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At least 811 records · Page 45Linked to original sources
PubMed2005

Transplantation of a polyembryonic wasp embryo: a technique for transferring endoparasitic embryo into the host egg.

Concealed development of many animal embryos prevents examination of development and limits the application of embryo manipulation techniques aimed at understanding developmental processes. In embryos developing in utero, such as in mammals, it is necessary to dissect embryos from the mother and, upon manipulative intervention, to implant them back into the recipient. Parasitic wasps present a promising system for understanding the evolution of early developmental processes. In basal ectoparasitic species that lay eggs on the surface of the host, it is possible to adapt embryo manipulation techniques developed in Drosophila. However, their derived endoparasitic relatives, which exhibit various modifications of developmental programs, undergo concealed development within the host body. For example, the parasitic polyembryonic wasp Copidosoma floridanum oviposits an egg into the egg of the host moth Trichoplusia ni. The host larva emerges and the parasite undergoes development within the host body, preventing embryo manipulation as a means of examining developmental regulation. Here we present a protocol for embryo transfer that allows the transplantation of C. floridanum egg into the host egg. This approach opens a new avenue in the application of various embryo manipulation techniques aimed at understanding the evolution of embryogenesis in endoparasitic Hymenoptera. In addition, this approach has potential for the development of other tools in C. floridanum, such as transgenesis and reverse genetics, which can also be extended to other endoparasitic species.

Animals↗
PubMed2002

The FAR proteins of parasitic nematodes: their possible involvement in the pathogenesis of infection and the use of Caenorhabditis elegans as a model system to evaluate their function.

Parasitic nematodes secrete a structurally novel class of fatty acid and retinol-binding (FAR) protein into the surrounding tissues of the host. These proteins are of interest because they may play an important role in scavenging fatty acids and retinoids from the host that are essential for the survival of the parasite and also because the localised depletion of such lipids may have immunomodulatory effects that compromise the host immune response. Research into the biological function(s) of the FAR proteins has been severely hampered by the difficulties associated with the life-cycle propagation of parasitic nematodes and the current intractability of the parasites to reverse-genetic studies. The genome of the free-living nematode Caenorhabditis elegans, however, encodes eight FAR-like proteins, and in this review we compare the FAR proteins of C. elegans and parasitic nematodes, and we discuss the suitability of C. elegans as a model system to investigate the biological function(s) of the FAR proteins of parasitic nematodes.

Amino Acid Sequence↗
PubMed2001

Leishmania infection and virulence.

Leishmaniasis is among the most important infectious diseases of the developing world. With the advent of effective culture conditions for the various stages of the life cycle, together with state of the art biochemical, genomics, and reverse genetic approaches, it has been possible to identify virulence factors required for the infection process. Several of these virulence factors are discussed within including lipophosphoglygan, A2, cysteine proteinases, and gp63.

Animals↗
PubMed2002

The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N-acetylglucosamine and N,N'-diacetylchitobiose.

During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.

ATP-Binding Cassette Transporters↗
PubMed2004

Mapping Ds insertions in barley using a sequence-based approach.

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgaresubsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions. The principal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

Base Sequence↗
PubMed2000

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker.

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas.

Amikacin↗
PubMed2002

NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells.

Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation.

Cells, Cultured↗
PubMed2003

Temperature sensitive mutations in the genes encoding the NS1, NS2A, NS3, and NS5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice.

Using reverse genetics, it is possible to readily add well-defined attenuating mutations to the genome of wild type or incompletely attenuated dengue (DEN) viruses to generate vaccine candidates that exhibit the desired balance between attenuation and immunogenicity. Here, we describe the identification of eight temperature sensitive missense mutations distributed in four non-structural protein genes that specify a 60- to 10,000-fold range of restricted replication in the suckling mouse brain compared to wild type recombinant DEN4 virus. The usefulness of such mutations in flavivirus vaccine design and development is discussed.

Animals↗
PubMed2005

Integration of mutations responsible for the attenuated phenotype of Pepper mild mottle virus strains results in a symptomless cross-protecting strain.

An enhanced attenuated strain of Pepper mild mottle virus (PMMoV) was constructed by incorporating mutations that affect viral attenuation from three reported attenuated strains of PMMoV, which causes serious economic losses in the production of green pepper in Japan. The new strain caused no symptoms on pepper plants and protected them from infection by a wild-type strain. The mutations responsible for viral attenuation were located in the intervening region (IR) of the 126-kDa/183-kDa proteins. The mutations had synergistic effects in terms of the attenuation of symptoms and decreased the accumulation of the viral coat protein in infected pepper plants. In this paper, we propose an efficient method for the improvement of attenuated viruses by reverse genetics in plant viruses.

Base Sequence↗
PubMed2005

Dimerization of glycoprotein E(rns) of classical swine fever virus is not essential for viral replication and infection.

The pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.

Animals↗
PubMed2005

Infectious bursal disease virus recovery from Vero cells transfected with RNA transcripts is enhanced by expression of the structural proteins in trans.

Positive sense RNA transcripts of infectious bursal disease (IBD) virus genome segments A and B have previously been shown to be infectious. In this study we demonstrate that recovery of IBD virus from the transfection of Vero cells with positive sense RNA transcripts of genome segments A and B was enhanced by expression of the viral structural proteins VP2 with VP3 or by expression of viral polyprotein VP243 from DNA plasmids in trans. Expression of individual viral proteins VP2, VP3, or VP4 alone from DNA plasmids did not enhance IBD virus recovery. Earliest virus recovery from transfection of positive sense RNA transcripts of genomic segments A and B was at 36 h and mean titers were 10(1.8) pfu/ml. IBD virus was recovered 6 hours after transfection in cells concurrently expressing either VP2 with VP3 or VP243 and mean titers were 10(8.5) pfu/ml or 10(9.2) pfu/ml, respectively. Likewise, expression of the viral polyprotein from DNA plasmid increased the permissiveness of Vero cells for infection with non-culture adapted IBD virus. The titer of recovered non-culture adapted virus from 10(3.3) pfu/ml to 10(10.3) pfu/ml with expression of the viral polyprotein. This report is the first to describe a reverse genetics model for IBD virus with high efficiency of virus recovery for non-culture adapted strains.

Animals↗
PubMed2001

Rescue of infectious bursal disease virus from mosaic full-length clones composed of serotype I and II cDNA.

Infectious Bursal Disease Virus (IBDV) is the causative agent of one of the most important and wide-spread infectious diseases among commercial chicken flocks. IBDV causes a depletion of B-lymphoid cells in the bursa of Fabricius, inducing immunosuppression, morbidity, or even acute mortality. Because currently used live IBDV vaccines are derivatives from field isolates no serologic discrimination between field isolates and live vaccines can be made. The recently developed reverse genetics techniques for IBDV allows one to generate genetically modified IBDVs which might have altered biological and antigenic properties. Here, we describe the rescue of mosaic serotype I IBDVs, of which the polyprotein encoding region was partly replaced by the corresponding region of a serotype II strain. A mosaic virus, containing the C-terminal part of serotype II VP3 showed only a slightly delayed release of progeny virus compared to unmodified serotype I virus, while maximum viral titers at 25 h post infection were equal. Since serotype specific epitope(s) are present in the C-terminal part of VP3, we were able to discriminate this rescued virus from serotype I and II IBDV strains. These findings make the use of a chimeric VP3 a promising approach to develop an IBDV marker vaccine.

Amino Acid Sequence↗
PubMed2002

A single amino acid substitution in 126-kDa protein of Pepper mild mottle virus associates with symptom attenuation in pepper; the complete nucleotide sequence of an attenuated strain, C-1421.

The complete nucleotide sequence of an attenuated Pepper mild mottle virus (PMMoV C-1421) RNA genome has been determined. There were two differences from the type isolate in Japan (PMMoV-J). The mutations were located in the middle of the 126-kDa protein (126 K) gene; one mutation influenced amino acid substitution at 649th Val to Ala (V649A), and the other was silent. The analyses using the reverse genetic system of PMMoV-J revealed that symptom attenuation on pepper related to V649A. Accumulations of 126 K and coat protein (CP) in V649A mutant-infected pepper were lower than those of PMMoV-J in immunoblotting. These results suggest that V649A substitution in 126 K affects the accumulation of 126 K leading to a limitation of CP accumulation.

Amino Acid Substitution↗
PubMed2001

Transposon tagging in maize.

Through recent government- and industry-sponsored efforts, several forward and reverse genetic screening programs have emerged over the past few years to aid in the genetic dissection of gene function in maize. Despite a US maize crop valued at $18.4 billion last year (http://www.ncga.com/03world/main/US_crop_value_2000.html) and rich genetic history, maize has taken a back seat to Arabidopsis thaliana as the model genetic system for plants over the past decade. With a fully sequenced genome, short generation time and small size, studies of Arabidopsis have provided plant scientists with a molecular framework for hormonal, developmental and environmental signaling pathways in plants. As investigations into Arabidopsis continue, our capacity to engineer biochemical pathways and alter plant physiological responses will become increasingly sophisticated. Nevertheless, approximately 130 million years have passed since monocot and higher eudicot lineages diverged. Thus, our ability to engineer agronomically important monocot grasses such as maize, rice and wheat will become increasingly limited by our lack of understanding of the physiological and morphological differences that have evolved in the monocots and higher eudicots. The sophisticated transposon collections now being generated for maize are but one of several recent projects (http://www.nsf.gov/bio/pubs/awards/genome01.htm) to provide grass researchers with essential tools for genome analysis. Because grain crops are such a closely related group, it is hoped that many of the findings made in one grass will be directly applicable to understanding the biology of another. The goal of this review is to highlight the recent developments in maize transposon-based gene characterization programs and provide a critical examination of the advantages and disadvantages each system offers.

DNA Transposable Elements↗
PubMed2005

The vertebrate phototransduction cascade: amplification and termination mechanisms.

The biochemical cascade which transduces light into a neuronal signal in retinal photoreceptors is a heterotrimeric GTP-binding protein (G protein) signaling pathway called phototransduction. Works from psychophysicists, electrophysiologists, biochemists, and geneticists over several decades have come together to shape our understanding of how photon absorption leads to photoreceptor membrane hyperpolarization. The insights of phototransduction provide the foundation for a mechanistic account of signaling from many other G protein-coupled receptors (GPCR) found throughout nature. The application of reverse genetic techniques has strengthened many historic findings and helped to describe this pathway at greater molecular details. However, many important questions remain to be answered.

Animals↗
PubMed2006

The WiscDsLox T-DNA collection: an arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline.

We have developed a new community resource, called the WiscDsLox collection, for performing reverse-genetic analysis in arabidopsis. This resource is composed of 10,459 T-DNA lines generated using the Arabidopsis thaliana ecotype Columbia. The flanking sequence tag for each T-DNA insertion has been deposited in public databases, and seed for each line is currently available from the Arabidopsis Biological Resource Center. The pDsLox vector used to create this new population contains a Ds transposon and Cre/Lox recombination sites. Each WiscDsLox line therefore has the potential to serve as a launch-pad for performing local saturation mutagenesis by mobilization of the Ds element. In addition, Cre-Lox recombination between the T-DNA and a transposed Ds element should enable targeted deletion of specific genomic regions. We generated the WiscDsLox collection using an improved high-throughput pipeline that streamlines analysis of large numbers of independent Arabidopsis thaliana (L.) Hyenh. lines. In this paper we describe the details of this novel method and also provide potential users of WiscDsLox T-DNA lines with useful background information about this collection. Experiments to characterize the utility of the Ds transposon and Cre/Lox elements present in the WiscDsLox lines are in progress and will be reported in the future.

Arabidopsis↗
PubMed2007

Changes in gene expression during male meiosis in Petunia hybrida.

We analyzed changes in gene expression during male meiosis in Petunia by combining the meiotic staging of pollen mother cells from a single anther with cDNA-AFLP transcript profiling of mRNA from the synchronously developing sister anthers. The transcript profiling experiments focused on the identification of genes with a modulated expression profile during meiosis, while premeiotic archesporial cells and postmeiotic microspores served as a reference. About 8000 transcript tags, estimated at 30% of the total transcriptome, were generated, of which around 6% exhibited a modulated gene expression pattern at meiosis. Cluster analysis revealed a transcriptional cascade that coincides with the initiation and progression through all stages of the two meiotic divisions. Fragments that exhibited high expression specifically during meiosis I were characterized further by sequencing; 90 out of the 293 sequenced fragments showed homology with known genes, belonging to a wide range of gene classes, including previously characterized meiotic genes. In-situ hybridization experiments were performed to determine the spatial expression pattern for five selected transcript tags. Its concurrence with cDNA-AFLP transcript profiles indicates that this is an excellent approach to study genes involved in specialized processes such as meiosis. Our data set provides the potential to unravel unique meiotic genes that are as yet elusive to reverse genetics approaches.

DNA, Complementary↗
PubMed2006

Mammalian Pol III promoter H1 can transcribe shRNA inducing RNAi in chicken cells.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. RNAi based on DNA vector is not sufficiently established in chicken species. The present study was performed to evaluate RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 in the chicken cells by using a dual fluorescence reporter assay, a plasmid encoding GFP and a plasmid encoding RFP. The evaluation of RNAi efficiency was performed in two kinds of chicken cell type: primary CEF cells and chicken DT-40 cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescent microscopy, and their mRNAs content were analyzed by quantitative RT-PCR. The intensity of the green fluorescence generated by GFP was greatly suppressed by human H1 promoter transcribed GFP-shRNA. Quantitative RT-PCR analysis showed that normalized GFP mRNA expression was reduced to 37 and 32 in primary CEF and DT-40 cells, respectively. In contrast to GFP, the intensity of the red fluorescence generated by RFP protein and the RFP mRNA levels remained unchanged. Consequently, it was concluded that the RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 is applicable to suppress the gene expression specifically and efficiently in chicken cells.

Animals↗