Rice Genetics IV - IRRI books - International Rice Research Institute

Gene detectionGene detection strategies have been developed to address the function of genes thatdo not directly reveal a knockout phenotype. One way is expression detection thatcan make use of inserts containing reporter gene constructs, such as enhancer traps orgene traps, whose expression depends on transcriptional regulatory sequences of theadjacent host gene. Another way is by creating misexpression mutants, such as activationtags that might reveal a gain-of-function phenotype.Enhancer detection was developed to detect enhancers in the genome, which arecapable of orientation-independent transcriptional activation from a distance, and thusidentify genes based on their expression pattern (Skarnes 1990) even though theymight not display an obvious mutant phenotype. Enhancer detection constructs containa reporter gene such as β-glucuronidase (GUS) with a weak or minimal promoter,for example, with a TATA box (transcription initiation signal), situated nearthe border of the insert and are expressed when integrated adjacent to an enhancer inthe genome. Gene trap-type inserts are designed to create fusion transcripts with thetarget gene. One type used effectively in plants is an exon trap that enables reportergene fusions to be created at various locations within a gene. By introduction ofsplice acceptor sites upstream of the reporter gene, transcriptional fusions are createdeven for insertions in introns, thus increasing the frequency of inserts expressing thereporter gene.A novel method for efficient selection of stable transpositions, using positive andnegative selection markers, has been developed for an Ac-Ds-based gene trap andenhancer trap system (Sundaresan et al 1995). Stable transposed Ds elements wererecovered and revealed GUS expression activity in about 50% of enhancer trap (DsE)and 25% of gene trap (DsG) inserts (Sundaresan et al 1995). A similar strategy forselection of stable transposed gene trap DsG elements has begun in rice (Chin et al1999) using greenhouse-selectable marker systems.Misexpression or gain-of-function mutants can be generated by insertion sequencesthat carry a strong enhancer element near the border and activate the expression of theadjacent genes. This method of “activation tagging” has been validated by an extensivescreen of more than 25,000 T-DNA tags (Weigel et al 2000), revealing a 1/1,000frequency of dominant mutants that were shown to be caused by the presence of anenhancer between 0.4 and 4 kb from the overexpressed gene. A transposon constructvariant to isolate dominant gain-of-function alleles employed the CaMV 35S promotertranscribing outward from the Ds transposon end and helped identify a semidominantoverexpression mutant (Wilson et al 1996). These examples demonstrate the use ofactivation tagging to generate dominant mutations by over-/misexpressing genes andgenerate phenotypes for processes or genes not uncovered by knockout mutants.Generation of rice transposon insertion populationsTo develop efficient transposon mutagenesis strategies in rice, we tested several parametersin order to select components for transposon constructs to be transformed.266 Greco et al