Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “reverse engineering”

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 1,585 records · Page 88Linked to original sources
PubMed2005

Behavior of transplanted bone marrow-derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation.

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.

Animals↗
PubMed2005

Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition.

PURPOSE: Tissue engineering has been used for bladder augmentations with small intestinal submucosa (SIS). Although favorable short-term outcomes have been reported, long-term followup has been poor. We investigate whether tissue engineering with stem cells improves the morphological and genetic composition. MATERIALS AND METHODS: A total of 33 Lewis rats (Harlan Laboratories, Indianapolis, Indiana) were used to investigate bladder augmentations with 4-layer SIS in certain groups, including the control group (sham operation), partial cystectomy with oversewn defect group (OG), augmentation with unseeded SIS group (USG) and augmentation with stem cell seeded SIS group (SSG). Bladders from 4 rats per group were harvested 1 and 3 months after surgery. Morphological analyses were performed using Masson's trichrome and immunohistochemical staining with cytokeratin AE1/AE3, smooth muscle alpha-actin and S100. Gene expression was evaluated using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for collagen I (CI), collagen III (CIII), cytokeratins 8 and 19, and smooth muscle myosin heavy chain (MHC). RESULTS: At 1 month trichrome staining revealed collagen admixed with indiscrete cells and morphology similar to that in controls in USG and SSG, respectively. Discrete smooth muscles fascicles and S100 staining were found in all groups except USG. Organized urothelium with increased basal cell layer staining was present in controls and SSG only. At 3 months increased collagen formation was present in OG and USG. Immunostaining showed hyperplasia of the urothelium with increased staining of the basal cell layer, discrete muscle fascicles and positive nerve staining in all groups. Using quantitative RT-PCR expression levels in SSG were more improved than in USG, especially for CI, CIII and MHC. This was further evident at 3 months when CI and CIII were over expressed in OG and USG but not in the control group or SSG. Furthermore, RT-PCR showed that cytokeratins 8 and 19, and MHC had greater expression levels in SSG than in USG. CONCLUSIONS: Bladder reconstitution occurs more rapidly using stem cell seeded SIS. Although in USG and SSG all 3 cellular constituents appear to develop by 3 months, only SSG had gene expression levels similar to those in controls. The results suggest an explanation for the fibrosis noted in unseeded SIS bladder augmentations and the possible solution using stem cells.

Animals↗
PubMed2026

Biomaterial-Integrated Electroporation for Therapeutic Delivery: From Gene Editing to Tumor Ablation and Immune Modulation.

Electroporation has evolved from a membrane-permeabilization method into a versatile therapeutic platform for intracellular delivery, locoregional tumor intervention, and bioelectrically regulated treatment. Depending on pulse intensity and duration, electroporation operates in two distinct modes: reversible electroporation (RE), which transiently permeabilizes the plasma membrane to enable delivery of nucleic acids, proteins, and small molecules while preserving cell viability, and irreversible electroporation (IRE), which causes permanent membrane damage for non-thermal tissue ablation. Increasingly, the therapeutic scope of electroporation is being expanded through integration with biomaterials, including nanocarriers, hydrogels, soft conductors, and micro/nanoengineered bioelectronic interfaces. These material-assisted strategies improve cargo protection, field confinement, local retention, tissue conformity, and spatiotemporal control, thereby extending electroporation beyond conventional transfection toward gene editing, engineered cell manufacturing, electrochemotherapy, tumor ablation, immune modulation, and transdermal or localized delivery. In this Review, we summarize the biophysical principles of RE and IRE, discuss how biomaterials reshape electroporation performance across therapeutic settings, compare the design logic of major biomaterial-assisted electroporation platforms, and highlight key translational challenges, including pulse-material compatibility, manufacturing scalability, in vivo dosimetry, and regulatory complexity.

Humans↗
PubMed1991

Mutagenic DNA repair in Escherichia coli. XIX. On the roles of RecA protein in ultraviolet light mutagenesis.

An experimental system was used in which His+ mutations induced by ultraviolet light (UV) arise from non-photo-reversible photoproducts whereas lethality is largely determined by photoreversible photoproducts. By exposing a strain with a deletion through recA to light immediately after UV, it was possible to examine mutagenesis under conditions where survival was not significantly different from 100%. No UV mutagenesis was seen in the absence of RecA protein even though the rest of the SOS system was fully expressed due to the presence of a defective LexA repressor and the active carboxy-terminal fragment of UmuD was present as a result of an engineered plasmid-borne gene. We conclude that RecA protein has a third essential function if UV mutagenesis is to be detected in excision-deficient-bacteria. Another experiment showed that in exerting this function RecA protein does not need activation by pyrimidine dimers elsewhere on the genome, in contrast to its protein-cleavage mediation functions with LexA and UmuD proteins. RecA1730 protein blocked UV mutagenesis unless delayed photoreversal was given showing that the third function of RecA protein is not in the misincorporation step. It is therefore most likely to be in the bypass step where UmuD' and UmuC are postulated to act, although the possibility cannot be excluded that RecA protein is required for some other survival function distinct from translesion synthesis.

Alleles↗
PubMed2005

DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions.

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.

Biosensing Techniques↗
PubMed2004

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes.

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.

Animals↗
PubMed2005

Accumulation of long-chain acylcarnitine and 3-hydroxy acylcarnitine molecular species in diabetic myocardium: identification of alterations in mitochondrial fatty acid processing in diabetic myocardium by shotgun lipidomics.

Diabetic cardiomyopathy is the result of maladaptive changes in energy homeostasis. However, the biochemical mechanisms underlying dysfunctional lipid metabolism in diabetic myocardium are incompletely understood. Herein, we exploit shotgun lipidomics to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which was reversible upon insulin treatment. Analysis of acylcarnitine molecular species in myocardium unexpectedly identified acylcarnitine molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species. Synthesis of 3-hydroxy acylcarnitine identified the natural products as the 3-hydroxylated acylcarnitines through comparisons of diagnostic fragmentation patterns of synthetic and naturally occurring constituents using tandem mass spectrometry. Diabetes induced an increase of both calcium-independent phospholipase A(2) (iPLA(2)) mRNA and iPLA(2) activity in rat myocardium. Cardiac ischemia in myocardium genetically engineered to overexpress iPLA(2) dramatically increased the amount of acylcarnitine present in myocardium. Moreover, mechanism-based inactivation of iPLA(2) in either wild-type or transgenic myocardium ablated a substantial portion of the acylcarnitine increase. Collectively, these results identify discrete insulin remediable abnormalities in mitochondrial fatty acid processing in diabetic myocardium and identify iPLA(2) as an important enzymatic contributor to the pool of fatty acids that can be used for acylcarnitine synthesis and energy production in myocardium.

Animals↗
PubMed1995

The effect of pulsatile frequency on wall shear in a compliant cast of a human aortic bifurcation.

A realistically compliant flow-through cast of a human aortic bifurcation was perfused with two almost identical physiological flow waves differing in pulsatile frequency. Near-wall fluid velocities were measured with a laser Doppler velocimeter at 14 sites along the flow divider and the lateral walls of the aorta and iliac arteries. The wall position at each site was tracked using a linescan camera. The temporal wall shear rate at each site was then calculated from the near-wall velocity profile and the instantaneous wall position. Increasing the frequency reduced the oscillatory component of shear rate at sites where it was greater than average, and increased it at sites where it was less, effectively reducing its site to site variability. Pulsatile frequency had no significant effect on mean shear rate at most sites. The phase shift between wall shear and radial strain was governed by the phase of the shear, and was linearly related to the extent of site dependent shear reversal. The mean shear rate was inversely related to the extent of shear reversal. If atherosclerotic development depends chiefly on mean shear rate, heart rate would not be expected to affect susceptibility, however, if only the wall sites experiencing the lowest maximum shears are vulnerable, then the effect of increasing the heart rate would seem to be beneficial.

Adult↗
PubMed2005

Protection from anthrax toxin-mediated killing of macrophages by the combined effects of furin inhibitors and chloroquine.

Cell surface proteolytic processing of anthrax protective antigen by furin or other furin-related proteases is required for its oligomerization, endocytosis, and function as a translocon for anthrax lethal and edema factors. Countering toxin lethality is essential to developing effective chemotherapies for anthrax infections that have proceeded beyond the stage at which antibiotics are effective. The primary target for toxin is the macrophage, which can be killed by lethal factor via both necrotic and apoptotic pathways. Here we show that three high-affinity inhibitors of furin efficiently blocked killing of murine J774A.1 macrophages by recombinant protective antigen plus lethal factor: RRD-eglin and RRDG-eglin, developed by engineering the protein protease inhibitor eglin c, and the peptide boronic acid inhibitor acetyl-Arg-Glu-Lys-boroArg pinanediol. Inhibition of killing was dose dependent and correlated with prevention of protective antigen processing. Previous studies have shown that weak bases, such as chloroquine, which neutralize acidic compartments, also interfere with toxin-dependent killing. Here we show that combining furin inhibitors and chloroquine strongly augments the inhibition of toxin-dependent killing, suggesting that combined use of antifurin drugs and chloroquine might provide enhanced therapeutic benefits. Reversible furin inhibitors protected against anthrax toxin killing for at least 5 h, but by 8 h, toxin-dependent killing resumed even though furin inhibitors were still active. An irreversible chloromethylketone inhibitor did not exhibit this loss of protection.

Algorithms↗
PubMed2004

A nanosensor for transmembrane capture and identification of single nucleic Acid molecules.

We have engineered a nanosensor for sequence-specific detection of single nucleic acid molecules across a lipid bilayer. The sensor is composed of a protein channel nanopore (alpha-hemolysin) housing a DNA probe with an avidin anchor at the 5' end and a nucleotide sequence designed to noncovalently bind a specific single-stranded oligonucleotide at the 3' end. The 3' end of the DNA probe is driven to the opposite side of the pore by an applied electric potential, where it can specifically bind to oligonucleotides. Reversal of the applied potential withdraws the probe from the pore, dissociating it from a bound oligonucleotide. The time required for dissociation of the probe-oligonucleotide duplex under this force yields identifying characteristics of the oligonucleotide. We demonstrate transmembrane detection of individual oligonucleotides, discriminate between molecules differing by a single nucleotide, and investigate the relationship between dissociation time and hybridization energy of the probe and analyte molecules. The detection method presented in this article is a candidate for in vivo single-molecule detection and, through parallelization in a synthetic device, for genotyping and global transcription profiling from small samples.

Avidin↗
PubMed2001

Telomeres and telomerase: implications for cancer and aging.

Maintenance of telomere stability is required for cells to escape from replicative senescence and proliferate indefinitely. Telomere length is maintained by a balance between processes that lengthen telomeres (telomerase) and processes that shorten telomeres (the end-replication problem). Telomerase is a cellular ribonucleoprotein reverse transcriptase which stabilizes telomere length by adding hexameric (TTAGGG) repeats to the telomeric ends of the chromosomes, thus compensating for the continued erosion of telomeres. Introduction of the telomerase catalytic protein component into normal telomerase-negative human cells results in restoration of telomerase activity and extension of cellular life span. Human cells with introduced telomerase maintain a normal chromosome complement and continue to grow in a normal manner. Telomerase-induced manipulations of telomere length may thus be important not only for cell and tissue engineering but also for dissecting the molecular mechanisms underlying inherited genetic diseases, as well as defining the genetic pathways leading to cancer. Because almost all human tumors express telomerase activity, inhibition of telomerase may result in gradual erosion of telomeres and eventual cessation of cell proliferation or induction of apoptosis. Thus telomerase may also be a promising target for cancer therapy.

Animals↗
PubMed2001

Engineering the prion protein using chemical synthesis.

In recent years, the technology of solid-phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared several modified and wild-type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, alpha-helical cellular protein (PrPc) into a beta-sheet-rich pathogenic isoform (PrP(Sc)). The ability to form PrP(Sc) in transgenic mice is retained by a 106 residue 'mini-prion' (PrP106), with the deletions 23-88 and 141-176. Synthetic PrP106 (sPrP106) and a His-tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N-(2-chlorobenzyloxycarbonyloxy) succinimide. A single reversed-phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90-200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t-Boc chemistry. To mimic the glycosylphosphatidyl inositol (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surface, where the conversion of PrPc is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences. These groups enabled sPrP to be immobilized on either the cell surface or a streptavidin-coated ELISA plate, respectively, in an orientation analogous to that of membrane-bound, GPI-anchored PrPc. The chemical manipulation of such biologically relevant forms of PrP by the introduction of point mutations or groups that mimic post-translational modifications should enhance our understanding of the processes that cause prion diseases and may lead to the chemical synthesis of an infectious agent.

Amino Acid Sequence↗
PubMedDate not supplied

Engineering the hepatocyte differentiation-proliferation balance by acellular cadherin micropresentation.

The successful development of bioartificial and cell-based liver support systems relies on the identification of molecular mechanisms controlling the balance between hepatocellular proliferation and differentiation. Although a definitive function-inductive role for the cell-cell adhesion molecule, E-cadherin, was established through lateral cadherin-cadherin engagement in hepatocyte cocultures (Brevia, T.A., and Moghe, P.V. Biotechnol. Bioeng. 76, 295, 2001), the roles of other modes of cadherin presentation are not well understood. Further, alternative cadherin display configurations promoting cell growth/proliferative pathways, a major requisite for sustainable engineered tissues, remain to be identified. In this report, we employed protein A-functionalized polymeric microsphere substrates that specifically bound self-dimerizing cadherin-IgG/Fc fusion chimeras via their Fc regions, thereby orienting them outward for active adhesion, and presented the E-cadherin chimeras basally to cultured rat hepatocytes to study the effects of cadherin display on cell proliferative potential and differentiated function. In contrast to the previously documented function-inductive roles of laterally expressed cadherin, basal acellular cadherin presentation resulted in an increase in hepatocyte DNA synthesis and cell divisions, accompanied by a decrease in the expression of a key marker of liver-specific function, albumin message levels. Next, we probed the relative effect of basal exogenous display of acellular cadherins on the inductive phase of differentiation within hepatocyte cocultures with cadherin-expressing L929 cells. When acellular cadherins were applied to hepatocyte cocultures involving chaperone cell-mediated cadherin presentation, the previously reported function-inductive effects of cadherins on hepatocyte function were reversed, resulting in lower levels of albumin and urea secretion indicating a dominance of acellular cadherin effects. Our results demonstrate that cadherins are important regulators of the balance between hepatocyte differentiation and proliferation, and furthermore that the direction of balance shift may be dependent on the method of cadherin presentation. Thus, the geometric display of cadherin could be a potential parameter to switch hepatocyte functional-proliferative balance and may aid in customizing scaffolds for regulating hepatic tissue dynamics.

Animals↗
PubMed1990

Manipulating yeast genome using plasmid vectors.

The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

Cloning, Molecular↗
PubMed2006

Encapsulated chondrocyte response in a pulsatile flow bioreactor.

We have developed a bioreactor-based millifluidic technique that allows for dynamic culture conditions and measurement of the fluid flow impinging upon a three-dimensional tissue engineering scaffold. Chondrocytes in scaffolds have been shown to require mechanical stimulation to produce an extracellular matrix that resembles native cartilage. This study investigates the effect of pulsatile flow on chondrocyte response in a model poly(ethylene glycol) dimethacrylate hydrogel. Bovine chondrocytes were encapsulated in the hydrogel and cultured for 7, 14 and 21 days at pulsatile flow frequencies of 0.5 Hz (15ml/min) and 1.5Hz (17ml/min). The scaffolds cultured under dynamic conditions were compared to those cultured under static (non-flow) conditions. Quantitative real-time reverse transcription polymerase chain reaction was used to quantify collagen type I, collagen type II and aggrecan gene copy numbers as markers for chondrocyte phenotypic expression. Histological sections stained with hematoxylin & eosin, and Alcian blue confirmed chondrocyte morphology and matrix formation. Interestingly, regulation of the collagen type II gene was particularly sensitive to the flow conditions. The understanding of the cell response to encapsulation and flow could be used to identify the appropriate culture conditions necessary to design and develop hydrogel carriers to promote the formation of extracellular matrix as well as to further our knowledge of chondrocyte mechanobiology.

Animals↗
PubMed2026

A chromosome-level genome assembly of Lycoris radiata reveals the evolutionary origin of Amaryllidaceae alkaloids and elucidates the complete galanthamine biosynthetic pathway.

Amaryllidaceae alkaloids (AmAs) comprise a structurally diverse group of specialized metabolites produced almost exclusively by species of the Amaryllidoideae subfamily and are of substantial pharmacological importance. However, the limited availability of high-quality genomes from Amaryllidoideae plants has constrained systematic investigations of the genes and evolutionary processes underlying AmA biosynthesis. Here, we present a chromosome-level genome assembly of Lycoris radiata, which enabled the discovery of key downstream enzymes in the galanthamine biosynthetic pathway and uncovered reversible reactions between two critical metabolite pairs. These findings provide new mechanistic insight into pathway architecture and enable reconstruction of the galanthamine biosynthetic pathway in Yarrowia lipolytica. Comparative genomic analyses indicate that several core genes for AmA biosynthesis originated in ancestral angiosperms, whereas the complete pathway was likely assembled in the Amaryllidoideae subfamily through gene duplication and neofunctionalization. Furthermore, integrated metabolomic and transcriptomic analyses suggest that roots contribute actively to AmA metabolism in Lycoris. Together, these findings provide a genomic and biochemical framework for understanding the evolution and engineering of AmA biosynthesis.

Lycoris↗
PubMed2000

A transgenic perspective on plant functional genomics.

Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype. To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops.

Crops, Agricultural↗
PubMed2004

Light-activated ion channels for remote control of neuronal firing.

Neurons have ion channels that are directly gated by voltage, ligands and temperature but not by light. Using structure-based design, we have developed a new chemical gate that confers light sensitivity to an ion channel. The gate includes a functional group for selective conjugation to an engineered K(+) channel, a pore blocker and a photoisomerizable azobenzene. Long-wavelength light drives the azobenzene moiety into its extended trans configuration, allowing the blocker to reach the pore. Short-wavelength light generates the shorter cis configuration, retracting the blocker and allowing conduction. Exogenous expression of these channels in rat hippocampal neurons, followed by chemical modification with the photoswitchable gate, enables different wavelengths of light to switch action potential firing on and off. These synthetic photoisomerizable azobenzene-regulated K(+) (SPARK) channels allow rapid, precise and reversible control over neuronal firing, with potential applications for dissecting neural circuits and controlling activity downstream from sites of neural damage or degeneration.

Action Potentials↗