Part:BBa_K2374002:Experience

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Applications of BBa_K2374002

The activity of GAL4 can be repressed by physical interaction with the yeast GAL80 protein . A dimer of GAL80 binds to the C-terminal ends of the GAL4 dimer so that, while it can still bind to a UAS sequence, it can no longer activate transcription . This interaction of GAL4 and GAL80 can be taken advantage of to refine the expression pattern of GAL4-dependent transgenes.

Spatial refinement can be achieved by combining distinct GAL4 and GAL80 expression patterns. Where the expression patterns overlap, the activity of GAL4 and thus the expression of the GAL4- responsive transgene will be inhibited.

Lines expressing GAL80 in different patterns can be produced in the same way as GAL4 lines, either by enhancer trapping or by cloning of known enhancer elements. Temporal specificity can be achieved using a temperature sensitive allele of GAL80.

The Temporal And Regional Gene Expression Targeting (TARGET) system takes advantage of a variant of GAL80 that while ubiquitously expressed under control of the tubulin 1α promoter is only active at permissive temperatures. Thought to be the result of a single glycine to arginine substitution at amino acid 203 of GAL80, GAL80ts is unable to bind GAL4 at restrictive temperatures above 29 °C but retains its repressive function at the permissive temperature of 18 °C. Controlling the activity of GAL80ts through temperature shifts provides temporal control of GAL4-dependent trans gene expression . Limiting expression to defined temporal windows can help to define critical periods for the effects of misexpression or rescue experiments.

Temperature- sensitive alleles of GAL4 itself have been generated and tested in Drosophila, however the ease of combining tubGAL80ts with already established GAL4 drivers cause the TARGET system to be more commonly used.

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