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Biomedical subjects

N J Kilby

Publications and source records attributed to N J Kilby.

8 recordsLinked to original sources
PubMed2004

Characteristics of RNA silencing in plants: similarities and differences across kingdoms.

RNA silencing is a collective term that encompasses the sequence of events that leads to the targeted degradation of cellular mRNA and thus to the silencing of corresponding gene expression. RNA silencing is initiated after introduction into the host genome of a gene that is homologous to an endogenous gene. Transcription of the introduced gene results in the formation of double-stranded RNA (dsRNA) that is cut into smaller dsRNA species termed small interfering RNAs (siRNAs) by an RNaseIII-like enzyme called 'Dicer'. siRNAs associate with a protein complex termed the 'RNA-induced silencing complex' (RISC), which mediates the binding of one strand of siRNAs with mRNAs transcribed from the native 'target' gene. The binding of siRNAs with native gene mRNAs earmarks native gene mRNAs for destruction, resulting in gene silencing. In plants, RNA silencing appears to serve as a defence mechanism against viral pathogens and also to suppress the activity of virus-like mobile genetic elements. In an apparent response to RNA silencing, some plant viruses express suppressors of RNA silencing. RNA silencing also is directly implicated in the regulation of the function(s) of microRNAs, which are the key determinants in an additional cellular mechanism related to the translational repression of genes, the effect of which ultimately impinges on development. The high degree of sequence similarity that exists between genes involved in RNA silencing in widely different organisms underscores the conserved nature of many aspects of the RNA silencing mechanism. However, depending (for example) on the precise nature of the target gene involved, there also are significant differences in the silencing pathways that are engaged by various organisms.

Gene Expression Regulation, Plant↗
PubMed2000

Mosaic analysis of GL2 gene expression and cell layer autonomy during the specification of Arabidopsis leaf trichomes.

Homozygous glabra2 (gl2) mutant Arabidopsis thaliana Landsberg erecta plants with only a few rudimentary single spiked trichomes on the leaf margin were transformed with a genomic clone of GL2, resulting in partial restoration of the normal leaf trichome phenotype. The introduced GL2 transgene was configured as part of an FLP recombinase-responsive gene switch, which permitted visibly marked gl2 mutant clonal sectors to be generated by FLP recombinase-mediated deletion of the GL2 transgene with concomitant activation of a previously silent beta-glucuronidase (GUS) marker gene. GUS marked sectors extending through all three leaf cell layers (L1, L2, and L3) displayed the anticipated gl2 mutant phenotype, whereas immediately adjacent unmarked tissue, and unmarked tissues overlaying GUS sectors restricted to the L2 and/or L3 cell layers, retained the GL2 restored phenotype. These data support the view that the GL2 gene product acts in a region-autonomous manner within a single cell layer and indicate that GL2 gene expression in the L1 layer is sufficient for GL2-directed outgrowth of trichomes.

Arabidopsis↗
PubMed2000

Controlled induction of GUS marked clonal sectors in Arabidopsis.

Stably transformed Arabidopsis lines in which GUS marked cell clones are readily produced in response to heat-shock have been established and characterized. Control of GUS activation is achieved by heat-shock-induced FLP recombinase activity which "switches on" expression of a GUS marker gene previously held transcriptionally silent. To obtain efficient GUS sectoring, single insert Arabidopsis lines carrying FLP recombinase under the control of a heat-shock-inducible promoter and an FLP-activatable GUS construct were generated. Analysis of GUS sectoring in lines hemizygous and homozygous for both inserts was conducted after various regimes of heat-shock were given at various developmental stages. It is shown that GUS sectoring events can be efficiently induced in most vegetative, aerial and sexual structures in Arabidopsis. Furthermore, the frequency of sectoring events, sector size and, to some extent, the tissues in which sectors are generated can be readily controlled by choice of the conditions and timing of heat-shock used.

Arabidopsis↗
PubMed1996

An Escherichia coli system for assay of F1p site-specific recombination on substrate plasmids.

We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the F1p site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40.pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying F1p-recognition targets (FRT) are transformed into BL-FLP, and the consequences of F1p-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect F1p-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used F1p substrate plasmid, pNEO beta GAL (O'Gorman et al. (1991) Science 251, 1351-1355).

Binding Sites↗
PubMed1995

FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis.

FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.

Arabidopsis↗
PubMed1993

Site-specific recombinases: tools for genome engineering.

Site-specific recombinases from bacteriophage and yeasts have been developed as novel tools for manipulating DNA both in the test-tube and in living organisms. We discuss the characteristics of these enzyme systems, review their application in genetic and developmental studies and speculate on their future potential for large-scale directed modifications of eukaryotic genomes.

Animals↗
PubMed1992

Promoter methylation and progressive transgene inactivation in Arabidopsis.

Agrobacterium-transformed Arabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of an Sst II site in the nos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes in Arabidopsis.

DNA, Bacterial↗