Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-06-05
2001-06-12
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S252300, C435S419000, C435S320100, C435S325000, C435S348000
Reexamination Certificate
active
06245531
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the use of recombinant DNA methods as applied to the nucleic acid sequences and polypeptides characteristic of insect steroid receptor superfamily members and, more particularly, to uses of such receptors and the DNA regulatory elements associated with genes whose expression they regulate for the production of proteins in cultured cells and, and to uses of such hormone receptor proteins and genes in identifying new hormones that control insect development.
BACKGROUND OF THE INVENTION
The temporal sequence of gene expression determines the nature and sequence of steps in the development of the adult animal from the fertilized egg. The common fruit fly,
Drosophila melanogaster
, provides a favorable model system for studying this genetic control of development. Various aspects of Drosophila development are representative of general insect and, in many respects, vertebrate development.
The steroid hormone 20-OH ecdysone, also known as &bgr;-ecdysone, controls timing of development in many insects. See. generally, Koolman (ed.),
Ecdysone: From Chemistry to Mode of Action
, Thieme Medical Pub., N.Y. (1989), which is hereby incorporated herein by reference. The generic term “ecdysone” is frequently used as an abbreviation for 20-OH ecdysone. Pulses, or rises and falls, of the ecdysone concentration over a short period of time in insect development are observed at various stages of Drosophila development.
These stages include embryogenesis, three larval stages and two pupal stages. The last pupal stage ends with the formation of the adult fly. One studied effect of ecdysone on development is that resulting from a pulse at the end of the third, or last, larval stage. This pulse triggers the beginning of the metamorphosis of the larva to the adult fly. Certain tissues, called imaginal tissues, are induced to begin their formation of adult structures such as eyes, wings and legs.
During the larval stages of development, giant polytene chromosomes develop in the non-imaginal larval tissues. These cable-like chromosomes consist of aggregates comprising up to about 2,000 chromosomal copies. These chromosome aggregates are extremely useful because they provide the means whereby the position of a given gene within a chromosome can be determined to a very high degree of resolution, several orders of magnitude higher than is typically possible for normal chromosomes.
A “puff” in the polytene chromosomes is a localized expansion or swelling of these cable-like polytene chromosome aggregates that is associated with the transcription of a gene at the puff locus. A puff is, therefore, an indicator of the transcription of a gene located at a particular position in the chromosome.
A genetic regulatory model was proposed to explain the temporal sequence of polytene puffs induced by the ecdysone pulse which triggers the larval-to-adult metamorphosis. See, Ashburner et al., “On the Temporal Control of Puffing Activity in Polytene Chromosomes,”
Cold Spring Harbor Symp. Quant. Biol
. 38:655-662 (1974). This model proposed that ecdysone interacts reversibly with a receptor protein, the ecdysone receptor, to form an ecdysone-receptor complex. This complex would directly induce the transcription of a small set of “early” genes responsible for a half dozen immediately induced “early” puffs. These early genes are postulated to encode regulatory proteins that induce the transcription of a second set of “late” genes responsible for the formation of the “late” puffs that appear after the early puffs. The model thus defines a genetic regulatory hierarchy of three ranks, where the ecdysone-receptor gene is in the first rank, the early genes in the second rank and the late genes in the third. While this model derived form the puffing pattern observed in a non-imaginal tissue, similar genetic regulatory hierarchies may also determine the metamorphic changes in development of the imaginal tissues that are also targets of ecdysone, as well as the changes in tissue development induced by the pulses of ecdysone that occur at other developmental stages.
Various structural data have been derived from vertebrate steroid and other lipophilic receptor proteins. A “superfamily” of such receptors has been defined on the basis of their structural similarities. See, Evans, “The Steroid and Thyroid Hormone Receptor Superfamily,”
Science
240:889-895 (1988); Green and Chambon, “Nuclear Receptors Enhance Our Understanding of Transcription Regulation,”
Trends in Genetics
4:309-314 (1988), both of which are hereby incorporated herein by reference. Where their functions have been defined, these receptors, complexed with their respective hormones, regulate the transcription of their primary target genes, as proposed for the ecdysone receptor in the above model.
Cultivated agriculture has greatly increased efficiency of food production in the world. However, various insect pests have found it advantageous to seek out and exploit cultivated sources of food to their own advantage. These insect pests typically develop by a temporal sequence of events which are characteristic of their order. Many, including Drosophila, initially develop in a caterpillar or maggot-like larval form. Thereafter, they undergo a significant metamorphosis from which an adult emerges having characteristic anatomical features. Anatomic similarity is a reflection of developmental, physiological and biochemical similarities shared by these creatures. In particular, the principles of the insect ecdysteroid-hormone receptors and development, as described by Ashburner above, likely would be shared by many different types of insects.
As one weapon against the destruction of cultivated crops by insects, organic molecules with pesticidal properties are used commonly in attempts to eliminate the insect populations. However, the ecological side effects of these pesticides, due in part to their broad activity and lack of specificity, and in part, to the fact that some of these pesticides are not easily biodegradable, significantly affect populations of both insect and other species of animals. Some of these organisms may be advantageous from an ecological or other perspective. Furthermore, as the insect populations evolve in directions to minimize the effects of the applied pesticides, the amounts of pesticides applied are often elevated so high as to cause significant effects on other animals, including humans, which are affected directly or indirectly by the application of the pesticides. Thus, an important need exists for both highly specific pesticides or highly active pesticides which have biological effects only on the species of animals targeted by the pesticides, and are biodegradable. Novel insect hormones which, like the ecdysteroids, act by complexing with insect members of the steroid receptor superfamily to control insect development, are likely candidates for pesticides with these desirable properties.
From a different perspective, many medically and commercially important proteins can be produced in a usable form by genetically engineered bacteria. However, many expressed proteins are processed incorrectly in bacteria and are preferably produced by genetically engineered eucaryotic cells. Typically, yeast cells or mammalian tissue-culture cells are used. Because it has been observed that protein processing of foreign proteins in yeast cells is also frequently inappropriate, mammalian cultured cells have become the central focus for protein production. It is common that the production of large amounts of foreign proteins makes these cells unhealthy, which may affect adversely the yield of the desired protein. This problem may be circumvented, in part, by using an inducible expression system. In such a system, the cells are engineered so that they do not express the foreign protein, and therefore are not unhealthy, until an inducing agent is added to the growth medium. In this way, large quantities of healthy cells can be produced and then induced to produce large amounts of the foreign protein. Un
Hogness David S.
Koelle Michael R.
Seagraves William A.
Board of Trustees of Leland Stanford University
Pak Michael
Reed Janet E.
Saul Ewing Remick & Saul, LLP
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