Allele-specific quantification of Drosophila engrailed and invected transcripts.
Changes in levels of transcription can be difficult to gauge in animals with lethal mutations. For example, mutations in a regulatory region of an essential gene can have secondary consequences that complicate attempts to quantify the transcripts produced by the mutant gene. We describe a method that circumvents this problem by revealing the relative amount of transcript produced from each allele in a heterozygote. With this method, recessive lethal mutations can be analyzed in animals that are phenotypically wild type. We used this technique to analyze mutations in the regulatory region of the Drosophila engrailed gene and found that truncations reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. We also found that these mutations affect expression of the linked invected gene, suggesting that engrailed and invected share a complex set of regulatory elements that operate over at least 85 kb. We suggest that this technique will have general utility for the quantitation of allele-specific transcripts, even when amounts of tissue are limiting.
Goldsborough
A S
AS
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Kornberg
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TB
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D005786:Gene Expression Regulation; D017344:Genes, Insect; D008040:Genetic Linkage; D006579:Heterozygote; D018398:Homeodomain Proteins; D007301:Insect Hormones / Q000235:genetics*; D007814:Larva; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D017931:DNA Primers; D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D007301:Insect Hormones; D012333:RNA, Messenger; D014157:Transcription Factors; C087983:inv protein, Drosophila