Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex that is composed of a heterodimer of the ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role of defining the repertoire of target genes that respond to the hormone. We report here the determination of the preferred core recognition motif by a binding site selection procedure. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif was observed. This core motif mediated the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. We further present evidence that the ligand of the ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression.

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.Keywords: Drosophila melanogaster; yeast; two-hybrid; ecdysone receptor; dimerization; ultraspiracle.Ecdysteroids are widespread steroid hormones found in invertebrates [1] and plants [2,3] that regulate a variety of developmental, physiological, and reproductive processes [1,3]. Among insects, these hormones regulate the expression of genes through a highly orchestrated and coordinated transcriptional network [4][5][6]. The widespread and diverse effects of ecdysteroids on transcriptional regulation have served as a powerful model for investigating the diverse mechanisms by which steroid hormones, acting via nuclear receptors, exert their effects on a variety of life processes [4,7].The ecdysone receptor (EcR) [8], responsible for mediating these responses, occupies a special position among nuclear hormone receptors because it shows a unique combination of characteristics [9]. Unlike the vertebrate steroid receptors [10][11][12], EcR heterodimerizes with the insect RXR orthologue, ultraspiracle (USP) [13][14][15]. Nevertheless, while other nuclear receptors that dimerize with RXR normally are bound to DNA response elements already in their nonliganded state [11,16], this apparently is not true for the EcR/USP heterodimer (see however, [17]). Immunostaining has shown that the polytene chromosomes of a Chironomid or Sciarid are devoid of EcR/USP signals when prepared from developmental stages associated with low ecdysteroid titers [18,19]. A short in vitro incubation of the tissues with 20-hydroxyecdysone, however, is followed by the appearance of immunostaining signals at known ecdysteroid-responsive gene loci [18,19]. The affinity of EcR/USP dimers for ecdysone response elements (EcREs) clearly increases in the presence of the ecdysteroid muristerone A as demonstrated...

Genetic instability in Streptomyces glaucescens characteristically involves the occurrence of gross genomic rearrangements including high-level sequence amplification and extensive deletion. We investigated the relationship of the unstable melC and strS loci and a 100 kb region of the chromosome which frequently gives rise to intense heterogeneous DNA amplification. Standard chromosome walking using a cosmid bank in conjunction with a "reverse-blot" procedure enabled us to construct a contiguous genomic BamHI map of the unstable region exceeding 900 kb. The unstable genes and the amplifiable region (AUD locus) are physically linked within a 600 kb segment of the chromosome. The previously characterized deletions which affect these loci are merely components of much larger deletions ranging from 270 to over 800 kb which are polar in nature, effecting the sequential loss of the strS and melC loci. The more extensive deletions terminate either adjacent to, or in the vicinity of DNA reiterations at the AUD locus. Additionally, a deletion junction fragment and the corresponding deletion ends were cloned and analysed at the sequence level.

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Earlier work has shown that the afsR genetic locus promotes formation of the pigmented antibiotics actinorhodin and undecylprodigiosin in Streptomyces lividans and its close relative, Streptomyces coelicolor. A protein designated as AfsR has been implicated in this activity. We report here the existence of a previously unknown gene, afsR2, which is separate from and adjacent to the AfsR-encoding sequence and which, when present at high copy number, (i) stimulates transcription of biosynthetic and regulatory genes in the actinorhodin gene cluster (act), and (ii) stimulates the synthesis of undecylprodigiosin. We show that the effects of afsR2 on actinorhodin synthesis are mediated through transcription of the actII-ORF4 locus, which encodes a transcriptional activator of other genes in the act cluster. Analysis of the cloned afsR2 gene indicates that its activity is the result of the 63-amino-acid protein it specifies.