a2,-Globulin is a rat protein of as yet unknown function whose synthesis can be induced by glucocorticoids and several other hormones. Induction by glucocorticoids is a secondary response to the hormone: protein synthesis is required before the hormone can exert its stimulatory effect on oL2.-globulin transcription. We have used the linker-scanning mutagenesis procedure, followed by transfer of the mutant genes into mouse L-cells for analysis of their phenotype, to determine sequences within a cloned a2u-globulin promoter that are required for its regulation by glucocorticoids. Mutations between positions -115 and -160 abolish or greatly reduce the inducibility of a2.-globulin by the hormone. Mutations just upstream from this region, between positions -177 and -220, have an opposite effect; they increase induction two-to fourfold.Steroid hormones are important regulators of gene expression in many organisms. In general, these hormones exert their effects at the transcriptional level, inducing or repressing the synthesis of specific mRNAs (reviewed in references 1, 61). These effects can be either direct or indirect. A direct or primary response is characterized by insensitivity of mRNA induction to inhibitors of protein synthesis and by a rapid increase in the transcription of the inducible gene (62). In the best-studied example of a primary response, the induction of mouse mammary tumor virus (MMTV) transcription by glucocorticoids, the hormone receptor complex binds to specific DNA sequences within the provirus and, by a still unknown mechanism, stimulates transcription from the viral promoter (reviewed in references 47, 48).Indirect or secondary responses to steroid hormones require protein synthesis for transcription of the induced RNA and usually shows a lag between administration of the hormone and initiation of the response. The classical example of such a response is the induction by ecdysterone of the late puffs on the chromosomes of Drosophila salivary glands (reviewed in reference 46). The late puffs, unlike the early puffs, do not appear if protein synthesis is inhibited when the glands are first treated with the hormone. It seems likely that during the lag period a regulatory protein(s) is synthesized in response to the hormone. This protein is required for the expression of the secondary response (2,13 Mutagenesis. An outline of the mutagenesis procedure is shown in Fig. 3. A library of deletions, extending into the promoter from the a2,-globulin structural gene (3' deletions), was made by Bal 31 nuclease digestion from the SalI site of plasmid p91FHS. A 10-jig portion of plasmid DNA was digested with SalI. The DNA was precipitated with ethanol and dissolved in 300 jil of buffer containing 12 mM CaCl2, 12 mM MgCl2, 20 mM Tris hydrochloride (pH 8), 1 mM EDTA, 0.6 M NaCl, and 3 U of Bal 31 nuclease and incubated at 15°C. After incubation for 5, 10, and 15 min, 100-jil samples of the mixture were added to a tube containing 50 ,ul of phenol-CHCl3 (1:1) and 10 jil of 0.25 M EDTA, and the tube was vortex...
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