C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress.

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell.

The significance of alternative transcripts for Caenorhabditis elegans transcription factor genes, based on expression pattern analysis.

Background: Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal's genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement.

Results: Of the 938 potential C.

A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones.

Background: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids.

Seamless replacement of Autographa californica multiple nucleopolyhedrovirus gp64 with each of five novel type II alphabaculovirus fusion sequences generates pseudotyped virus that fails to transduce mammalian cells.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the type I alphabaculoviruses, is able to transduce and deliver a functional gene to a range of non-host cells, including many mammalian lines and primary cells, a property mediated by the envelope fusion protein GP64. AcMNPV is non-cytopathic and inherently replication deficient in non-host cells. As such, AcMNPV represents a possible new class of gene therapy vector with potential future clinical utility.

Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs.

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics.

Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.

Background: Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.

Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones.

By determining spatial-temporal expression patterns, reporter constructs provide significant insights into gene function. Although additionally providing information on subcellular distribution, translational reporters, where the reporter is fused to the gene coding sequence, are used less frequently than simpler constructs containing only putative promoter sequences. Because these latter constructs may not contain all necessary regulatory elements, resulting expression patterns must be interpreted cautiously.

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described.

The fmo genes of Caenorhabditis elegans and C. briggsae: characterisation, gene expression and comparative genomic analysis.

The flavin-containing monooxygenase (FMO) gene family is conserved and ancient with representatives present in almost all phyla so far examined. The genes encode FAD-, NADP- and O(2)-dependent enzymes that catalyse oxygenation of soft-nucleophilic heteroatom centres in a range of substrates. Although usually classified as xenobiotic-metabolising enzymes, examples of FMOs exist that have evolved to metabolise specific endogenous substrates as part of a discrete physiological process.

Genetic polymorphisms of flavin-containing monooxygenase (FMO).

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU).

The peroxisome proliferator-activated receptor alpha-selective activator ciprofibrate upregulates expression of genes encoding fatty acid oxidation and ketogenesis enzymes in rat brain.

Activated peroxisome proliferator activated receptor alpha (PPAR alpha) protects against the cellular inflammatory response, and is central to fatty acid-mediated upregulation of the gene encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). We have previously demonstrated both PPAR alpha and mHS expression in brain, implying that brain-targeted PPAR alpha activators may likewise up-regulate mHS expression in brain. Thus, to attempt pharmacological activation of brain PPAR alpha in vivo, we have administered to rats two drugs with previously defined actions in rat brain, namely the PPAR alpha-selective activator ciprofibrate and the pan-PPAR activator valproate.