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Results for “reverse genetics”

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At least 217 records · Page 12Linked to original sources
PubMed2006

Bone-marrow-derived stem cells repair basement membrane collagen defects and reverse genetic kidney disease.

Type IV collagen is a predominant component of basement membranes, and glomeruli of a kidney filter approximately 70-90 liters of plasma every day through a specialized glomerular basement membrane (GBM). In Alport syndrome, a progressive disease primarily affecting kidneys, mutations in GBM-associated type IV collagen genes (COL4A3, COL4A4, or COL4A5) lead to basement membrane structural defects, proteinuria, renal failure, and an absence of all three GBM collagen triple helical chains because of obligatory posttranslational assembly requirements. Here, we demonstrate that transplantation of wild-type bone marrow (BM) into irradiated COL4A3(-/-) mice results in a possible recruitment of BM-derived progenitor cells as epithelial cells (podocytes) and mesangial cells within the damaged glomerulus, leading to a partial restoration of expression of the type IV collagen alpha3 chain with concomitant emergence of alpha4 and alpha5 chain expression, improved glomerular architecture associated with a significant reduction in proteinuria, and improvement in overall kidney histology compared with untreated COL4A3(-/-) mice or irradiated COL4A3(-/-) mice with BM from adult COL4A3(-/-) mice. The alpha3(IV) collagen produced by BM-derived podocytes integrates into the GBM and associates with other alpha-chains to form type IV collagen triple helical networks. This study demonstrates that BM-derived stem cells can offer a viable strategy for repairing basement membrane defects and conferring therapeutic benefit for patients with Alport syndrome.

Animals↗
PubMed2003

Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus.

A previously undescribed coronavirus (CoV) is the etiologic agent responsible for severe acute respiratory syndrome (SARS). Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor (2S,3S)-transepoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester. In addition, subgenomic transcripts were initiated from the consensus sequence ACGAAC in both the WT and infectious clone SARS-CoV. Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

Animals↗
PubMed1998

Generation of up-regulated allosteric variants of potato ADP-glucose pyrophosphorylase by reversion genetics.

Mutagenesis of the large subunit (LS) of the potato ADP-glucose pyrophosphorylase generated an enzyme, P52L, that was insensitive to 3-phosphoglycerate (3-PGA). To identify additional residues involved in 3-PGA interaction, we subjected P52L LS DNA to a second round of mutagenesis and identified second-site revertants by their ability to restore glycogen accumulation as assessed by iodine (I2) staining. Enzymes from class I revertants with normal I2-staining had an 11- to 49-fold greater affinity for the activator 3-PGA compared with the P52L mutant and a decreased sensitivity to the inhibitor orthophosphate. Sequence analysis of these class I revertants identified a P66L mutation in R4, an E38K mutation in R20, and a G101N mutation in R10 and R32. These mutations appear to restore 3-PGA binding by counteracting the effect of the P52L mutation because introducing E38K or G101N into the wild-type LS led to enzyme variants with higher affinity for the activator 3-PGA and increased resistance to the inhibitor orthophosphate. The generation of these revertant enzymes provides additional structure-function information on the allosteric regulation of higher plant ADP-glucose pyrophosphorylases and validates a strategy for developing novel variants of the enzyme that may be useful in manipulating starch biosynthesis in higher plants.

Allosteric Site↗
PubMed1995

The role of cytochrome c-550 as studied through reverse genetics and mutant characterization in Synechocystis sp. PCC 6803.

The gene coding for cytochrome c-550 in Synechocystis sp. PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide. Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues. This includes a cleavable N-terminal leader sequence of 25 residues. This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes. Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues. These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen. Using the cloned gene, insertion and deletion mutants of Synechocystis sp. PCC 6803 were constructed. In the absence of cytochrome c-550, both mutants were capable of photoautotrophic growth but at a significantly reduced rate. Atrazine bindng and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53-67% of the amount of photosystem II as compared with wild type. The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40% of that in the wild type strain. Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria. This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y. (1992) FEBS Lett. 301, 145-149; Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832). Based on these results, the gene for cytochrome c-550 was named psbV. The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed.

Amino Acid Sequence↗
PubMed2005

The application of reverse genetics technology in the study of rabies virus (RV) pathogenesis and for the development of novel RV vaccines.

Rabies is a central nervous system (CNS) disease that is almost invariably fatal. Neurotropism, neuroinvasiveness, and transsynaptic spread are the main features that determine the pathogenesis of rabies. Recent advances in rabies virus (RV) research, which made direct genetic manipulations of the RV genome possible, greatly improved the understanding of the role of different viral and host cell factors in the pathogenesis of rabies. Here the authors discuss molecular mechanisms associated with rabies RV infection and its spread to the CNS.

Brain↗
PubMed2002

Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene.

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector-parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.

Animals↗
PubMed2001

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics.

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.

Acids↗
PubMed2002

A high-throughput Arabidopsis reverse genetics system.

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

Agrobacterium tumefaciens↗
PubMed2004

Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis.

Acetyl-CoA provides organisms with the chemical flexibility to biosynthesize a plethora of natural products that constitute much of the structural and functional diversity in nature. Recent studies have characterized a novel ATP-citrate lyase (ACL) in the cytosol of Arabidopsis thaliana. In this study, we report the use of antisense RNA technology to generate a series of Arabidopsis lines with a range of ACL activity. Plants with even moderately reduced ACL activity have a complex, bonsai phenotype, with miniaturized organs, smaller cells, aberrant plastid morphology, reduced cuticular wax deposition, and hyperaccumulation of starch, anthocyanin, and stress-related mRNAs in vegetative tissue. The degree of this phenotype correlates with the level of reduction in ACL activity. These data indicate that ACL is required for normal growth and development and that no other source of acetyl-CoA can compensate for ACL-derived acetyl-CoA. Exogenous malonate, which feeds into the carboxylation pathway of acetyl-CoA metabolism, chemically complements the morphological and chemical alterations associated with reduced ACL expression, indicating that the observed metabolic alterations are related to the carboxylation pathway of cytosolic acetyl-CoA metabolism. The observations that limiting the expression of the cytosolic enzyme ACL reduces the accumulation of cytosolic acetyl-CoA-derived metabolites and that these deficiencies can be alleviated by exogenous malonate indicate that ACL is a nonredundant source of cytosolic acetyl-CoA.

ATP Citrate (pro-S)-Lyase↗
PubMed2003

Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system.

To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.

Cell Line↗
PubMed2004

Accelerating aging by mouse reverse genetics: a rational approach to understanding longevity.

Investigating the molecular basis of aging has been difficult, primarily owing to the pleiotropic and segmental nature of the aging phenotype. There are many often interacting symptoms of aging, some of which are obvious and appear to be common to every aged individual, whereas others affect only a subset of the elderly population. Although at first sight this would suggest multiple molecular mechanisms of aging, there now appears to be almost universal consensus that aging is ultimately the result of the accumulation of somatic damage in cellular macromolecules, with reactive oxygen species likely to be the main damage-inducing agent. What remains significant is unravelling how such damage can give rise to the large variety of aging symptoms and how these can be controlled. Although humans, with over a century of clinical observations, remain the obvious target of study, the mouse, with a relatively short lifespan, easy genetic accessibility and close relatedness to humans, is the tool par excellence to model aging-related phenotypes and test strategies of intervention. Here we present the argument that mouse models with engineered defects in genome maintenance systems are especially important because they often exhibit a premature appearance of aging symptoms. Confirming studies on human segmental progeroid syndromes, most of which are based on heritable mutations in genes involved in genome maintenance, the results thus far obtained with mouse models strongly suggest that lifespan and onset of aging are directly related to the quality of DNA metabolism. This may be in keeping with the recent discovery of a possible 'universal survival' pathway that improves antioxidant defence and genome maintenance and simultaneously extends lifespan in the mouse and several invertebrate species.

Aging, Premature↗
PubMed2000

Prions: pathogenesis and reverse genetics.

Spongiform encephalopathies are a group of infectious neurodegenerative diseases. The infectious agent that causes transmissible spongiform encephalopathies was termed prion by Stanley Prusiner. The prion hypothesis states that the partially protease-resistant and detergent-insoluble prion protein (PrPsc) is identical with the infectious agent, and lacks any detectable nucleic acids. Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology. The prion protein (PrPc) is encoded by the Prnp gene, and disruption of Prnp leads to resistance to infection by prions. Introduction of mutant PrPc genes into PrPc-deficient mice was used to investigate structure-activity relationships of the PrPc gene with regard to scrapie susceptibility. Ectopic expression of PrPc in PrPc knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrPc knockout and transgenic mice overexpressing PrPc allowed selective reconstitution experiments aimed at expressing PrPc in neurografts or in specific populations of hemato- and lymphopoietic cells. The latter studies helped in elucidating some of the mechanisms of prion spread and disease pathogenesis.

Animals↗