Page 1

United States Patent [191

Hogness et al.

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US005514578A
[11] Patent Number:
5,514,578
[45] Date of Patent:
May 7, 1996
[54] POLYNUCLEOTIDES ENCODING INSECT
STEROID HORMONE RECEIVER
POLYPEPTIDES AND CELLS
TRANSFORMED WITH SAME
[75] Inventors: David S. Hogness, Stanford; Michael
R. Koelle, Menlo Park; William A.
Segraves, San Diego, all of Calif.
[73] Assignee: The Board of Trustees of Leland
Stanford University, Stanford, Calif.
[21] Appl. No.: 954,937
[22] Filed:
Sep. 30, 1992
Related US Application Data
[63] Continuation of Ser. No. 485,749, Feb. 26, 1990, abandoned.
[51] Int. Cl.6 ................................... C12N 5/00; C12N 1/20;
CO7H 04/21
[52] US Cl. ........................................ 435/2402; 435/252.3;
536/235
[58] Field of Search.
935/9, 33, 34; 435/691, 172.3, 320.1, 240.2
[56]
References Cited
US PATENT DOCUMENTS
4,704,362 11/1987 Itakura et a1. ........................... .. 435/253
4,818,684
4/1989 Edelmann et al. ............................... 435/7

FOREIGN PATENT DOCUMENTS

WO90/06364 6/1990 WIPO.
OTHER PUBLICATIONS
Ashburner et al. (1974) Cold Spring Harbor Symp. Quantity
Biol, 38:655-662. The Temporal Control of Pouf?ng Activity
in Polytene Chromosomes.
Evans (1988) Science 240:889-895 The Steroid and Thyroid
Hormone Receptor Superfamily.
Green and Chambon (1988) Trends in Genetics 4:309—3l4
Nuclear Receivers Enhance 0ur Understanding of Tran~
scription Regulation.
Segraves (1988) Ph.D. Thesis, Stanford University Molecu
home and Genetic Analysis of the E75 Ecdysone-Responsive
Gene of Drosophila melanagasten
Krust et al. (1986) EMBO J. 5:891-897 The chicken oestrus
gene receptor sequence: homology with v-erbA and the
human estrogen and glucocorticoid receptors.
M. Kanehisa (1984) Nucleic Acids Res. 122203-213 Use of
statistical criteria for screening potential homologies in
nucleic acid sequences.
Hershko and Ciechanover (1982) Ann. Rev. Bioch.
51:335—364 Mechanisms of Intracellular Protein Break
down.
Miller et al. (1985) EMBO J. 4:1609-1614 Receiver
zinc—binding domains in the protein transcription factor IHA
from Xenopus oocytes.
'
Freedman et al. (1988) Nature 334:543-546 The function
and structure of the metal coordination sites within the
glucocorticoid receptor DNA binding domain.
Seveme et al. (1988) EMBO J. 9:2503-2508 Metal binding
?nger” structures in the glucocorticoid receptor de?ned by
site-directed mutagenesis.
Giguere et al. (1986) Cell 46:645-652 Functional Domains
of the Human Glucocorticoid Receptor.
Danielson et a1. (1987) Mol. Endocrinol. 1:816-822
Domains of the Glucocorticoid Receptor Involved in Spe
ci?c and Nonspeci?c Deoxyribonucleic Acid Binding, Hor
mone Activation, and Transcriptional Enhancement.
Rusconi et al. (1987) EMBO J. 6:1309-1315 Functional
dissection of the hormone and DNA binding activities of the
glucocorticoid receptor.
Mader et al. (1989) Nature 338:271-274 Three amino acids
of the estrogen receptor are essential to its ability to
distinguish an estrogen from a glucocorticoid-responsive
element.
Umesono and Evans (1989) Cell 57:1139-46 Determinants
of Target Gene Speci?city for Steroid/Thyroid Hormone
Receivers.
Umesono et al (1988) Nature 336:262—265 Retinoic acid
and thyroid hormone induce gene expression through a
common responsive element.
.
Kumar and Chambon (1988) Cell 55: 145-156 The Estrogen
Receiver Binds Tightly to Its Responsive Element as a
Ligand-Induced Homodimer.
Guiochon et al. (1989) Cell 57:1147-1154 Mechanisms of
Nuclear Localization of the Progesterone Receiver: Evi
dence for Interaction between Monomers.
Picard and Yamamoto (1987) EMBO J. 6:3333-3340 Two
signals mediate hormone-dependent nuclear localization of
the glucocorticoid receptor.
Pratt et al. (1988) J. Biol. Chem. 263:267-273 A Region in
the Steroid Binding Domain Determines Formation of the
Non-DNA-Binding, 9S Glucocorticoid Receptor Complex.
Nauber et al. (1988) Nature 336:489-492 Abdominal sec
mentation of the Drosophila embryo requires a hormone
receptor—like protein encoded by the knirps gene gap.
Oro et al. (1988) Nature 336z493-496 The Drosophila gene
knirps-related is a member of the steroid receptor gene
superfamily.
Rothe et al. (1989) EMBO J. 8:3097-3094 Three hormone
receptor~like Drosophila genes encode an identical DNA-b
inding nger.
Petkovich et al. (1987) Nature 330:444-450 A human ret
inoic acid receiver which belongs to the family of nuclear
receivers.
Dieckmann and Tzagalo?" (1985) J. Biol. Chem.
260:1513-1520 Assembly of the Mitochondrial Membrane
System.
Riddihough and Pelham (1987) EMBO J. 613729-3734 An
ecdysone response element in the Drosophila hsp27 pro
moter.
(List continued on next page.)
Primary Examiner—Jacqueline M. Stone
Assistant Examiner—Deborah Crouch
Attorney, Agent, or Firm-Townsend and Townsend and
Crew
[57]
ABSTRACT
Polynucleotide sequences which encode ecdysone receivers
have been isolated and expressed in host cells.
8 Claims, 6 Drawing Sheets

Page 2
5,514,578
Page 2
OTHER PUBLICATIONS
Strangmarm-Diekmann et a1. (1990) Eur. J. Biochem.
189:137-143. A?inity Labeling of Partially Puri?ed Ecdys
teroid Receptor with Bromoacetylated 20-OH-ecdysone
Derivative.
Lehmann et a1. (1988) Molecular and Cellular Endocrinol
ogy 57:239-249 Ecdysteroid Receptors of the blow?y.
Poole et a1. (1985) Cell 40:37-40 The Engrailed Locus of
Drosophila: Structural Analysis of an Embryonic Transcript.
Feigl et a1. (1989) Nucleic Acids Research
17(1 8):7167—7178 A member of the steroid hormone recep
tor gene family is expressed in the 20—OH—ecdysone induc
ible pu?” 75B in Drosophila melanogaster.
Ronald M. Evans (1988) Science 240:889—895 The Steroid
and Thyroid Hormone Receptor Superfarnily.
Bidmon and Koolman (1989) Experience 45:106-109
Ecdysteroid receptors located in the central nervous system
of an insect.
Meyerowitz and Hogness (1982) Cell 28:165-176 Molecu
home Organization of a Drosophila Pullc Site That Responds to
Ecdysone.
Lehmann et al (1988) Molec. & Cell. Endocrinol. Ecdys
teroid receivers of the blow?y, 57, 239-249.
Suggs et al (1981) Proced. Nat. Sci. 78, 6613-6617.
Reeck et a1. (1987) Cell 50, 667.

Page 3
US Patent
May 7, 1996
Sheet 1 of 6
5,514,578
EcR ORF
SV40
intron/
poly A
HYGr
OHF
Copy LTR'
Page 4
US Patent
May 7, 1996
Sheet 2 of 6
5,514,578
r)
PDAdh -34 +53 '
BamHI BglII ScaI lSacI
SalI
SalI
UBX leader and AUG
pUC18
pEcHE/Adh/Bgal
Bgal
7.9 kb
ORF
EcoRI
SV40
splice
Page 5
US Patent
May 7, 1996
_Sheet 3 0f 6
5,514,578
EcR ORF
Page 6

Page 7

Page 8

Page 9
5,514,578
1
POLYNUCLEOTIDES ENCODING INSECT
STEROID HORMONE RECEIVER
POLYPEPTIDES AND CELLS
TRANSFORMED WITH SAME
This invention was made in part with Government sup
port under Grant DCB 8405370 from the National Science
Foundation. The Government may have certain rights in this
invention.
This is a continuation Ser. No. 07/485,749 ?led Feb. 26,
1990, now abandoned.
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 super
family members and more particularly to uses of such
- receptors and the DNA regulatory elements associated with
whose expression genes they regulate for the production of
proteins in cultured cells and, and to uses of such hormone
receptor proteins and genes 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 ?y, Droso
phila 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 develop
ment.
The steroid hormone 20-0H ecdysone, also known as
B-ecdysone, controls timing of development in many
insects. See generally, Koolman (ed.), Ecdysane: From
Chemistry to Mode of Action, Thieme Medical Pub., NY.
(1989), which is hereby incorporated 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 Droso
phila development.
These stages include embryogenesis, three larval stages
and two pupal stages. The last pupal stage ends with the
formation of the adult ?y. 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 ?y. 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 with
prising up to about 2,000 chromosomal copies. these chro
mosome aggregates are extremely useful because they pro
see the means by which 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 typi
cally possible for normal chromosomes.
A “puif' in the polytene chromosomes is a localized
expansion or swelling of these cable-like polytene chromo
add aggregates that are associated with the transcription of
a gene at the puff locus. A pu?' is an indicator of
the transcription of a gene located at a particular position in
the chromosome.
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60
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two
A genetic regulatory model was proposed to explain the
temporal sequence of polytene puffs induced by the ecdys
one pulse which triggers the larval-to~adult metamorphosis.
See, Ashburner et al., “On the Temporal Control of Pu?ing
Activity in Polytene Chromosomes,” Cold Spring Harbor
Symp. Quantity Biol. 38:655-662 (1974). this model pro
posed that ecdysone interacts reversibly with a receiver
protein, the ecdysone receptor, to form an ecdysone-receptor
complex. This complex would directly induce the transcript
tion 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 de?nes a genetic regulatory hierarchy
of three ranks, where the ecdysonereceptor gene is in the
?rst rank, the early genes in the second rank and the late
genes in the third. While this model derived form the pu?ing
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 devel
opment 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. The “superfam
ily” of such receivers has been de?ned 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 4z309~3l4 (1988), both of which are hereby incor
porated herein by reference. Where their functions have
been de?ned, these receivers, complexed with their respec
I had hormones, regulate the transcription of their primary
target genes, as proposed for the ecdysone receptor in the
above model.
Cultivated agriculture has greatly increased and?iciency of
food production in the world. However, various insect pests
have found it advantageous to seek out and exploit culture
vated 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 signi?cant metamor
phosis from which an adult emerges having 'characteristic'
anatomical features. Anatomic similarity is a reaction 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 pesticide properties are
used commonly in attempts to eliminate the insect population
tions. However, the ecological side effects of these pesti
cides, due in part to their broad activity and lack of speci
?city, and in part, to the fact that some of these pesticides are
not easily biodegradable, signi?cantly 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 applied pesticides are often elevated so high
as to cause signi?cant effects on other animals, including
humans, which are affected directly or indirectly by the
application of the pesticides. Thus, an important need exists

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5,514,578
3
for both highly speci?c 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 com
plexing with insect members of the super receptor steroid
family to control insect development, are likely candidates
for pesticides with these desirable properties.
From a different perspective, many medically and with
mercially important proteins can be produced in a usable
form by genetically engineered bacteria. However, many
expressed proteins are processed incorrectly in bacteria and
are preferred produced by genetically engineered eucary
otic 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 fre
warmly inappropriate, mammalian cultured cells have
become the central focus for protein production. it is with
mon that the production of large amounts of foreign proteins
makes these cells unhealthy, which may affectly affect the
yield of the desired protein. this problem may be circumspect
vented, 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. Unfortunately, in the presently available systems,
the inducing agents themselves, such as metal ions or high
temperature, adversely affect the cells, thus again lowering
the yield of the desired foreign protein the cells produce. THE
need therefore exists for the development of innocuous
inducing factors for efficient production of recombinant
proteins. Such innocuous factors could also prove invaluable
for human therapy, where the individual suffers from lack of
the ability to produce particular proteins. by using methods
similar to those for producing proteins in cultured cells, such
innocuous factors for inducing the synthesis of the required
protein could be used for controlling both the timing and the
abundance of the protein produced in the affected individual.
The hormones that complex with mammalian or other
vertebrate members of the steroid receptor superfamily are
unlikely candidates for such innocuous factors, nor have
they have been found to satisfy the required properties of such
factors, because mammalian cells contain these receptors, or
highly homologous proteins, which would alter the expression
of many target genes in the presence of the respective
hormone, adversely affecting the host cells.
For these and other reasons, Getting steroid receptors or
nucleic acid information about them has been a goal of
researchers for several years. Unfortunately, efforts have
been unsuccessful despite signi?cant investment of
resources. The absence of information on the structure and
molecular biology of steroid receptors has signi?cantly
hindered the ability to produce such products.
Thus, there exists a need for detailed sequence informs
tion on insect members of the steroid receptor superfamily,
and the genes that encode these receptors and for resulting
reagents useful in nding new molecules which may act as
agonists or antagonists of natural insect members of the
steroid receptor superfamily, or as components of systems
for highly speci?c regulation of recombinant proteins in
mammalian cells.
SUMMARY OF THE INVENTION
In accordance with the present invention, isolated recom
binant nucleic acids are provided which, upon expression,
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4
are capable of coding for other than native vertebrate
steroid receptor or fragment thereof. These nucleic acids
typically comprise a segment having a sequence substan
tially homologous to one or more coding regions of domains
A, B, D, E or F from an insect steroid receptor superfamily
member gene having steroid binding domain homology.
home, the nucleic acids encode a polypeptide capable of
binding to a ligand for an insect steroid receptor superfamily
member and are capable of hybridizing to an insect steroid
receptor superfamily member gene segment under selective
hybridization conditions, usually stringent hybridization
conditions. Mammalian cells transform with the nucleic
acids are also provided.
In another embodiment, isolated recombinant nucleic
acids are included that have sequence exhibiting identity
over about 20 nucleotides of a coding segment of an insect
steroid receptor superfamily member having steroid binding
domain homology. The nucleic acids can be transformed
into cells to express a polypeptide which binds to a control
element responsive to a ligand of an insect steroid receptor
superfarrrily.
Alternatively, an isolated DNA molecule is provided
having a DNA sequence capable of binding to an insect
steroid receptor superfamily member other than 20-OH
ecdysone receiver, such as DHR3, E75A or E75B. the DNA
sequence may be present in an expression vector and pro
motto transcription of an operably linked sequence (eg,
encoding a polypeptide) in response to binding by an insect
steroid receptor superfamily member. Also contemplated are
recombinant nucleic acids comprising a controlling element
responsive to a ligand of an insect steroid super receiver
family member linking responsive controlling element (eg,
an alcohol dehydrogenase promoter), a non-heat shock pro
moter sequence (eg, an alcohol dehydrogenase promoter)
and a sequence comprising a reporter gene.
Additional embodiments of the present invention include
polypeptides comprising an insect steroid super receptor
family member or fragment thereof, wherein such polypep
tide is substantially free of naturally-associated insect cell
components and exhibits a biological activity characteristic
of an insect steroid receptor superfamily member with a
hormone binding domain. world, the insect steroid
receiver superfamily member or fragment thereof also com
prises a DNA binding domain and the polypeptide is capable
of binding to a analogue hormone selected from the group
consisted of an insect hormone, an insect hormone agonist
and an insect hormone antagonist. The polypeptide can
comprises a zinc-?nger domain and usually is capable of
binding to a DNA controlling element responsive to an
insect hormone. As desired, the polypeptide may be fused to
a second polypeptide, typically a heterologous polypeptide
which comprises a second steroid receiver superfamily
member.
Fragments of such polypeptides can have a sequence
substantially homologous to consensus El, E2 or E3 region
sequences. By way of example, a preferred fragment has a
sequence comprising:
a segment at least about 25% homologous to a consensus
El region sequence;
a segment at least about 30% homologous to a consensus
E2 region sequence; and
a segment at least about 30% homologous to a consensus
E3 region sequence.
The polypeptides of the present invention have a variety
of utilities. For example, a method for selecting DNA
sequences capable of being speci?cally bound by an insect

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5,514,578
5
steroid receptor superfamily member can comprise the steps
of screening DNA sequences for binding to such a polypep
tides and selecting DNA sequences exhibiting such binding.
Alternatively, methods for selecting ligands speci?c for
binding to a hormone binding domain of an insect steroid
superfamily member receiver can comprise the steps of
screening compounds for binding to one or more superfam
ily members and selecting compounds exhibiting speci?c
binding to the members. Also included are methods for
modulating insect physiology or development (eg, killing)
comprising the steps of screening compounds for binding to
an insect steroid receptor superfamily member, selecting
compounds exhibiting said binding and administering the
binding to an insect.
.

Additionally provided are methods for selecting ligands

speci?c for binding to a ligand binding domain of an insect
steroid receptor superfamily member comprising combin
mg:
(i) a fusion polypeptide which comprises a ligand binding
domain functionally linked to a DNA binding domain
of a second steroid receptor superfamily member; and
(ii) the second nucleic acid sequence encoding the second
polypeptide, wherein expression of the second nucleic
acid sequence is responsive to DNA binding
binding domain;
screening compounds for an activity of inducing expressions
sion of said second polypeptide; and
selecting said compounds.
Also provided are methods for producing a polypeptide
including the steps of:
selecting a cell, typically a mammalian or plant cell which
is substantially insensitive to exposure of an insect steroid
superfamily ligand receptor;
introducing into said cell:
(i) a receptor for the ligand; and
(ii) the nucleic acid sequence encoding the polypeptide, the
nucleic acid sequence operable linked to controlling
element responsive to the presence of the selected ligand,
wherein a transformed cell is produced; and
exposing the transformed cell to the ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. pMTEcR, a Cu2+-inducible EcR expression plas
mid. The PMT, EcR ORF and Act 50 poly A elements are
de?ned in Experimental Example I[[, part A. The HYG'
ORF confers hygromycin resistance and is under control of
the promoter in the LTR of Drosophila transposable he
ments, copies. The SV40 intron/poly A element provides an
intron for a possible splicing requirement, as well as a
polyadenylation/cleavage sequence for the HYG' ORF
mRNA. The pATl53 DNA derived from a bacterial plasmid.
FIG. 2. The ecdysone~inducible pEcRE/Adh/[Sgal reporter
plasmid. See the text of Experimental Example 'III, part B,
for the construction of this plasmid and the de?nitions of all
symbols (except the SV40 splice and poly A) which are
de?ned in the ?gure legend.
FIG. 3. The constitutive EcR expression plasmid, pAct
EcR. The construction of this plasmid and the de?nition of
the symbols are given in Experimental Example III, part B.
FIG. 4(AC). The cDNA sequence of the EcR gene.
Numerals at the left refer to the nucleotide sequences; those
on the right to the amino acid sequence in the EcR protein.
Nucleotides l~5 194 are the sequence of EcR-17 eDNA,
while nucleotides 5195-5534 derive from the EcR-9 cDNA.
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40
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65

6
The underlined sequences in the 5' and 3' untranslated
regions refer, respectively, to the ATG codons and the
AATAAA consensus polyadenylation signals. Positions of
the introns and the donor and acceptor splice sequences are
indicated above the cDNA sequence in small type. The
amino acid sequences homologous to the conceived DNA
binding (C region) and hormone-binding (E region) domains
of the steroid receptor superfamily are underlined.
DESCRIPTION OF THE PREFERRED
EMBODIMENTS
The present invention provides novel isolated nucleic acid
sequences encoding polypeptide products exhibiting the
structure and/or activities of insect members of the steroid
superfamily receiver. Having elucidated the structures of
these insect steroid receptors from their genes, the separate
ligand-binding domains and DNA-binding domains are used
individually or in combination to screen for new ligands or
DNA sequences which bind to these domains. Thus, for
example, receivers may be used to control expression of
reporter genes for which sensitive assays exist. Or, the
hormone-binding domains serves as reagents for screening
new molecules, useful as either agonists or antagonists of
steroid receptor superfamily members. Either new classes of
molecules may be screened, or selected modifications from
known ligands may be used. These new ligands ?nd use as
highly speci?c and highly active, naturally occurring pesti
cides. Alternatively, structural information about interaction
tions between the ligand and binding domains directs meth
ods for mutagenizing or substituting particular residues in
the binding domains, thereby providing for altered binding
speci?city. Thus, inter alia, the present invention provides
for screening for new ligand molecules, for the design of
new ligand-binding domain interactions, for producing
novel chimaeric steroid receiver superfamily members and
for generating new combinations of ligands and binding
domains.
The present invention also provides for the isolation or
identification of new steroid hormone-responsive elements
and associated genes. By appropriate operable linkage of
selected sequences to DNA controlling elements which are
responsive to binding by the DNA-binding domains of
steroid receiver superfamily members, new regulatory com
combinations result. The present invention further provides for
the design of either a binding domain in a member of the
insect steroid receptor superfamily that will recognize given
DNA sequences, or conversely for the modification of DNA
sequences which will bind to particular DNA-binding
domains. Both the DNA-binding domain of a superfamily
member polypeptide and its DNA recognition sequence can
be coordinately modified to produce wholly new receiver
DNA interactions.
In an alternative embodiment, a DNA-binding sequence
recognized by a selected receiver may be operable linked to
a desired genetic sequence for inducible expression. Thus,
upon administration of a ligand speci?c for that selected
receiver, the desired genetic sequence is appropriately regu»
lated. expression systems are constructed that are respon
sive to administration of insect steroid receptor superfamily
specific ligands. By identifying and isolating new members
of the insect steroid receptor superfamily, new regulatory
reagents become available, both with respect to usable
hormones, and with respect to useable controlling elements.
In another embodiment, highly regulatory expression of
a gene may be achieved by use of regulatory elements
responsive to speci?c ligands to the superfamily members.

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5,514,578
7
If transformed cells are grown under conditions where
expression is repressed or not induced, the cells may grow
to higher densities and enjoy less stressful conditions. upon
reaching high density, the regulatory ligand molecule will
adjust to cause high expression. If the selected cells are
otherwise insensitive to the inducing ligand, the cells will
not affected by exposure to the ligand used to regulate
expression. This provides a means both for highly efficient
regulatable expression of genes, and for introduction of
these genes into intact organisms.
In accordance with speci?c embodiments of the present
invention, nucleic acid sequences encoding portions of
insect steroid hormone receptor hormone receptor superfam
ily members have been elucidated. For example, certain
ecdysone receptor polypeptides have been isolated and
characterized; speci?cally, DNA's encoding four different
members of the Drosophila steroid receptor superfamily
have been characterized. One is the 20-OH ecdysone recep
tor, also called the ecdysone receiver (EcR), for which a
full-length encoding sequence has been determined. To sec
where member is Drosophila hormone receptor 3 (DHR3), a
protein with sequence homology to various steroid receptor
superfamily members. The third and fourth members of the
superfamily are E75A and E75B, closely related proteins.
These members are encoded by segments of the same gene,
and each possesses sequence homology to other steroid
receiver superfamily members.
The DNA sequences encoding each of these members of
the insect steroid receptor superfamily provide probes for
screening for homologous nucleic acid sequences, both in
Drosophila and other genetic sources. This screening allows
isolation of homologous genes from both vertebrates and
invertebrates. Production of large amounts of the encoded
proteins are effected by inserting those sequences into expresses
sion systems.
The EcR, DHR3, E75A and E75B genes are each linked
to similar DNA sequences which likely function as control
ling, or regulatory, elements. These controlling elements are
in a regulated fashion characteristic of response to binding
by proteins homologous to members of the steroid receptor
superfamily. The present invention provides for the isolation
of these hormone-responsive control elements, and for their
use in regulating gene expression. One embodiment of a
DNA construct comprises: (1) multiple copies of an insect
steroid receptor superfamily controlling element linked to
(2) a minimal gene promoter, preferably not a heat shock
promoter gene, which provides highly inducible expression
of (3) an operably linked gene. This construct provides a
very sensitive, assay for the presence of the controlling
molecule of the receptor.
5
Another aspect of the present invention involves cells
comprising: (1) isolated recombinant gene segments encod
ing biologically active fragments of insect steroid receptor
superfamily proteins; (2) DNA sequences which bind insect
steroid receptors, eg, the elements involved in hormone
responsive control; or (3) modified receptor proteins. Prog
eny of cells which are transformed are included within
transformed cells generally. In particular, the present invention
tion provides for a system available expression of polypep
tides is responsive to steroid induction. For instance, the
system which expresses the desired protein in response to
exposure to ecdysone analogues is constructed by operaably
linking an ecdysone-responsive enhancer to a peptide encod
ing segment.
The present invention also provides insect steroid receiver
substantially free proteins from naturally-associated insect
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cell components. Such receivers will typically be either
full-length proteins, functional fragments, or fusion proteins
comprising segments from an insect steroid receptor protein
fused to a heterologous, or normally non-contiguous, protein
domain.
The present invention further provides a number of meth
ods proteins for utilizing the receptor subject. One aspect' of
the present invention is a method for selecting new hormone
analogues. The isolated hormone-binding domains speci?
cally bind hormone ligands, thereby providing a means to
screen for new molecules possessing the property of binding
with high a?inity to the ligand-binding region. Thus, the
binding domain of an insect steroid receptor superfamily
member may be used as a reagent to develop a binding assay.
On one level, the binding domains can be used as af?nity
reagents for a batch or in a column selective process, to
selectively retain ligands which ?nd. Alternatively, the function
tional assay is preferred for its greater sensitivity to ligand
binding. By using a reporter molecule for binding, either
through a direct assay for binding, or through an expression
or other functional linkage between binding and another
function, an assay for binding may be developed. for
example, by operable linkage of an easily assayable reporter
gene to a controlling element responsive to binding by an
insect steroid receptor superfamily member, and where
ligand-binding is functionally linked to protein induction, an
extremely sensitive assay for the presence of a ligand or of
the receiver results. Such a construct useful for assaying the
presence of 20-OH ecdysone is described below. this with
struct is useful for screening for agonists or antagonists of
the 20-OH ecdysone ligand.
In particular, this method may be used to detect the ligand
which bind to a receiver, ie, an “orphan receiver,” whose
ligand is unknown. Binding domains with “unknown”
ligands may originate from either newly identified insect
steroid receptor superfamily members, or from mutagenesis.
A hybrid receiver may be created with a ligand-binding
domain and DNA-binding domain from di?'erent sources.
This would allow screening for ligands for "orphan recep
tor” binding domains functionally linked to known DNA
binding domains which will control known reporter gene
constructs as described below. This system for calling
recipient binding provides and extremely sensitive assay for
ligand-receptor interactions.
Alternatively, the tertiary structure and spatial interactions
between a ligand-binding domain from an insect steroid
superfamily member and its ligand receiver will direct
design for new combinations of ligand-binding domains
with ligands. Either method provides for highly selecting
speci?c and unusual ligands which may be bound only by a
modification of a natural polypeptide-binding receptor
domain. Alternatively, novel steroid hormone analogues
may be selected which exhibit modi?ed speci?city for
binding to a limited group of steroid receptors.
The present invention also provides for new and useful
combinations of the various related components. the recom
binant nucleic acid sequences encoding the polypeptides, the
polypeptide sequences, and the DNA sites to which the
bind receivers (ie, the regulatory, or control, elements)
together provide for combining particular components in
novel fashions. For instance, upon expression, fusing
nucleic acid sequences encoding peptides from different
sources will provide polypeptides exhibiting hybrid proper
ties. In particular, hybrid receivers comprising segments
from other members of the superfamily, or from other
sources, will be made. hybrid genetic constructs provide for
exhibiting unusual control and expression character genes

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9
istics. Combining an insect steroid receptor-responsive
enhancer segment with a different polypeptide encoding
segment will produce a steroid-responsive expression sys
has for that polypeptide.
The isolation of insect steroid receivers provides for
isolation or screening of new ligands for receptor binding.
Some of these will interfere with, or disrupt, normal insect
development. It may sometimes be important to either
accelerate or decelerate insect development, for instance, in
preparing sterile adults for release. Alternatively, in certain
circumstances, a delay or change in the timing of develop
ment may be lethal or may dramatically modify the ability
of an insect to affect an agricultural crop. Thus, naturally
recurrent, biodegradable and highly active molecules to
disrupt the timing of insect development will result.
Furthermore, these polypeptides provide the means by
which antibodies have been raised. These possessions
speci?city for binding to particular steroid receptor classes.
Thus, reagents for determining qualitative or quantitative
presence of these or homologous polypeptides may be
produced. Alternatively, these antibodies may be used to
separate or purify receptor polypeptides.
Transcription sequences of insect steroid receptor superfam
ily members
The ecdysone receptor gene is a member of the steroid
and thyroid hormone receptor gene superfamily. the steroid
receptors and thyroid hormone receptors are components of
a collective group of ligand-responsive transcription factors.
See, Evans, Science 240:889-895 (1988), and Segraves,
Molecular and Genetic Analysis of the E75 Ecdys0ne~
Responsive Gene of Drosophila melanogaster (Ph.D. thesis,
Stanford University 1988), both of which are hereby incor
porated herein by reference for all purposes. these receivers
show extensive sequence similarity, especially in their “zinc
?nger” DNA-binding domains, and also in a ligand, or
hormone, binding domain. Modulation of gene expression
Apparently occurs in response to receiver binding to speci?c
control, or regulatory, elements in the DNA. the cloning of
receptor cDNAs provides the ?rst opportunity to study the
molecular bases of steroid action. the steroid receptor
superfamily is a class of receivers which exhibit similarities
in structural and functional features. While the term insect is
used herein, it will be recognized that the same methods and
molecules may be derived form other species of animals, in
particular, within the class Insecta, but more broadly should
be applicable to all members of the phylum Arthropoda,
which use ecdysteroids as hormones. Thus, although the
term insect is used herein, it will be recognized that in some
circumstances the larger group of arthropods may be also
included. Members of the superfam receiving insect steroid
ily (superfamily) are characterized by functional domains
involved in ligand-binding and DNA binding, both of which
interact to efect a change in the regulatory state of a gene
operably linked to the DNA-binding site of the receiver.
Thus, the receptors of the insect steroid receptor superfamily
seem to be linking-responsive transcription factors. The
receptors of the present invention exhibit at least a hormone
binding domain characterized by sequence homology to
particular regions, labeled El, E2 and E3.
The members of the insect steroid receptor superfamily
are typically characterized by structural homology of par
particular domains, such as de?ned initially in the estrogen
receiver. Speci?cally, a DNA-binding domain, C, and a
ligand-binding domain, E, are separated and ?anked by
additional domains as identi?ed by Krust et a1. (Krust et al.
(1986), EMBO J. 5:891-897), which is incorporated herein
by reference.
The C domain, or zinc-?nger DNA-binding domain, is
usually hydrophilic, having high cysteine, lysine and argi
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nine content—a sequence suitable for the required tight
binding. The E domain is usually hydrophobic and charac
terized regions E1, E2 and E3. the binding-binding
domains of the present invention are typically characterized
by having signi?cant homology in sequence and structure to
these three regions. Amino proximal to the C domain is a
region initially de?ned as separate A and B domains. Region
D separates the more conserved domains C and E. Region D
typically has a hydrophilic region whose predicted second“
ary structure is rich in turns and coils. The F region is
carboxy proximal to the E region (see, Krust et al., supra).
The ligand-binding domain of the members of the insect
steroid receptor superfamily is typically carboxyl-proximal,
relative to the DNA-binding domain described below. See,
Evans, Science 240:889-895. The entire hormone-binding
domain is typically between about 200 and 250 amino acids
but may be less. This domain has the subregions of high
homology, termed the El, E2 and E3 regions. See Table 4.
The El region is 19 amino acids long with a consensus
sequence AKX(L/I)PGFX)?3T(L/I)(D/E)DQITLL, where X
represents any amino acid andv the other letters are the
standard single-letter code. Positions in parentheses are
alternatives. Typically, members of the insect steroid recep
tor superfamily will have at least about ?ve matches out of
the sixteen assigned positions, preferably at least about nine
matches, and in preferred embodiments, at least about ten
matches. Alternatively, these insect steroid super receptor
family members will have homologous sequences exhibiting
at least about 35% homology, preferably at least about 55%
homology and more preferably at least about 60% to 70%
homology at positions assigned preferred amino acids.
The E2 region is a 19 amino-acid segment with a con
sensus sequence:
where - represents an optional absence of an amino acid.
Typically, an insect steroid receptor superfamily member
exhibit at least about six matches, will preferably at least
about eight matches and more preferably at least about nine
matches. Alternatively, E2 sequences of insect steroid recep
tor superfamily members exhibit at least about 30% homol
ogy, preferably at least about 40% homology, and more
preferably at least about 45% homology.
The E3 region is a 12 amino-acid segment with a con
sensus sequence
The insect steroid receptor superfamily members will typi
cally show at least about four matches out of nine
assigned preferences in the E3 region, preferably at least
about ?ve matches and more at least about six
matches. Alternatively, over the assigned positions, mem
bers of the insect steroid receptor superfamily will typically
exhibit at least about 45% homology, usually at least about
55% homology and preferably at least about 65% homology.
In preferred embodiments, the insect steroid receptor
superfamily members will exhibit matching of at least about
?ve positions in an El region, at least about six positions in
an E3 region and at least about four positions in an E3
region. Thus, the combination of all three regional sequence
constraints are especially preferred.
The DNA-binding domain of these insect steroid receptor
superfamily members is characterized by "zinc ?ngers"
motif. See, Evans, Science 240:889-895. The domain is
typically arnino proximal to the ligand, or hormone, binding

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site. Typically, the DNA-binding domain of the insect ste~
roid receiver superfamily members is characterized by clus
tering of basic residues, a cysrich composition and homol
ogy in sequence. See, Evans, RM (1988), Science
240:889-89; and Experimental section below. Signi?cant
sequence homology among superfamily members exists.
Typically, the insect steroid receptor superfamily members
will exhibit at least about [30]% homology in the 67il
amino acid region of this domain, usually at least about 40%
homology, and preferably at least about 45% homology.
Steroids are derivatives of the saturated tetracyclic hydro
carbon perhydrocyclopentanophenanthrene. among the
molecules in the group “steroids” are the bile acids, cholic
acid and deoxycholic acid, the adrenocortical steroids, such
as corticosterone and aldosterone, the estrogens such as
estrone and [i-estradiol, the androgens, such as testosterone
and progesterone, and the ecdysteroids. the terms steroid or
steroid hormones are used interchangeably herein and are
intended to include all steroid analogues. Typically, steroid
analogues molecules are which have minor modifications of
various peripheral chemical groups. See, Koolman (ed.)
(1989), cited above, for details on ecdysteroids.
Although ligands for the insect steroid receptor superfam
ily members have historically been characterized as steroids,
the term “steroid” in the labe “insect steroid receptor
superfamily” is not meant literally. The use of “steroid” has
resulted from a historical label of members of a group
recognized initially to include only steroids. However, the
limitation is no longer applicable. Thus, there may be
members of the insect steroid receptor superfarrrily, as
de?ned herein, whose binding-binding speci?city is not
directed to "steroids." Typically, the ligands for members of
the insect steroid receptor superfamily are lipophilic mol
ecules.
The term "ligand" is meant herein to exclude the DNA
sequence to which the DNA-binding domain binds. Thus,
the term ligand is meant to refer to the molecules that bind
the domain described here as the “hormone-binding
domain.” Also, a ligand for an insect steroid super receptor
family member is a link which serves either as the natural
binding to which the member binds, or a functional analogue
which may serve as an agonist or antagonist. However, the
functional term “hormone” is used, again, because of the
historic usage to describe the receivers, but is meant to apply
to virtually any chemical messenger used to communicate
between cell types. These molecules are typically used in
intercellular signal transduction, but are not limited to those
having molecules slow or systemic effects.
Substantial homology in the nucleic acid context means
either that the segments, or their complementary strands,
when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least
about 60% of the residues, usually at least about 80% and
preferably at least 90% of the nucleotides. Alternatively,
substantial homology exists when the segments will hybrid
ize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence derived from
Table 1, 2 or 3. Selectivity of hybridization exists when
hybridization occurs which is more selective than total lack
of speci?city. Typically, selective hybridization will occur
when there is at least about 55% homology over a stretch of
at least about 14/25 nucleotides, preferably at least about
65%, more preferably at least about 75%, and most
ably at least about 90%. See, Kanehisa, M. (1984), Nucleic
Acids Res. 121203-213, which is incorporated herein by
reference. Stringent hybridization conditions will typically
include salt concentrations of less than about 1M, more
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less than about 500 mM and usually less than
about 200 mM. Temperature conditions will typically be
greater than 20° C., more usually greater than about 30° C.
and preferably in excess of about 37° C. Other factors
may signi?cantly affect the stringency of hybridization,
including, among others, base composition and size of the
complementary strands, presence of organic solvents and
extent of base mismatching, the combination of parameters
is more important than the absolute measure of any one.
A gene for an insect steroid receptor superfamily member
gene includes its upstream (eg, promoter) and downstream
operably linked controlling elements as well as the complex
mentary strands. It also comprises the segment encoding the
transcription unit, including both introns and exons. Thus, an
isolated gene allows for screening for new steroid receptor
genes by probing for genetic sequences which hybridize to
either controlling or transcribed segments of a receptor gene
of the present invention. Three segments of particular inter
are the controlling elements, both upstream and down
stream, and segments encoding the DNA-binding segments
and the hormone-binding segments.
Insect steroid receptor superfamily member polypeptides
The polypeptide sequence of the ecdysone receptor is rep
resented in Table 2. Other insect steroid receptor superfam
ily member polypeptide sequences are set forth in Tables 1
and 3. Preferred nucleic acid sequences of the cDNAs
encoding these insect steroid receiver superfamily member
polypeptides are also provided in the corresponding tables.
Other nucleic acids may be used to encode the proteins,
making use of the degeneracy or non-universality of the
genetic code.
As used herein, the term “substantially pure” describes a
protein or other material which has been separated from its
native contaminants. Typically, monomeric protein is
substantially pure when at least about 60 to 75% of a sample
exhibits a single polypeptide backbone. Minor variants or
chemical modifications typically share the same polypeptide
sequence. Usually a essentially pure protein will comprise
over about 85 to 90% of a protein sample, and preferably
will be over about 99% pure. Normally, purity is measured
on a polyacrylamide gel, with homogeneity determined by
staining. Alternatively, for certain purposes high resolution
will be necessary and HPLC or a similar means for puri?
cation will be used. For most purposes, the simple chroma
tography column or polyacrylamide gel will be used to
determine purity.
The term "substantially free of naturally-associated insect
cell components” describes a protein or other material which
is separated from the native contaminants which accompany
it in its natural insect cell state. Thus, the protein which is
chemically synthesized or synthesized in a cell system
different from the insect cell from which it naturally originates
nates will be free from its naturally-associated insect cell
components. The term is used to describe insect steroid
receiver superfamily members and nucleic acids which have
been synthesized in mammalian cells or plant cells, E. coli
and other procaryotes.
The present invention also provides for analogues of the
insect steroid receptor superfamily members. such ana
logs include both modifications to a polypeptide backbone
and variants and mutants of the polypeptides. Modifications
include chemical derivatizations of polypeptides, such as
acetylations, carboxylations and the like. They also include
glycosylation modifications and processing variants of a
typical polypeptide. These processing steps speci?cally
include enzymatic modi?cations, such as ubiquinization.
See, eg, Hershko and Ciechanover (1982), “Mechanisms of

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Intracellular Protein Breakdown,” Ann. Rev. Bioch.,
5 1:335-364.
Other analogues include genetic variants, both natural and
induced. Induced mutants may be derived from various
techniques including both random mutagenesis using
reagents such as irradiation or exposure to EMS, or may take
the form of engineered changes by site-speci?c mutagenesis
or other techniques of modern molecular biology. See,
Sambrook, Fritsch and Maniatis (1989), Molecular Cloning:
A Laboratory Manual (2d ed.), CSH Press.
As described above, the DNA-binding zinc ngers sec
ment of a receiver shows high speci?city of recognition of
speci?c target DNA sequences. An understanding of the
DNA protein-binding interactions provides for the modi?
cation in a rational manner either DNA or protein charac
teristics, or both, to effect speci?city of binding for modu
link of enhancer activity. More importantly, isolation of
genes for new members of the insect steroid receptor super
farnily allows their use to produce the receptor polypeptides
and to isolate and isolate new controlling elements. By using
the DNA-binding domains, as described above, controlling
elements which are responsive to the ligands bound by the
corresponding superfarnily members may be identi?ed and
isolated. This shall yield a variety of controlling elements
responsive to ligands. By the methods described above, the
ligands for any particular member of the insect steroid
receiver superfarnily may be identi?ed.
The controlling elements typically are enhancers, but may
also include silencers or various other types of ligand
responsive elements. They may operate at large distances,
but will typically be within about 50 kb, usually within about
35 kb, more usually within about 20 kb and preferably
within about 7 kb of the genes that these elements regulate.
Polypeptide fragments and fusions
Besides substantially full-length polypeptides, the present
provides for biologically active fragments of the
polypeptides. Significant biological activities include
ligand-binding, DNA binding, immunological activity and
other biological activities characteristic of steroid receptor
superfarnily members. Immunological activities include
both immunogenic function in a target immune system, as
as well as sharing of immunological epitopes for binding,
serving as either a competitor or substitute antigen for
asteroid receptor epitope.
For example, ligand-binding or DNA-binding domains
may be “swapped” between different new fusion polypep
tides or fragments. Thus, new chimaeric polypeptides exhib
iting new combinations of speci?cities result from the
functional linkage of ligand~binding speci?cities are DNA
binding domains. This is extremely useful in the design of
inducible expression systems.
For immunological purposes, immunogens may be pro
duced which repeat polypeptide segments, thereby tandemly
producing highly antigenic proteins. Alternatively, such
polypeptides will serve as highly efficient competitors for
specific binding. Production of antibodies to insect steroid
receiver superfarnily members is described below.
The present invention also provides for other polypeptides
comprising fragments of steroid receptor superfarnily mem
bers. Thus, fusion polypeptides between the steroid receptor
segments and other homologous or heterologous proteins are
provided. Homologous polypeptides may be fusions
between different receptor steroid superfarnily members,
resulting in, for instance, a hybrid protein exhibiting ligand
speci?city of one member and DNA-binding speci?city of
another. Likewise, heterologous fusions may be constructed
which would exhibit a combination of properties or activi
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ties of the derivative proteins. Typical examples are fusions
of a reporter polypeptide, eg, luciferase, with another
domain of a receiver, eg, a DNA-binding domain, so that
the presence or location of a desired linking may be easily
determined. See, eg, Dull et al., US. Pat. No. 4,859,609,
which is hereby incorporated herein by reference. other
typical gene ?ision partners include "zinc ?nger" segment
swapping between DNA-binding proteins, bacterial [i-ga
lactosidase, trpE Protein A, l3-lactamase, alpha anylase,
dehydrogenase and yeast alpha mating factor. See, .
eg, Godowski et al. (1988), Science 241:812-816; and
Experimental section below.
Insect steroid receptor superfamily member expression
With the sequence of the receptor polypeptides and the
recombinant DNA sequences encoding them, large amount
ties of members of the insect steroid receptor superfarnily
will be prepared. By the appropriate expression of vectors in
cells, high e?iciency production may be achieved. Thereaf~
ter, standard purification methods may be used, such as
ammonium sulfate precipitations, column chromatography,
electrophoresis, centrifugation, crystallization and others.
See various volumes of Methods in Enzymology for tech
niques typically used for protein purification. Alternatively,
in some embodiments high efficiency of production is
unnecessary, but the presence of a known inducing protein
within a carefully engineered expression system is quite
valuable. For instance, a combination of: (l) a ligand
responsive enhancer of this type operable linked to (2) a
desired gene sequence with (3) the corresponding insect
steroid receptor superfarnily member together in an expres
sion system provides a speci?cally inducible expression
system. Typically, the expression system will be a cell, but
an in vitro expression system may also be constructed.
The desired genes will be inserted into any of a wide
selection of expression vectors. The selection of an app
priate vector and cell line depends upon the constraints of
the desired product. Typical expression vectors are described
in Sambrook et al. (1989). Suitable cell lines may be selected
from a depository, such as the ATCC. See, ATCC Catalog
of Cell Lines and Hybridomas (6th ed.) (1988); ATCC Cell
Lines, Viruses, and Antisera, each of which is hereby
incorporated herein by reference. The vectors are introduced
to the desired cells by standard transformation or transfection
tion procedures as described, for instance, in Sambrook et al.
(1989).
»

Fusion will proteins typically be made by either recom

binant nucleic acid methods or by synthetic polypeptide
methods. Techniques for nucleic acid manipulation are
described generally, for example, in Sambrook et al. (1989),
Molecular Cloning: A Laboratory Manual (2nd ed.), Vols.
1-3, Cold Spring Harbor Laboratory, which are incorporated
herein by reference. Techniques for Synthesis of Polypep
tides are described, for example, in Meni?eld, J. Amer.
Chem. Soc. 85:2149-2156 (1963).
The recombinant nucleic acid sequences used to produce
fusion proteins of the present invention may be derived from
natural or synthetic sequences. Many natural gene sequences
are obtainable from various (DNA or from genomic libraries
using appropriate probes. See, GenBankTM, National Institute
Tutes of Health. Typical probes for steroid receptors may be
selected from the sequences of Tables 1, 2 or 3 in accordance
' with standard procedures. suitable synthetic DNA fragments
may be prepared by the phosphorarnidite method described
by Beaucage and Carruthers, Tetra. Letts 22:1859-1862
(1981). A double stranded fragment may then be obtained
either by synthesizing the complementary strand and anneal
ing the strand together under appropriate conditions or by

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15
adding the complementary strand using DNA polymerase
with an appropriate primer sequence.
With the isolated steroid receptor genes, segments of the
transcribed segments may be used as probes for isolating
homologous sequences from different sources, either diifer
ent animals, or different but homologous genes exhibiting
sequence homology. By selection of the segment used as a
probe, particular functionally associated segments will be
isolated. Thus, for example, other nucleic acid segments
encoding either binding-binding or DNA-binding domains of
new receivers will be isolated. Alternatively, by using ste
mid-responsive controlling elements as a probe, new ste
roid-responsive elements will be isolated, along with the
associated segment of DNA whose expression is regulated.
This method allows for the isolation of ligand-responsive
genes, many of which are themselves, also members of the
insect steroid receptor superfamily.
The natural or synthetic DNA fragments coding for a
desired steroid receiver fragment will be incorporated into
DNA constructs capable of introduction to and expression in
an in vitro cell culture. Usually the DNA constructs will be
suitable for replication in a unicellular host, such as yeast or
bacteria, but may also be intended for introduction to, with
and without and integration within the genome, cultured
mammalian or plant or other eukaryotic cell lines. DNA
constructs prepared for introduction into bacteria or yeast
will typically include a replication system recognized by the
host, the intended DNA fragment encoding the desired
polypeptide receptor, transcription and translational initiation
tion regulatory sequences operably linked to the polypeptide
encoding segment and transcriptional and translational ter
mination regulatory sequences operably linked to the
polypeptide encoding segment. The transcriptional regulates
tory sequences will typically include a heterologous
enhancer or promoter which is recognized by the host. The
selection of an appropriate promoter will depend upon the
host, but promoters such as the trp, lac and phage promoters,
tRNA promoters and glycolytic enzyme promoters are
known. See, Sambrook et al. (1989). Conveniently available
expression vectors which include the replication system and
transcriptional and translational regulatory sequences
together with the insertion site for the steroid receptor DNA _
sequence may be employed. Examples of workable combi
nations of cell lines and expression vectors are described in
Sambrook et al. (1989); see also, Metzger et al. (1988),
Nature 334:31-36.
genetic constructs
The DNA segments encoding the members of the insect
steroid receptor superfamily will typically be used in a
plasmid vector. Two separate embodiments exist, the ?rst
having an expression control DNA sequence operable linked
to the insect steroid receiver superfamily member coding
sequences for expression of the insect steroid receptor
superfamily member alone. A second includes an insect
steroid receptor superfamily member as a component of an
expression system for another gene to make expression of
that other gene ligand responsive. This latter embodiment is
separately described just below. the expression control
sequences will be commonly eucaryotic enhancer or pro
moter systems in vectors capable of transforming or trans
fecting eukaryotic host cells. Once the vector has been
incorporated into the appropriate host, the host, depending
on the use, will be maintained under suitable conditions for
high level expression of the nucleotide sequences.
Steroid-responsive expression of selected genes
For steroid-responsive expression of other genes, the
steroid receptor gene will typically be cotransformed with a
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recombinant construct comprising a desired gene for expression
sion operaably linked to the steroid-responsive enhancer or
promoter element. In this use, a single expression system
will typically comprise a combination of (1) a controlling
element responsive to a ligand of an insect steroid receptor
superfamily member, (2) a desired gene for expression,
operably linked to the controlling element, and (3) an insect
steroid receiver superfamily member which can bind to the
controlling element. Usually, this system will be within a
cell, but an in vitro system is also possible. the insect steroid
receiver superfamily member will typically be provided by
expression of a nucleic acid encoding it, though it need not
be expressed at particularly high levels. Thus, in one pre
ferred embodiment, the system will be achieved through
cotransformation of a cell with both the regulatable with
struct and another segment encoding the insect steroid
superfamily member receiver. Usually, the controlling it
ment will be an enhancer element, but it may work in reverse
and be used to repress expression. In this embodiment, the
ligand for the insect steroid receptor superfamily member
will be provided or withheld as appropriate for the desired
expression properties.
A particularly useful genetic construct comprises an alcohol
hol dehydrogenase promoter operably linked to an easily
assayable reporter gene, eg, B-galactosidase. In a preferred
embodiment of this construct, a multiplicity of copies of the
insect steroid receptor superfamily member is used. for
example, operable linkage of controlling elements response
sive to insect steroid receptor superfamily members, e.g.,
EcR, DHR3, E75A and E75B, to the alcohol dehydrogenase
(ADH) promoter, or others as described above, and protein
coding sequences for a particular reporter protein, as
described above leads to steroid-responsive expression of
that protein. This controlling element responsive to the
construct provides a very sensitive system for the detection
of responsive expression. This will be used in sensitive
assays for the presence of a receiver-ligand interaction,
Allowing for detection of either ligand or receptor or both.
DNA sequences will normally be expressed in hosts after
the sequences have been operably linked to (ie, positioned
to ensure the functioning of) an expression control sequence.
These expression vectors are typically replicable in the host
organisms either as episomes or as an integral part of the
chromosomal host DNA. Commonly, expression vectors
will contain selection markers, eg, tetracycline or neomy
cin, to allow detection of those cells transformed with the
desired DNA sequences (see, eg, US Pat. No. 4,704,362,
which is incorporated herein by reference).
E. coli is one procaryotic host useful for cloning the DNA
sequences of the present invention. other microbial hosts
suitable for use include bacilli, such as Bacillus subtilis, and
other enterobacteriaceae, such as Salmonella, Serratia, and
various Pseudomonas species.
Other eukaryotic cells may be used, including yeast cells,
insect tissue culture cells, avian cells or the like. home,
mammalian tissue cell culture will be used to produce the
inducible polypeptides of the present invention (see, Win
nacker, From Genes to Clones, VCH Publishers, N.Y.
(1987), which is incorporated herein by reference). mom
malian cells are preferred cells in which to use the insect
steroid receptor superfamily member ligand-responsive
gene constructs because they naturally lack the molecules
which check responses to the ligands for insect steroid
receiver superfamily members.
Since mammalian cells are insensitive to many ligands for
insect steroid receptor superfamily members, exposure of
these cells to the ligands of the insect steroid receptor

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superfamily members typically will have negligible physi
logical or other effects on the cells, or on a whole organism.
This insensitivity of the cells to the ligands provides pre
ferred combination of ligand induction with an otherwise
insensitive cell. This provides for transformation of insen
sitive cells with the controlling element operably linked to a
derived gene, resulting in an expression system whose
ligand for eliciting response causes minimal physiological
effects. Therefore, cells can grow and express substantially
unaffected by the presence of the ligand. the caller may
cause response either in the positive or negative direction.
For example, cells might be desired to be grown to high
density before expression. In a positive induction system,
the inducing ligand would be added upon reaching high
density, but since the ligand itself is innocuous to the cells,
the only physiological imbalances result from the expression
itself. Alternatively, in a negative repression system, the
binding is supplied until the cells reach a high density, but
again, the presence of the ligand is innocuous. upon reach
ing a high density, the ligand would be removed. Introduction
tion of these cells into whole organisms may be performed
so that the products of expression may be provided to the
whole organism. In this circumstance, the natural insensi
tivity of cells to the ligands will also be advantageous.
Expression vectors for these cells can include expression
control sequences, such as an origin of replication, a pro
moter, an enhancer and necessary processing information
sites, such as ribosome-binding sites, RNA splice sites,
polyadenylation ' sites, and transcriptional terminator
sequences. past, the enhancers or promoters will be
those naturally associated with genes encoding the steroid
receivers, although it will be understood that in many cases
others will be equally or more appropriate. Other preferred
expression control sequences are enhancers or promoters
derived from viruses, such as SV40, Adenovirus, Bovine
Papilloma Virus, and the like.
Similarly, preferred promoters are those found naturally
in immunoglobulin-producing cells (see, US Pat.
4,663,281, which is incorporated herein by reference), but
SV40, polyoma virus, cytomegalovirus (human or murine)
and the LTR from various retroviruses (such as murine
leukemia virus, murine or Rous sarcoma virus and I'HV)
may be used. See, Enhancers and Eukaryotic Gene
Expression, Cold Spring Harbor Press, NY, 1983, which is
incorporated herein by reference.
The vectors containing the DNA segments of interest
(eg, the steroid receptor gene, the recombinant steroid~
responsive gene, or both) can be transferred into the host cell
by well-known methods, which vary depending on the type
of cellular host. For example, calcium chloride transfection
is commonly used for procaryotic cells, whereas calcium
phosphate treatment may be used for other cellular hosts.
(See, generally, Sarnbrook et al. (1989), Molecular Cloning:
A Laboratory Manual (2d ed.), Cold Spring Harbor Press,
which is incorporated herein by reference.) The term ','trans
formed cell” is meant to also include the progeny of a
transformed cell.
As with the purified polypeptides, the nucleic acid sec
ments associated with the ligand-binding segment and the
DNA-binding segment are particularly useful. these gene
segments will be used as probes for screening for new genes
exhibiting similar biological activities, though the control~
ling elements of these genes are of equal importance, as
described below.
,

Many types of proteins are preferentially produced in

eukaryotic cell types because of abnormal processing or
modification in other cell types. Thus, mammalian proteins
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are preferred expressed in mammalian cell cultures. E?i~
scientific expression of a desired protein may be achieved, as
described above, by placing: (l) a desired protein encoding
DNA sequence adjacent to controlling elements responsive
to ligands for insect steroid receptor superfamily members
and an appropriate promoter. Furthermore, unhealthy cells
are particularly difficult to maintain alive and efficiency of
expression of exogenous proteins falls. inducible expression
systems partly solve this problem, but the presently avail
able inducing molecules have direct side effects on the cells.
By selecting an inducing molecule which otherwise has no
effects on the cell, a more natural physiological state of the
cells may be achieved in growing the cells to high density.
Upon exposure to such an inducing molecule, the cells
initially in a healthy state will produce the desired protein at
high levels without the harmful effects from the
action of the inducing molecule itself. Ecdysteroids and
other ligands for insect steroid receptor superfamily mem
bers are not normally found in mammalian cells, and thus
serves as favorable candidates for a role as innocuous induc
ing molecules. Cyclic pulses of ligands in a cell culture may
provide periods for cells to recover from effects of production
tion of large amounts of exogenous protein.
Additional steroid responsive gene elements have also
been isolated using the techniques of the present invention.
Other genes adjacent to, and operably linked to, steroid
responsive gene controlling elements are selectable by locat
ing DNA segments to which steroid receptors speci?cally
bind or by hybridization to homologous controlling it~
ments. For example, other steroid responsive genes have
been isolated. Many of the genes which are ligand-respon
sive may also be new members of the insect steroid receptor
superfarnily.
Having provided for the essential pure polypeptides,
biologically active fragments thereof and recombinant
nucleic acids comprising genes for them, the present invention_
tion also provides cells comprising each of them. By Appro
priate introduction techniques well known in the ?eld, cells
including them may be produced. See, eg, Sambrook et
al. (1989).
In particular, cells comprising the steroid responsive
controlling elements are provided, and operable linkage of
standard protein encoding segments to said controlling it
ments produce steroid responsive systems for gene expression
sion. Cells so produced may be introduced into intact
organisms, for example, plants, insects (including caterpil
lars and larvae) and animals. This may provide for the form of
regulable expression of desired genes but where the regu
lating linking has no other e?ects on the cells because they
otherwise lack the receptors and responsive genes. for
example, plants the receivers and responsive genes. for
example, plants may be induced to fruit at desired times by
administration of the appropriate ligand, or animals may be
ligand-responsive in production of particular products. And,
in fact, biochemical de?sciences may be overcome by
ligand-responsive expression of cells introduced into an
intact organism which, itself, otherwise lacks genes
responsive to the presence of such a ligand. Cells containing
these expression systems may be used in gene therapy
procedures, including in humans.
Once a sufficient quantity of the desired steroid receiver
polypeptide has been obtained, the protein may be used for
various purposes. A typical use is the production of anti
bodies speci?c for binding to steroid receptors. These anti
bodies may be either polyclonal or monoclonal and may be
produced by in vitro or in vivo techniques.
For production of polyclonal antibodies, an appropriate
target immune system is selected, typically a mouse or

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19
rabbit. The essentially purified antigen is presented to the
immune system in a fashion determined by methods appro
priate for the animal and other parameters well known to
immunologists. Typical sites for injection are in the foot
pads, intramuscularly, intraperitoneally, or intradermally. of
course, another species may be substituted for a mouse or
rabbit.
An immunological response is usually assayed with an
immunoassay. Normally such immunoassays involve some
purification of a source of antigen, for example, produced by
the same cells and in the same fashion as the antigen was
produced. The immunoassay may be a radioimmunoassay,
an enzyme-linked assay (ELISA), a ?uorescent assay, or any
of many other choices, most of which are functionally
equivalent but may exhibit advantages under speci?c con
editions.
Monoclonal antibodies with affinities of 108 M“1 prefer
ably 109 to 101°, or stronger will typically be made by
standard procedures as described, eg, in Harlow and Lane
(1988), Antibodies: A laboratory Manual, Cold Spring
Harbor Laboratory; or Goding (1986), Monoclonal Antibod
ie: Principles and Practice (2d ed) Academic Press, New
York, which are hereby incorporated herein by reference.
Brie?y, appropriate animals will be selected and the desired
immunization protocol followed. After the appropriate
period of time, the spleens of such animals are excised and
individual spleen cells fused, typically, to immortalized
myeloma cells under appropriate selection conditions.
Thereafter the cells are clonally separated and the supema~
tants of each clone are tested for their production of an
appropriate antibody speci?c for the desired region of the
antigen.
Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar
vectors. See, Huse et al., (1989) “Generation of a Large
Combinatorial Library of the Immunoglobulin Repertoire in
Phage Lambda,” Science 246:1275-1281, hereby embody
rated herein by reference.
The polypeptides and antibodies of the present invention
may be used with or without modification. Frequently, the
polypeptides and antibodies will be labeled by joining,
either covalently or non-covalently, a substance which pro
vides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported exten
sively in both the scienti?c and patent literature. suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescens, chernilurninescers, magnetic pair
tickles and the like. Patents, teaching the use of such labels
include US. Pat. US. 3,817,837; 3,850.752; 3,939,350;
3,996.345; 4,277,437; 4,275,149; and 4,366,241. Also,
recombinant immunoglobulins may be produced, see
Cabilly, US. Pat. No. 4,816,567.
Another use of purified receptor polypeptides is for deter
mination of the structural and biosynthetic aspects of the
polypeptides. Structural studies of interactions of the ligand
binding domains with selected ligands may be performed by
various methods. The preferred method for structural detain
mination is X-ray crystallography but may include various
other forms of spectroscopy or chromatography. See, eg,
Connolly, ML, J. Appl. Crystall, 16:548 (1983); and'
Connolly, ML, Science 2212709 (1983), which are hereby
incorporated herein by reference. For example, the structure
of the interaction between hormone ligand and hormone
binding segments may be determined to high resolution.
From this information, minor substitutions or modifications
to either or both of the ligand and ligand-binding segment
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20
may be made. This information enables the generation of
modi?ed interactions between a ligand and its binding
segment to either increase or decrease affinity of binding and
perhaps increase or decrease in response to binding. Likewise,
the interaction between the zinc ?ngers DNA-binding sec
ments with the speci?c nucleic acid-binding sequence may
is similarly modi?ed.
As a separate and additional approach, isolated linking
binding polypeptide domains may be used to screen for
new ligands. This perrrrits screening for new agonists or
antagonists of a particular steroid receptor. isolated DNA
binding segments may be used to screen for new DNA
sequences which will speci?cally bind to a particular recep
tor~binding segment. Typically, these receptor-speci?c bind
ing sites will be controlling elements for steroid responsive
genes. Thus, having isolated these DNA-binding sequences,
genes which are responsive to the binding of a given
receiver can be isolated. This provides a method for isolation
ing genes which are responsive to induction or inhibition by
a given hormone receptor.
In another aspect of the present invention, means for
disrupting insect development are provided where new
ligand agonists or antagonists are discovered. these with
pounds are prime candidate as agonists or antagonists to
interferes with the normal insect development. By application
of new steroid analogues of ligands for insect steroid
receiver superfamily members, it is possible to modify the
normal temporal sequence of developmental events. for
example, accelerating insect development will minimize
generation time. This may be very important in circum
stances where large numbers of insects are desired ?nally,
for instance, in producing sterile males in Mediterranean ?y
infestations. Alternatively, it may be useful to slow devel
opment in a pest infestation, such that the insects reach
destructive stages of development only after commercial
crops may have passed sensitive stages.
In another commercial application, ligands discovered by
methods provided by the present invention may be used in
the silk-production industry. Here, the silkworms are arti?
cially maintained in a silk-producing larvae stage, thereby
being silk productive for extended time periods. the devel
option of larvae may also be accelerated to reach the
silk-producing stage in their life cycle earlier than naturally. ,
Other analogues of ligands for insect steriod receptor
superfarnily members may be selected which, upon appli
cation, may be completely disruptive of normal develop
ment, leading to a lethal result. However, the use of slightly
modi?ed natural substances will often have greater speci
City of action and much higher activities, thus allowing for
lower levels of application. Also, because the ligands may be
more lipophilic, they may be more readily absorbed directly
into the insect surface or article. Extremely low amounts of
natural ligands may be effective in controlling pests. fur
thermore, many of these ligands are likely top be relatively
easily manufacture, perhaps by biological methods using
enzymatic production methods. There may be new ligands
for insect steroid receptor superfarrrily members which may
be more species speci?c or may exhibit a particularly useful
spectrum of effectiveness, for example, being lethal to
harmful insects. The greater speci?city of the hormones will
allow avoidance of use of non-speci?c pesticides possessing
undesired deleterious ecological side effects. For instantaneous
residue of pesticides accumulate in food, often having
deleterious effects on humans. Furthermore, compounds
having structures closely analogous to natural compounds
May be susceptible to natural mechanisms of biological
degradation.

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21
Another aspect of the present invention provides for the
isolation or design of new gene segments which are respon
sive to ligands for insect steroid receptor superfamily mem
bers. For example, use of the nucleic acids to screen for
homologous sequences by standard techniques will provide
genes having similar structural features. Assessed
intron structures will typically be characteristic of larger
superfamily categories. The preferred domains for screening
will be the ligand-binding or DNA-binding segments, how
ever, the DNA segments which are recognized by the
DNA-binding domains, ie, the controlling elements, will
also be of particular interest. By screening for new control
ling elements, by either sequence homology to other known
ones, or by screening with the DNA zinc ?nger-binding
domains of other receivers, additional receivers can be
isolated. Receptors and genes important in the general
developmental sequence of expression will be discovered.
Using this set of developmentally regulated genes will allow
selection of particular molecules which are responsible for
controlling expression of developmentally regulated genes.
The following experimental section is offered by way of
example and not by limitation.
EXPERIMENTAL
EXAMPLE I
CLONING STRUCTURE AND EXPRESSION OF
THE DROSOPHILA E75 GENE THAT ENCODES
TWO MEMBERS OF THE STEROID
SUPERFAMILY RECEIVER
A. Cloning of Genomic DNA Encompassing the Ecdys
one-Inducible 75B Pu?" Locus
Methods
Genomic DNA libraries
In situ hybridization
B. Identification of a 50~kb Region of Cloned Genomic
DNA that Contains Sequences Homologous to Ecdysone
induced transcripts
Methods
organ culture and RNA isolation
Southern blot analysis
C. The E75 Gene Contains Two Overlapping Transcript
tion Units: E75A and E75B
Methods
cDNA libraries
Northern blot analysis
S1 nuclease protection and primer extension analysis
DNA sequence analysis
D. The E75 Gene Encodes Two Members of the Steroid
Superfamily receiver
Protein sequence analysis
E. Expression Vectors for E75 Proteins
EXAMPLE II
CLONING, STRUCTURE AND EXPRESSION
OF THE ECR AND DHR3 GENES THAT
ENCODE ADDITIONAL MEMBERS OF THE
STEROID SUPERFAMILY RECEIVER
Identification and Chromosomal Mapping of EcR and
DHR3 Genomic Clones
Structure of the EcR and DHR3 Genes and Their cDNAs
Methods
I

Isolation of cDNA and additional genomic clones

22
DNA sequence analysis
C. The Predicted Amino Acid Sequence of the EcR and
DHR3 Proteins and their Implications
D. In Situ Labeling of the EcR and DHR3 Proteins with
5 Antibodies Induced by Proteins Produced in E. coli
EXAMPLE III.
THE ECDYSTEROID-BINDING, DNA-BINDING
AND GENETIC REGULATORY PROPERTIES
OF THE ECR PROTEIN DEMONSTRATE THAT
IT IS AN ECDYSONE RECEIVER
A. The EcR Protein Binds Ecdysteroids
Methods
Extracts
Hormone-binding assays
B. Genetic Regulatory Activity of the EcR Protein in vivo
Methods
Construction of the pAdh/Bgal, pEcRE/Adh/Bgal and
pActEcR plasmids
Transfection and generation of the cell line SRS 1.5
C. Speci?c Binding of the EcR Protein to Ecdysone
Response Elements
Methods
Conditions for the DNA binding assay
10
EXAMPLE IV
30
GENE MUTAGENESIS RECEIVER
A. Deletion Mutations
B. E75 Mutations Generated by Ethyl Methane Sulfonate
Methods
Strains, markers and chromosomes
Quantitative Southern blot mapping for detection of
mutant
Molecular cloning of mutant lesions
gamma ray mutagenesis
EMS mutagenesis
In situ hybridization and cytological analysis
35
40
EXPERIMENTAL
45
EXAMPLE I
CLONING STRUCTURE AND EXPRESSION OF
THE DROSOPHILA E75 GENE THAT ENCODES
TWO MEMBERS OF THE STEROID
SUPERFAMILY RECEIVER.
The following experiments demonstrate that the E75 gene
encodes two members of the steroid receptor superfamily.
This is due to the amino acid sequence homology receptor
to the conserved DNA-binding and ligand-binding domains
of this superfamily, and that E75 is an ecdysone-inducible
gene that accounts for and is responsible for the ecdysone
inducible early puff at the 75B locus in the Drosophila
polytene chromosome.
A. Cloning of Genomic DNA Encompassing the Ecdys
one-Inducible 75B Pullc Locus
We have used the method of chromosomal walking
(Bender, W., P. Spierer, and DS Hogness, 1983. Chromo
Somali walking and jumping to isolate DNA from the Ace
and rosy loci and the Bithorax complex in Drosophila
melanogaster. J. Mol. Biol. 168:17-33) to isolate the
genomic DNA encompassing the 75B puff region. The
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5,514,578
23
starting point for the walk was a genomic clone, X8253 (a
gift of J. Burke), which had been localized by in situ
hybridization to the proximal end of 75B. isolated restriction
fragments of 18253 were used to screen a library of genomic
DNA from the Canton s (cs) strain of D. melanogaster
(Maniatis, T., RC Hardison, E. Lacy, J. Lauer, C.
O'Connell, D. Quon, GK Sim, and A. Efstradiatis, 1978.
The isolation of structural genes from libraries of eukaryotic
DNA. Cell 15:687-701). Genomic clones kcDm3504 and
7tcDm3505 were isolated by homology to A8253.
The walk was then extended in both directions until ~100
kb of genomic DNA had been isolated, when the orientation
of the walk was determined by in situ hybridization of the
terminal segments to polytene chromosomes. Thereafter, the
walk was extended in the rightward direction on the molecu
lar map, or distally relative to the centromere. The 350 kb of
genomic DNA encompassed by the walk corresponding to the
chromosomal region between bands 75A6~7 and 75B1l-13,
as determined by in situ hybridization. This region includes
the 75B puff, which appears to initiate by simultaneous
decondensation of chromosomal bands 75B3-5 and then
spreads to surrounding bands.
Methods
Genomic DNA libraries
Canton S genomic DNAs were isolated from a library of
sheared, EcoRI-linkered Canton S DNA cloned into the
Charon 47t phage vector (Maniatis, T., RC Hardison, E.
Lacy, J. Lauer, C. O'Connell, D. Quon, GK Sim, and A.
Efstradiatis, 1978. The isolation of structural genes from
libraries of eukaryotic DNA. Cell 15:687-701). 0' genomic
DNAs were isolated from the library of sheared DNA, GC
tailed into the sep6 A vector (Meyerowitz, FM, and DS
Hogness, 1982. Molecular organization of a Drosophila pull'
site that responds to ecdysone. Cell 28:165-176). one step
in the chromosomal walk was taken using the cosmid library
(prepared in collaboration with S. Gemeraad) of Sau Illa
partially digested 0' DNA cloned into the cosmid p14Bl by
the method of Ish-Horowicz and Burke (Ish-Horowicz, D.,
and JF Burke, 1982. Rapid and efficient cosmid cloning.
Nucleic Acids Res. 9:2989-2998).
In Situ hybridization
In situ hybridization to polytene chromosomes was car
ried out with DNA probes that were nick-translated in the
presence of 3H-labeled 'ITP (NEN), as described by Bonner
and Pardue (Bonner, JJ, and ML Pardue, 1976. Ecdys
one-stimulated RNA synthesis in imaginal discs of Droso
phila melanogaster. Assay by in situ hybridization. chro
mosoma 58:87—99), with the following modi?cations: Heat
and RNAase treatments of the slides were omitted, and
hybridization and washing were at 63°C. in ZXSSPE for 18
and 2 hours, respectively.
B. Identification of a 50 kb Region of Cloned Genomic
DNA that Contains Sequences Homologous to Ecdysone
induced transcripts
Restriction fragments of the above genomic clones were
tested for their ability to hybridize with each of two cDNA
probes: one derived from the RNA in ecdysone-induced
cells, and the other from the RNA in noninduced cells. two
such differential screens were carried out. In the beginning,
genomic DNA covering the entire 350 kb walk was exam
ined with cDNA probes synthesized with reverse tran
scriptase from an oligo(dT) primer annealed to poly(A)+
RNA. The poly(A)+ RNA was prepared from total inner
tissues that were mass-isolated from late third instar larvae
and incubated in the presence of ecdysone plus cyclohex
imide, or cycloheximide alone. (See Methods, below. Cyclo
hexinride was included because higher levels of ecdysone
induced transcripts accumulate in its presence.)
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65

24
Each of the 32P-labeled cDNA probes made from these
two poly(A)+ RNAs was applied to one of two duplicate
Southern blots that contained, in addition to the genomic
fragments from the walk, a control DNA consisted of
sequences from the ribosomal protein 49 gene (O'Connell,
P., and M. Rosbash, 1984. Sequence, structure and codon
preference of the Drosophila ribosomal protein 49 gene.
Nucleic Acids Res. 12:5495-5513), which was used to
normalize the hybridization intensities of the duplicate blots.
This screen revealed sequences speci?c to ecdysone~in~
duced RNAs only within the ltcDm3522 genomic clone that
is centered at approximately +220 kb on the molecular map.
Because the above probes will preferentially detect
sequences near the 3' termini of the RNAs, particularly in the
case of long transcripts, the second differential screen was
carried out with cDNA probes primed with random hexarn
ers (see Methods, below). This screen, which was restricted
to the 135 kb of genomic DNA between +105 kb and +240
kb, revealed ecdysone-inducible sequences in fragments
spread out over an ~50 kb region between +170 kb and +220
kb. This region represents the E75 gene.
Methods
organ culture and RNA isolation
Late third instar O' larvae were harvested, washed in
0.7% NaCl, resuspended in Robb's phosphate-buffered
saline (PBS) (Robb, JA, 1968. Maintenance of imaginal
discs of Drosophila melanogaster in chemically de?ned
average. J. Cell. Biol. 41:876-885), preaerated with a blender,
and passed through a set of rollers to extrude the organs.
This “grindate” was changed through a coarse Nitex screen to
remove carcasses, and settled ?ve times (3-5 minutes per
settling) by gravity to remove ?oating and microscopic
debris. Isolated tissues (primarily salivary glands, imaginal
discs, gut, and Malphigian tubules) were cultured at 25°C.
in plastic petri dishes in aerated Robb's PBS. B-ecdysone
(Sigma) (0.2 pl/ml of 10 mg/ml) in ethanol and/or cyclo
heximide (2 pl/ml of 35 mM) in water was added to the
appropriate cultures. Incubations in the presence of cyclo
heximide were for ~8 hours. isolated tissues were homo
enized in 10 volumes of 6M guanidine-HCl/0.6M sodium
acetate (pH 5.2), centrifuged at 5000 g for 10 minutes to
removes debris, and layered onto a 5.7M CaCl shelf, as
described previously (Chirgwin, JM, AE Przbyla, RJ
MacDonald, and WJ Rutter, 1979. Isolation of biologically
active ribonucleic acid from sources enriched in ribonu~
release. Biochemistry 18:5294-5299). Poly(A)+ RNA was
purified by oligo(dT) chromatography.
Southern blot analysis
_
Southern blots were performed on nitrocellulose, as
described previously (Segraves, WA, C. Louis, S. Tsubota,
P. Schedl, JM Rawls, and BP Jarry, 1984. The Rudimen
tary locus of Drosophila melanogaster. J. Mol. Biol.
175:1-17). cDNA probes were prepared by reverse tran
scription (AMV reverse transcriptase; Seikagaku) of 2 pg of
poly(A)+ RNA with 700 ng of oligo(dT)12_16 (Collaborative
Research) or 15 pg of random hexamers (Pharrnacia) in a 20
pl reaction mixture containing 80 mM Tris Cl (pH 8.3 at 42°
C.), 10 mM MgCl'2, 100 mM KCl, 0.4 mM DTT, 0.25 mM
each of dATP, dGTP, and dTTP, and 100 pCi of [32PldCTP
(800 Ci/mole; Amersham). After incubation at 37°C. for 45
minutes, 80 pl of 10 mM EDTA and 2 pl of 5 N NaOH were
added before incubation at 70°C. for 10 minutes to denature
the products and hydrolyze the RNA. After the addition of
10 pl of 1M Tris Cl (pH 7.5) and 5 pl of 1N HCl,
unincorporated label was removed by chromatography on
Biogel P60.

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