70 kilobases. No differences were detected between revertant and wild-type DNAs. Thus, the reversion in phenotype of p(un) mice is coupled with the loss of one copy of an approximately 70-kilobase duplicated segment. Testable models are presented to account for p(un) reversion.

Alleles↗
PubMed2002

Genetic reversion of inherited skin disorders.

Human epidermis is a squamous stratified epithelium whose integrity relies on balanced processes of cell attachment, proliferation, and differentiation. In monogenic skin dermatoses, such as mecano-bullous diseases, or DNA repair deficiencies such as the xeroderma pigmentosum (XP), alterations of skin integrity may have devastating consequences as illustrated by the extremely high epidermal cancer proneness of XP patients. The lack of efficient pharmacological treatments, the easy accessibility of skin, and the possibility of long term culture and genetic manipulations ex vivo of epidermal keratinocytes, have encouraged approaches toward gene transfer and skin therapy prospects. We review here some of the human genetic disorders that exhibit major traits in skin, as well as requirements and difficulties inherent to approaches aimed at stable phenotypic correction.

DNA Repair↗