Biomedical subjects

Graham Thow

Publications and source records attributed to Graham Thow.

4 recordsLinked to original sources

PubMed2004

Dual functionality of a plant U-rich intronic sequence element.

In potato invertase genes, the constitutively included, 9-nucleotide (nt)-long mini-exon requires a strong branchpoint and U-rich polypyrimidine tract for inclusion. The strength of these splicing signals was demonstrated by greatly enhanced splicing of a poorly spliced intron and by their ability to support splicing of an artificial mini-exon, following their introduction. Plant introns also require a second splicing signal, UA-rich intronic elements, for efficient intron splicing. Mutation of the branchpoint caused loss of mini-exon inclusion without loss of splicing enhancement, showing that the same U-rich sequence can function as either a polypyrimidine tract or a UA-rich intronic element. The distinction between the splicing signals depended on intron context (the presence or absence of an upstream, adjacent and functional branchpoint), and on the sequence context of the U-rich elements. Polypyrimidine tracts tolerated C residues while UA-rich intronic elements tolerated As. Thus, in plant introns, U-rich splicing elements can have dual roles as either a general plant U-rich splicing signal or a polypyrimidine tract. Finally, overexpression of two different U-rich binding proteins enhanced intron recognition significantly. These results highlight the importance of co-operation between splicing signals, the importance of other nucleotides within U-rich elements for optimal binding of competing splicing factors and effects on splicing efficiency of U-rich binding proteins.

Base Sequence↗

PubMed2003

High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata.

A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.

Base Sequence↗

PubMed2002

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris.

A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.

Amino Acid Substitution↗

PubMed2002

Mutational analysis of a plant branchpoint and polypyrimidine tract required for constitutive splicing of a mini-exon.

The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422-433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3-4 nt long (<6 nt apart) that, within the context of the mini-exon system, required being close (<14 nt) to the branchpoint sequence. The functional characterization of the branchpoint sequence and polypyrimidine tract defines these sequences in plants for the first time, and firmly establishes polypyrimidine tracts as important signals in splicing of at least some plant introns.

Base Composition↗