Gene switch

Details Gene switch Gene switch

1996-05-24

2002-04-30

Pak, Michael (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

C435S254100, C435S254110, C435S325000, C435S410000, C435S419000, C536S023500

Reexamination Certificate

active

06379945

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to the identification and characterisation of insect steroid receptors from the Lepidoptera species
Heliothis virescens,
and the nucleic acid encoding therefor. The present invention also relates to the use of such receptors, and such nucleic acid, particularly, but not exclusively, in screening methods, and gene switches. By gene switch we mean a gene sequence which is responsive to an applied exogenous chemical inducer enabling external control of expression of the gene controlled by said gene sequence.
Lipophilic hormones such as steroids induce changes in gene expression to elicit profound effects on growth, cellular differentiation, and homeostasis. These hormones recognise intracellular receptors that share a common modular structure consisting of three main functional domains: a variable amino terminal region that contains a transactivation domain, a DNA binding domain, and a ligand binding domain on the carboxyl side of the molecule. The DNA binding domain contains nine invariant cysteines, eight of which are involved in zinc coordination to form a two-finger structure. In the nucleus the hormone-receptor complex binds to specific enhancer-like sequences called hormone response elements (HREs) to modulate transcription of target genes.
The field of insect steroid research has undergone a revolution in the last three years as a result of the cloning and preliminary characterisation of the first steroid receptor member genes. These developments suggest the time is ripe to try to use this knowledge to improve our tools in the constant fight against insect pests. Most of the research carried out on the molecular biology of the steroid receptor superfamily has been on
Drosophila melanogaster
(Diptera), see for example International Patent Publication No WO91/13167, with some in Manduca and Galleria (Lepidoptera).
It has been three decades since 20-hydroxyecdysone was first isolated and shown to be involved in the regulation of development of insects. Since then work has been carried out to try to understand the pathway by which this small hydrophobic molecule regulates a number of activities. By the early 1970s, through the studies of Clever and Ashburner, it was clear that at least in the salivary glands of third instar Drosophila larvae, the application of ecdysone lead to the reproducible activation of over a hundred genes. The ecdysone receptor in this pathway is involved in the regulation of two classes of genes: a small class (early genes) which are induced by the ecdysone receptor and a large class (late genes) which are repressed by the ecdysone receptor. The early class of genes are thought to have two functions reciprocal to those of the ecdysone receptor; the repression of the early transcripts and the induction of late gene transcription. Members of the early genes so far isolated and characterised belong to the class of molecules with characteristics similar to known transcription factors. They are thus predicted to behave as expected by the model of ecdysone action (Ashburner, 1991). More recently, the early genes E74 and E75 have been shown to bind both types of ecdysone inducible genes (Thummel et al., 1990; Segraves and Hogness, 1991), thus supporting their proposed dual activities. It should be noted however, that the activation of a hierarchy of genes is not limited to third instar larvae salivary glands, but that the response to the ecdysone peak at the end of larval life is observed in many other tissues, such as the imaginal disks (i.e. those tissues which metamorphose to adult structures) and other larval tissues which histolyse at the end of larval life (eg. larval fat body). The model for ecdysone action as deduced by studying the third instar chromosome puffing may not apply to the activation of ecdysone regulated genes in adults. In other words, the requirement for other factors in addition to the active ecdysone receptor must be satisfied for correct developmental expression (e.g. the Drosophila yolk protein gene expression in adults is under control of doublesex, the last gene in the sex determination gene hierarchy).
The ecdysone receptor and the early gene E75 belong to the steroid receptor superfamily. Other Drosophila genes, including ultraspiracle, tailless, sevenup and FTZ-FI, also belong to this family. However, of all these genes only the ecdysone receptor is known to have a ligand, and thus the others are known as orphan receptors. Interestingly, despite the ultraspiracle protein ligand binding region sharing 49% identity with the vertebrate retinoic X receptor (RXR) ligand binding region (Oro et al., 1990), they do not share the same ligand (i.e. the RXR ligand is 9-cis retinoic acid) (Heymann et al., 1992 and Mangelsdorf et al., 1992). All the Drosophila genes mentioned are involved in development, ultraspiracle for example, is required for embryonic and larval abdominal development. The protein products of these genes all fit the main features of the steroid receptor superfamily (Evans, 1988; Green and Chambon, 1988, Beato, 1989) i.e. they have a variable N terminus region involved in ligand independent transactivation (Domains A and B), a highly conserved 66-68 amino acid region which is responsible for the binding of DNA at specific sites (Domain C), a hinge region thought to contain a nuclear translocation signal (Domain D), and a well conserved region containing the ligand binding region, transactivation sequences and the dimerisation phase (Domain E). The last region, domain F, is also very variable and its function is unknown.
Steroid receptor action has been elucidated in considerable detail in vertebrate systems at both the cellular and molecular levels. In the absence of ligand, the receptor molecule resides in the cytoplasm where it is bound by Hsp90, Hsp70 and p59 to form the inactive complex (Evans, 1988). Upon binding of the ligand molecule by the receptor a conformational change takes place which releases the Hsp90, Hsp70 and p59 molecules, while exposing the nuclear translocation signals in the receptor. The ligand dependent conformational change is seen in the ligand binding domain of both progesterone and retinoic acid receptors (Allan et al., 1992a). This conformational change has been further characterised in the progesterone receptor and was found to be indispensable for gene transactivation (Allan et al., 1992b). Once inside the nucleus the receptor dimer binds to the receptor responsive element at a specific site on the DNA resulting in the activation or repression of a target gene. The receptor responsive elements usually consist of degenerate direct repeats, with a spacer between 1 and 5 nucleotides, which are bound by a receptor dimer through the DNA binding region (Domain C).
Whereas some steroid hormone receptors are active as homodimers others act as heterodimers. For example, in vertebrates, the retinoic acid receptor (RAR) forms heterodimers with the retinoic X receptor (RXR). RXR can also form heterodimers with the thyroid receptor, vitamin D receptor (Yu et al., 1991; Leid et al., 1992) and peroxisome activator receptor (Kliewer et al., 1992). Functionally the main difference between homodimers and heterodimers is increased specificity of binding to specific response elements. This indicates that different pathways can be linked, co-ordinated and modulated, and more importantly this observation begins to explain the molecular basis of the pleotropic activity of retinoic acid in vertebrate development (Leid et al., 1992b). Similarly, the Drosophila ultraspiracle gene product was recently shown to be capable of forming heterodimers with retinoic acid, thyroid, vitamin D and peroxisome activator receptors and to stimulate the binding of these receptors to their target responsive elements (Yao et al., 1993). More significantly, the ultraspiracle gene product has also been shown to form heterodimers with the ecdysone receptor, resulting in cooperative binding to the ecdysone response element and capable of rendering mammalian cells ecdysone resp

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