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Molecular analysis of the Methorprene-tolerant gene region of Drosophila melanogaster.

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Archives of Insect Biochemistry and Physiology 30:133-147 (1 995)
Molecular Analysis of the Methoprene-tolerant
Gene Region of Drosophila melanogaster
Christopher Turner and Thomas G. Wilson
Department of Biology, University of Virginia, Charlottesville, Virginia (C.T.);Department of
Biology, Colorado State University, Fort Collins, Colorado (T.G.W.)
Adult functions of juvenile hormone (JH)have been described for Drosophila
melanogaster and other dipteran insects, but preadult function for this hormone remains largely unknown in this order of insects. We have identified a
mutation of Drosophila, Methoprene-tolerant (Met), which appears to alter JH
reception during late larval development. The molecular cloning of M e t will be
a step toward understanding this gene and possibly identifying a preadult role(s)
for JH. Molecular cloning was initiated using the technique of transposon-tagging with a transposable P element. P-element insertional alleles of M e t were
generated, and genomic libraries were constructed from two of these alleles.
From these libraries P-element-bearing clones were isolated that in situ hybridized to the cytogenetic region where M e t had been previously localized by
genetic methods. Two of the alleles were shown to have complete P-elements
inserted in similar, but not identical, locations in the predicted cytogenetic
region where M e t is located. A late-larval cDNA library was screened to identify transcriptional units in this region, and clones were recovered with homology to a D N A fragment abutting the P-element insertion site. These clones may
represent M e t cDNA molecules. @ 1995 Wiley-Liss, Inc.
Key words: juvenile hormone, methoprene, hormone receptor gene, insecticide resistance
Understanding the role of juvenile hormone (JH*)in dipteran insects continues to be a challenge for insect physiologists. Functions for this hormone
have been identified during adult development, and Professor L.I. Gilbert
Acknowledgments: We thank Laura Hill-Eubanks for technical assistance with cDNA library screening and subsequent cDNA characterization. We thank Carl Thummel, University of Utah, Salt Lake
City, Scott Hawley, University of California, Davis, and Alan Spradling, Carnegie Institute, Baltimore,
for clones. This work was funded in part by support from American Cyanamid Company.
Received July 21, 1994; accepted January 23, 1995.
Address reprint requests to Thomas G. Wilson, Department of Biology, Colorado State University, Fort Collins, CO 80523.
*Abbreviations used: ap = apterous; bp = basepair; EMS = ethyl methane sulfonate; JH = juvenile hormone; kb = kilobase; Met = Methoprene-tolerant; nod = no distributive disjunction;
v = vermilion.
0 1995 Wiley-Liss, Inc.
Turner and Wilson
and his colleagues have added much to our understanding of JH chemistry
and biology during adult development. However, progress toward elucidating JH function in preadult dipteran insects has been frustratingly slow. The
classical endocrinological methodology of allatectomy and hormone replacement that revealed the involvement of JH during preadult development in
insects from other orders has been unsuccessful in Diptera because of the
close association of the corpus allatal cells with unrelated cells in the ring
gland (King et al., 1966).The technical difficulty of unambiguous allatectomy
thus has thwarted this approach to JH removal from larvae.
Another approach is the use of the anti-JH compound, precocene (Bowers
et al., 1976),which acts in many insects as a suicide substrate resulting in cell
death and subsequent dysfunction of the corpus allatum (Pratt et al., 1980).
In Drosopkila melanogasteu adults, precocene results in apparent JH deficiency
(Landers and Happ, 1980; Wilson, 1982; Wilson et al., 1983), but in larvae
physiological amounts of precocene have little effect, and higher, probably
pharmacological, concentrations resulted in morbidity (Wilson, unpublished
data). This latter effect was probably due to general toxicity instead of a specific effect of this compound to produce JH deficiency. Why precocene is effective in adults but not in larvae is unclear, but this chemical means of
allatectomy has proven uninsightful in Drosopkila larvae.
A third approach that holds promise is genetic, that of identifying mutants which have blocked JH synthesis, rapid JH degradation, or insensitive JH reception during larval development. This approach is most feasible
for genetically manipulable dipteran insects such as Drosopkila melanogasteu,
Lucilia cuprim, and Musca domestica. One Drosopkila mutant, apterous (ap),
has two phenotypic characters suggestive of JH deficiency: non-vitellogenic
oocyte development and failure of larval fat body histolysis following eclosion (Butterworth and King, 1965).Subsequent work showed that the ovary
defect could be rescued by u p ovary transplants into wild-type adult hosts
or by treatment with a JH analog (Postlethwait and Weiser, 1973; Postlethwait et al., 1976). The larval fat body defect could be rescued by application of a JH analog (Postlethwait and Jones, 1978). The remaining
phenotypic characters of up, such as the wing and haltere defects (Butterworth and King, 19651, were unresponsive to treatment with a JH analog.
More recently, up4 adults have been shown to possess a lowered JHIII titer
(Bownes, 1989) and drastically lowered JH synthesis in cultured adult corpora allata (Altaratz et al., 1991; Dai and Gilbert, 1993).However, up4 thirdinstar larvae were shown to have more normal ( 5 0 4 0 % wild-type) JH
synthesis (Altaratz et al., 1991; Dai and Gilbert, 1993). Therefore, while
this mutation has proven useful for examining JH function in adult Drosopkiln (see Discussion), it has not helped our understanding of preadult
roles of this hormone.
To elucidate any preadult role(s) of JH of Dvosopkila, we are using a genetic
approach of identifying an insensitive JH receptor mutant. Such a mutant
would be expected to display a phenotype similar to that of a JH biosynthetic mutant, and JH function might be gleaned from a detailed examination of the phenotype. In our approach we made use of the high toxicity of
methoprene, a JH analog insecticide (Staal, 1975), to Dvosopkila (Wilson and
Methoprene-tolerant From Drosophila
Fabian, 1986; Riddiford and Ashburner, 1991).Reasoning that the phenotype
of such a mutant would include resistance to methoprene, we screened the
progeny of Drosophila males that were mutagenized by ethyl methanesulfonate
or X-rays on a dose of methoprene that is toxic to susceptible flies (Wilson
and Fabian, 1987). We recovered a total of 8 lines with high resistance to
methoprene (Wilson and Fabian, 1986, 19871, all of which proved to be alleles at a locus we termed Methopuene-tolerant (Met).
We have genetically characterized Met as follows (Wilson and Fabian,
1986): (1) Met results in as much as 100-fold resistance to both the toxic
and morphogenetic effects of methoprene; (2) Met maps by recombination
to 35.4 on the X-chromosome and by deficiency mapping to 10C5-D2, (3)
Met is expressed as a semidominant mutation; resistance is present in heterozygotes at a level intermediate between that in homozygotes and in
wild-type; (4) Met results in resistance to topical application of both the
natural hormones, JH I11 and JH bisepoxide, as well as to each of two
additional JH analogs that we have tested. However, Met flies are not resistant to other classes of insecticides; (5) in genetic mosaics, Met is expressed autonomously. This last result ruled out a circulating factor
responsible for Met resistance.
The biochemistry of Met resistance has been explored. Possible mechanisms
of enhanced secretion or metabolism, tissue sequestration, and reduced cuticular penetration of JH were ruled out by direct experimentation (Shemshedini
and Wilson, 1990).However, when binding of JH to a target tissue was examined, Met flies were found to possess a high-affinity JH cytosolic binding
protein that has a 10-fold lower binding affinity for JH I11 than that from
Met' flies; similar binding results were obtained upon examination of two
additional Met alleles (Shemshedini and Wilson, 1990). We have presented
indirect evidence that this binding protein is a JH receptor (Shemshedini et
al., 1990), but direct evidence is lacking. Binding in MetlMet' heterozygotes
is biphasic, suggesting that these flies possess two binding proteins, each having binding characteristics of either Met homozygotes or wild-type flies
(Shemshedini and Wilson, 1990); this result is also consistent with the semidominant expression of Met. Thus, the Met'gene may encode the receptor; if
not, then it encodes a protein that must be intimately and stoichiometrically
involved in JH reception.
We have begun to clone the Met gene by transposon tagging (Bingham et
al., 1981) with P-element transposable genetic elements. This method required
a P-element insertion either in or near the Met' gene. A screen was devised
to recover P-element insertional Met alleles following P-element mutagenesis,
and four alleles were recovered. Two of these were shown to be P-element insertions by both genetic reversion experiments and in situ hybridization of a
P-element DNA probe to the expected cytogenetic region of Met, 1OC (Wilson and Turner, 1992; Wilson, 1993).
In this report we have constructed genomic libraries from these alleles to
isolate the region containing the Met gene. This region has been characterized by restriction mapping. In addition, we isolated cDNA molecules from
this region. One transcriptional unit is located very close to the P-element
insertion site and may represent Met.
Turner and Wilson
Drosophila Strains and Culture Conditions
Oregon-RC was used as the wild-type strain in this work. The Met allele was derived following ethyl methanesulfonate mutagenesis of OregonRC as described (Wilson and Fabian, 1986). The MetA3and MetK17alleles
are described in Wilson and Turner (1992). MetDz9and MetN6are alleles
recovered in a screen (Wilson and Fabian, 1987) following X-ray treatment
of males with 3,000 rads (Wilson and Fabian, unpublished data). Met153is
a lethal revertant and Met14’ is a methoprene-susceptible viable revertant
of MetA3recovered in a revertant screen as previously described (Wilson
and Turner, 1992). The vermilion (v) mutant was obtained from the MidAmerica Drosophila Stock Center, Bowling Green State University, OH, and
was used as a genetically marked Met’ strain. Flies were raised in uncrowded conditions at 25 1°C on a standard agar-yeast-cornmeal diet
with propionic acid added to retard fungal growth. All cultures were maintained under a LD12:12 photoperiod.
Genomic Library Constructions and Probing
Separate genomic libraries were prepared from MeP3 and MetK1’ flies. DNA
was prepared from adults that were starved for 2 h to ensure digestion of
yeast in their gut. Flies were frozen in liquid nitrogen, and DNA was extracted by the procedure described by Jowett (1986). Each preparation of DNA
from MetA3and MetK17flies was partially digested with Sau3A and fractionated on a 1 0 4 0 % linear sucrose gradient. Fractions containing fragments in
the 15-30 kb size range were pooled and dialyzed against TE (pH 8). The
DNA was concentrated by ethanol precipitation and resuspended in TE. The
Sau3A ends of the genomic fragments were partially filled by addition of
dGTP and dATP using the Klenow Fill-In kit (Strategene, La Jolla, CA) and
cloned into the Lambda Fix vector (Stratagene). Each library had a titer of
about 3 x lo7pfu/ml. Approximately 50,000 phage, representing five genomic
equivalents of insert DNA, were plated from each library and screened initially with [32PldCTP-labelledpn25.1, a plasmid containing a P element, to
detect P-element-bearing clones.
To identify those P-element-bearing clones from the 1OC region, a probe
for this region was required. The nearest cloned gene to Met is no distributive
disjunction (nod), located just distal to the Met gene based on genetic studies
with deficiency chromosomes from this region (Voelker et al., 1985; Zhang et
al., 1990). Cosmids c6.01 and c6.02 (c6.02 includes the nod gene) were obtained from S. Hawley. The library filters were reprobed with the more proximal c6.01 cosmid, and clones that were positive with both px25.1 and c6.01
were picked, replated, and rescreened. DNA was prepared from positive
clones by the small-scale method (Maniatis et al., 1982).
In Situ Hybridization
A 2 kb Hind111 fragment (coordinates 1-3 on the map in Fig. 1)was isolated
from the A3(15-3) clone and labeled with digoxigenin-dUTP by the random
primer method as described in de Frutos et al. (1990).Chromosome squashes
Mefhoprene-tolerant From Drosophila
J i ;
2.9 kb P l c m e u
Fig. 1. Restriction map of the Met region. Relative to the centromere of the X-chromosome, left i s distal; right, proximal. The positions of the clones isolated from the M e p 3
[A3(7-2) and A3(15-3)] and MetK” [DI 7(1) and K17(6)] libraries are shown above the map.
The insertional point of the complete 2.9 kb P-element located in the M e p 3 allele i s indicated by the arrow; the insertional point of the P-element in the M e p ” allele i s about 300
bp distal to this. Positions on the map occupied by the c6.01 cosmid and the distal portion
of the c6.02 cosmid are shown below the map. B = BamHI, D = HindlII, E = EcoR1, H =
Hpal, K = Kpnl, S = Sall, X = Xhol .
of v/v females raised at 18°C were prepared by the method of Pardue (1986).
In situ hybridization was carried out by the procedure of de Frutos et al.
Southern Analysis
DNA from partially starved adult flies was isolated (Jowett, 1986) and
digested to completion with Hind111 and electrophoresed in 0.8% agarose
gels. Gel lanes were loaded with 5-10 l g of DNA from flies of the indicated genotype and electrophoresed in 0.8% agarose gels in 1 x TBE (0.45M
Tris, 0.45 M boric acid, 2 mM EDTA) at 1 V/cm for 12-18 h. Fragments
were sized using molecular weight standards obtained from Stratagene.
Gels were blotted onto nylon membranes (Hybond N) and probed with a
[32P]dCTP-labeledK ~ Mfragment
(coordinates -3,to -11, Fig. 1) prepared
from the phage clone A3(15-3).
cDNA Library Screening
The cDNA library used was a LZAP (Stratagene) library with cDNA inserts derived from mRNA prepared from late third-instar wild-type larvae.
The library was plated out at a density of about lo4 plaques/plate and
screened with [32P]dCTP-labelledA3(15-3) clone. Positive clones were purified by replating and reprobing.
Turner and Wilson
Strategy for Cloning Met
There are several approaches for cloning a gene of interest. One method
involves cloning after microsequence determination of the gene product, as
has been done with the putative JH receptor from Manduca (Palli et al., 1994).
However, although the Met gene product has been implicated in JH reception (Shemshedini and Wilson, 1990), its identity is unknown, and so this
approach was not possible. Another method, cloning by homology to another JH-analog resistance gene isolated from another insect, likewise was
not possible since Met is the only JH-analog resistance gene to be described
in any insect. A third method is that of transposon-tagging the gene of interest with a transposable genetic element. Since the P-family of transposable
elements in Drosophila is useful for transposon-tagging (Bingham et al., 19811,
we elected to clone Met using this approach. Transposon-tagging is initiated
using flies carrying a P-element insertional allele of the gene of interest to
construct a genomic library. One or more clones are recovered from the library by probing with a P-element, and the cytogenetic location of a positive
clone for the gene is confirmed by in situ hybridization to be the region where
the gene has been previously localized by a genetic method. This clone should
either contain the gene of interest (if small) or provide an entry point for a
short walk to clone the entire gene.
Transposon tagging requires transposon insertional alleles. We recovered
four putative P-element insertional alleles of Met in a screen and confirmed
that at least two of these, MetA3and MetK”, (1)carried a P-element in the Met
region, lOC, by in situ hybridization and (2) were revertable to wild-type
(methoprene-susceptibility)at high frequency (-0.5-1 %) under the appropriate genetic conditions (Wilson and Turner, 1992). Therefore, each possessed
characteristics expected of a P-element insertional allele. The recovery of each
of these alleles was separated in time by about a year, so they represent separate mutational events.
Isolation of Putative Met-Containing Clones
Separate genomic libraries were constructed from MetA3and MetKI7.P-element-bearing clones were identified from each of these libraries by probing
with ~ ~ 2 5 .a1 plasmid
containing an entire P-element. Identification of Pelement-bearing clones from the 1OC region required a probe for this region.
The nearest cloned gene to Met is no distributive disjunction (nod),located just
distal (toward the non-centromere end of the chromosome) to the Met gene
based on genetic studies with deficiency chromosomes from this region. This
gene was cytogenetically located at 1OC2-5 (Voelker et al., 1985; Zhang et al.,
1990),and Met was located proximally (toward the centromere) at 10C5-10D2
based on deficiency chromosomes that include each gene. Zhang et al. (1990)
isolated two cosmids, c6.01 and c6.02, from the nod region, and c6.01, the
more proximal cosmid, was used to reprobe the library filters. From each
library two clones that were positive for each probe were isolated, purified,
and restriction-enzyme mapped. A restriction map of the region defined by
the cosmids and phage clones is shown in Figure 1.This map was constructed
Methoprene-tolerant From Drosophila
based on restriction mapping of the cosmid clones. The phage clones were
then aligned on the map after restriction mapping of each with HindIII, KpnI,
and SalI. Based on the orientation of the c6.01 and c6.02 cosmids determined
by Hawley (unpublished data) and confirmed by restriction mapping in our
lab, the distal end of the chromosome extends to the left in Figure 1.
P-elements found in Drosophila can be either full-length (2.9 kb) or smaller
internally deleted elements (OHare and Rubin, 1983).Both of the P-elements
contained in the four clones recovered from the MetA3 and Metm7 genomic
DNA were full-length. This determination is based on the presence of a SalI
site and two HindIII sites found in the intact element (OHare and Rubin,
1983) as well as the expected 0.8 kb fragment between the HindIII sites seen
on a Southern blot of HindIII digests of clones A3(15-3) and K17(1) probed
with a P-element probe. This same blot showed that the P-elements are inserted into the MetA3and MetK1’ alleles in the orientation shown in Figure 1
(Turner, 1992).This determination was possible because the 2.1 HindIII fragment of the P-element was found to be attached to the small HindIII fragment proximal to the insertion site instead of the much larger 6.6 kb HindIII
fragment distal to the insertion site (Fig. 1).
The insertional locations of the P-element in each of the two alleles is separated by about 300 bp. This determination could be made because the Southern
blot of HindIII digests of clones A3(15-3) and K17(1)showed P-element-bearing
HindIII fragment sizes of 2.4 kb for A3(15-3) and 2.7 kb for K17(1). Since the
genomic HindIII site located at position 1.2 on the map (Fig. 1) is stationary,
then the insertion sites for the P-elements must differ by about 300 bp.
Southern Analysis of Met Alleles and Chromosomal Variants
To further localize the Met gene within the cloned region, a Southern analysis of various Met alleles was carried out. It is known that certain types of
mutagenesis, including X-ray and transposable element insertional, result in
DNA size changes in or very near the mutant gene. These size changes can
be caused by deletions, insertions, or translocations. Such changes would be
expected to result in restriction fragment size alterations in or near the gene,
whereas other types of mutagenesis, such as point mutations, should not result in restriction pattern changes unless the base-pair change happened to
occur within a restriction site. Therefore, the location of any restriction fragment size changes helps to locate the Met gene.
DNA was isolated from each of the two P-element alleles, from two X-ra
induced alleles (MetD2’and MetN6),and from two revertants of MetA3(Met74;
and Met153,a lethal revertant) and digested to completion with HindIII. When
the 7.5 KpnI fragment (located at map coordinates -3 to -11) was used as a
probe to recognize both the 6.8 and 8.5 kb HindIII fragments on a Southern
blot, the 6.8 kb band (coordinates -5.5 to 1.3) showed size changes in all of
the alleles except MetN6(Fig. 2). No such size changes were evident in the 8.5
kb flanking fragment (coordinates -6 to -14.5, Fig. 2) or when the blot was
probed with another DNA fragment proximal to the P-element insertion site
(Turner, 1992).Therefore, size changes in the DNA are associated with a fragment abutting the P-element insertional site.
This Southern blot also provided molecular information about the Met al-
Turner and Wilson
8.5 kb-,
6.8 kb-t
Fig. 2. Top: Kpnl fragment (coordinates -3 to -11; Fig. 1 ) probing of a Southern blot of
Hindlll-digested genomic D N A isolated from various M e t alleles. Two fragments are recognized: a 6.8 kb fragment abutting the P-element insertional site, and an 8.5 kb fragment
distal to the 6.8 kb fragment (see Fig. 1). Note the size variation for 6.8 and size constancy
for 8.5. The Met' strain examined is vermilion (v). Met'53 i s heterozygous with v since the
hemizygous or homozygous Met'53 genotype i s lethal. Y represents the Y-chromosome, and
the slash separates homologous chromosomes. Bottom: In situ hybridization of a Hindlll
fragment from clone 15-3 to v/v polytene chromosomes. The arrow indicates the hybridization site at 10C5.
Methoprene-tolerant From Drosophila
leles. Jud in from the HindIII fragment sizes shown in Figure 2, it appears
a deletion of about 0.5-0.7 kb. Presumably, a vital gene,
that Met' k contains
either Met or another gene, is partially or wholly contained in the deleted
DNA. Both of the P-element insertional alleles have a HindIII fragment that
is slightly smaller than the wild-type 6.8 kb fragment. This is due to the new
HindIII site located about 100 bp into the proximal end of the P-element
(OHare and Rubin, 1983) that replaces the HindIII site (coordinate 1.3 on the
map in Fig. 1)in wild-type. This fragment is slightly smaller in MetKI7than in
MetA3,again indicating that the P-element insertional site is different for the
two alleles. The Met141revertant has a fragment size similar to MetA3and suggests that instead of a precise excision of the P-element, only a portion of the
P-element was excised, leaving the proximal HindIII site intact in the remnant remaining in the revertant. Imprecise excisions are common in Drosophila revertant studies; for example, Searles et al. (1986) found phenotype
reversion for the P-element insertional allele of the Rpll gene, even though a
remnant of the element remained in the insertion site. The MetD2' allele could
be due to any of several chromosomal changes, including an inversion,
translocation, or insertion, which results in a chromosomal break in the 6.8
kb fragment region. Finally, the 6.8 kb fragment in the MetN6allele appears
wild-type in size; the DNA lesion in this allele, perhaps a point mutation or
small deletion, appears to be too small to be discernable on an agarose gel.
In Situ Hybridization
To confirm the location of Met in the 1OC region, in situ hybridization to
polytene chromosomes was carried out with a 2 kb HindIII fragment from
A3(15-3) located at map coordinates 2.8-4.8. Hybridization occurred specifically at the cytogenetic location 10C5, as expected based on the map position
of the nod gene and the proximity of Met to nod both by genetic recombination and deficiency mapping (Voelker et al., 1985; Wilson and Fabian, 1986)
and by molecular proximity on the c6.01 clone.
cDNA Library Probing
To identify transcriptional units in the Met region, we screened a cDNA
library using clone A3(15-3) as a probe. The cDNA library was templated
from mRNA from wild-type late third-instar larvae. This developmental stage
is during the sensitive period for JH analog toxicity and morphogenetic effects on Drosophila (Ashburner, 1970). Since Met results in resistance to these
effects of JH analogs (Wilson and Fabian, 1986), then Met transcripts would
be expected to be present at this time, and consequently Met cDNA molecules represented in a librar
Screening of about 2 x 10 clones resulted in seven that gave strong hybridization. Two of these were recognized by the 8.5 kb HindIII fragment
(coordinates -5.5 to -14) and one clone mapped proximal to the P-element
insertional site; none of these clones was considered further. However, four
of the clones hybridized with only the 6.8 kb HindIII fragment and are being
further studied. Three of these contain a 3.2 kb, perhaps identical, insert and
one contains a 2.1 kb insert that appears to be a truncated cDNA similar to
the 3.2 kb cDNAs. All four clones hybridized strongly with one another. We
Turner and Wilson
have restriction-mapped the clones with the enzymes shown in Figure 1 and
found only one KpnI and one BarnHl site in the 3.2 kb insert DNA. This restriction map is similar to that for the genomic region located at map coordinates -3.5 and 1 (Fig. l), and suggests a transcriptional unit that abutts the
P-element insertional site. Studies are in progress to determine if these clones
represent Met transcripts.
In this work we have isolated overlapping clones which cover a map distance of over 50 kb. This region of course includes the P-element, which is
likely to be inserted either close to or within the Met gene, judging from
results obtained with other P-element insertional mutations (Searles et al.,
1986; Kelley et al., 1987).When a 7.5 kb KpnI fragment distal to the P-element
insertional site was used to recognize two HindIII fragments on a genomic
Southern blot of various alleles of Met, the 6.8 kb fragment abutting the
P-element insertional site showed size variation in the alleles, but the 8.5
kb fragment located away from the P-element insertional site showed size
constancy as did another fragment located proximal to the P-element insertional site. These results suggest that DNA size changes were induced
by the X-ray and P-element mutagenesis, presumably in or very near the
Met gene.
Judging from the cDNA isolation results, it appears that the genomic region represented by the 15-3 clone encodes at least three transcriptional units
during late larval development. Four of the seven cDNAs isolated are apparently transcribed from the 6.8 kb HindIII fragment region, and they may represent Met cDNAs. The close proximity of this transcriptional unit is consistent
with the mutagenic effect of P-elements. Generally, they insert within several
hundred bp of the transcriptional start site of the gene affected and consequently either diminish or abolish transcription of the gene (e.g., Kelly et al.,
1987). We may therefore expect loss of Met gene product to result in resistance to JH analogs, perhaps in a manner similar to loss of ecdysone receptors resulting in loss of Dvosophila Kc cell sensitivity to ecdysone (Stevens et
al., 1980). Whether the three alleles of Met that resulted from chemical (EMS)
mutagenesis and are therefore likely to be point mutations (Wilson and Fabian,
1986) have a similar predicted loss of Met gene product remains to be seen.
All of the Met alleles recovered to date by the three mutagenesis methodologies are semidominant and have a similar phenotype, suggesting that resistance can occur either by alteration (the EMS-induced Met alleles and perhaps
or by loss of Met gene product.
also MetD2g)
These genomic fragments and cDNA molecules can now be used as probes
to recognize Met transcripts on Northern blots. A combination of cDNA mapping and transcript analysis should allow us to select a genomic fragment for
transformation of Met flies to unambiguously identify the Met region.
It is hoped that a molecular analysis of Met will shed light on the function
of JH during preadult development. For example, if as we believe Met encodes a JH receptor protein, then in situ hybridization of Met transcripts or
protein to preadult tissues should reveal those tissues important in the re-
Methoprene-tolerant From Drosophila
ception of this hormone. This information should be useful for understanding the tissues that are target tissues for JH.
Met as a putative JH receptor mutant should also be useful for elucidating
JH function during development. If indeed Met encodes a JH receptor protein involved in insect development and reproduction, then one might expect a severe phenotype from Met mutants having altered JH binding. To
examine this possibility, we carried out a detailed analysis of the developmental and reproductive phenotype of several Met alleles and found the Met
phenotype to be surprisingly mild (Wilson and Fabian, 1986; Minkoff and
Wilson, 1992).Nevertheless, in the absence of methoprene Met flies were rapidly outcompeted by Met' flies in a population cage (Minkoff and Wilson,
1992).Although we have offered several explanations for the lack of a severe
Met phenotype (discussed in Minkoff and Wilson, 1992), we favor the "functional redundancy" explanation: flies protect critical pathways and mechanisms with alternatives that allow survival and limited reproduction.
If Met analysis is useful for elucidating preadult functions for JH, then this
knowledge will complement our understanding of JH in adults, knowledge
which has been focused by Professor Gilbert and his colleagues. They have
explored the chemistry and biology of JH and have made three important
contributions to our understanding of this hormone. The first is that they
identified a new JH, juvenile hormone bisepoxide (JHB,), which is produced
in vitro from cultured corpora allata of cyclorrhaphous dipteran insects, but not
in more primitive Dipterans, mosquitoes (Richard et al., 1989).JHIII has been the
only JH assayed from intact Drosophila (Sliter et al., 1987; Bownes and Rembold,
1987).Although JHB, has lowered JH activity relative to JHIII in a topical application assay (Richard et al., 1989; Wilson, unpublished data), it seems to be the
predominant JH found after gland culture in Drosophila (Richard et al., 1989),
Calliphora (Duve et al., 1992), and Lucilia (Lefevere et al., 1993). JHB, does not
compete with JHIII for binding to a hemolymph JH binding protein in either
Lucilia or Drosophila (Trowel1 et al., 1994; Wilson, unpublished data), but
methoprene also fails to compete for binding (e.g.,Shemshediniand Wilson, 1988),
so this result is perhaps not surprising. To our knowledge, JHB3has never been
detected in vivo, and in vivo detection would certainly clarify our understanding of the fate of this hormone after biosynthesis.
A second novel contribution has been the detection of ovarian diapause
in adult Drosopkila melanogaster. Although JH has been implicated in diapause in a variety of insect species, including cave-dwelling Drosophila
species (Kambysellis and Heed, 1974), Drosophila melanogaster had previously not been shown to exhibit diapause. Saunders et al. (1990) were able
to induce reproductive diapause in females placed at 12°C and short-day
photoperiod shortly after eclosion. Diapause could be broken by application of either JHIII or JHB3, the latter apparently more effective. This effect of JHB3 is the most dramatic to date for this hormone and suggests
that Drosophila may use the two hormones for separate functions. If true,
then separate binding proteins may exist for the hormones, a possibility
that remains to be explored. If diapause induction in Drosophila melanogaster
has a genetic basis, then the identity of the gene(s) involved could be undertaken. This gene(s) could then be used to identify a homologous gene(s)
Turner and Wilson
involved in diapause in other insects and could open a novel approach
toward the control of certain insect pests.
Finally, Professor Gilbert’s lab efforts have made us aware that a Drosophila mutant sometimes isn’t as straightforward as it appears. Although it
has a complex phenotype (Butterworth and King, 1965; Wilson, 19801, the
apterous mutant has been shown to be JH deficient in the adult stage
(Postlethwait et al., 1976; Bownes, 1989; Altaratz et al., 1991). The juvenile
hormone deficiency is not a direct effect of the mutation on the corpus
allatum, as shown earlier by mosaic analysis (Wilson, 1981) and more recently by the nature and tissue localization of the a p gene product, a member of the LIM family of regulatory proteins (Cohen et al., 1992). The most
well-studied phenotypic characteristics correlated with JH deficiency in
Drosophila are nonvitellogenic oocyte development and histolysis of larval fat body during the first 2 days of adult life. JH has been implicated,
either directly or indirectly, in both the synthesis and uptake of vitellogenin
into the oocytes (Postlethwait and Handler, 1979; Giorgi, 1979; Jowett and
Postlethwait, 1980) as well as in acceleratinp larval fat body histolysis
(Postlethwait and Jones, 1978). The severe up allele clearly demonstrates
these phenotypes and appropriately shows a depressed JHIII titer and JH
biosynthetic rate in adults, but flies carrying the up56fallele are vitellogenic
and have wild-type fat body histolysis (Butterworth and King, 1965; Wilson, 1980; Richard et al., 1993). However, the JH biosynthetic rate is unexpectedly low in
(Altaratz et al., 1991). Recently, Bownes has examined
the JHIII titer of a p adults and has found little correlation of JH titer with
vitellogenic oocyte development (unpublished data). The simplest interpretation of these results is that the JH titer in apterotrs adults, albeit low
relative to wild-type, is sufficient for vitellogenesis. Nevertheless, these
results have called into question the role of JH during oogenesis in Drosophila, a role once thought to be rather firmly established.
Altaratz M, Applebaum SW, Richard DS, Gilbert LI, Segal D (1991): Regulation of juvenile
hormone synthesis in wild-type and apterous mutant Duosopkila. Mol Cell Endocrinol
Ashburner M (1970): Effects of juvenile hormone on adult differentiation of Drosopkila rnelanogaster. Nature 227:187-189.
Bingham PM, Levis R, Rubin GM (1981): Cloning of DNA sequences from the white locus of
D. melanogaster by a novel and general method. Cell 25:693-704.
Bowers WS, Ohta T, Cleere JS, MarselIa PA (19761: Discovery of insect anti-juvenile hormones
in plants. Science 193:542-547.
Bownes M (1989): The roles of juvenile hormone, ecdysone and the ovary in the control of
Duosophila vitellogenesis. J Insect Physiol35:409-413.
Bownes M, Rembold H (1987): The titre of juvenile hormone during the pupal and adult
stages of the life cycle of Drosopkila melanogasfeu. Eur J Biochem 164:709-712.
Methoprene-tolerant From Drosophila
Butterworth €34, King RC (1965): The developmental genetics of u p mutants of Drosopkila
melanogaster. Genetics 52:1153-1174.
Cohen B, McGuffin E, Pfeifle C, Segal D, Cohen SM (1992):apterous, a gene required for imaginal
disc development in Drosopkila encodes a member of the LIM family of developmental
regulatory proteins. Genes Dev 6:715-729.
Dai J-D, Gilbert LI (1993): An ultrastructural and developmental analysis of the corpus allatum of juvenile hormone deficient mutants of Drosopkila melanogas feu. Wilhelm Roux Arch
Entwicklungsmech Org 202:85-94.
de Frutos R, Kimura K, Peterson K (1990): In situ hybridization of Drosopkila polytene chromosome with digoxigenin-dUTP labeled probes. Methods Mol Cell Biol232-36.
Duve H, Thorpe A, Yagi KJ, Yu CG, Tobe SS (1992): Factors affecting the biosynthesis and
release of juvenile hormone bisepoxide in the adult blowfly Callipkora vomitoria. J Insect
Giorgi F (1979): In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosopkila melanoguster. Cell Tissue Res 203:241-247.
Jowett T (1986): Preparation of nucleic acids. In Roberts DB (ed): Drosopkila: A Practical Approach. Oxford: IRL Press, pp 275-286.
Jowett T, Postlethwait JH (1980): The regulation of yolk polypeptide synthesis in Drosopkila
ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog. Dev Biol
Kambysellis MP, Heed WB (1974): Juvenile hormone induces ovarian development in diapausing cave-dwelling Drosopkila species. J Insect Physiol20:1779-1786.
Kelley MR, Kidd S, Berg RL, Young MW (1987): Restriction of P-element insertions at the
Notch locus of Drosopkila melanoguster. Mol Cell Biol7:1545-1548.
King RC, Aggarwal SK, Bodenstein D (1966): The comparative submicroscopic cytology of
the corpus allatum-corpus cardiacum complex of wild type and fes adult female Drosopkila
melanogaster. J Exp Zoo1 161:151-175.
Landers MH, Happ GM (1980): Precocene inhibition of vitellogenesis in Drosopkila melanogaster. Experientia 36:619-620.
Lefevere KS, Lacey MJ, Smith PH, Roberts B (1993): Identification and quantification of juvenile hormone biosynthesized by larval and adult Australian sheep blowfly Lucilia cuprina
(Diptera, Calliphoridae). Insect Biochem Mol Biol23:713-720.
Maniatis T, Fritsch EF, Sambrook J (1982): Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Minkoff C 111, Wilson TG (1992): The competitive ability and fitness components of the
Metkoprene-tolerant (Met) Drosopkila mutant resistant to juvenile hormone analog insecticides. Genetics 131:91-97.
OHare K, Rubin GM (1983):Structures of P transposable elements and their sites of insertion
and excision in the Drosopkila melanoguster genome. Cell 34:25-35.
Palli SR, Touhara K, Charles J-P, Bonning BC, Atkinson JK, Trowel1 SC, Hiruma K, Goodman
WG, Kyriakides T, Prestwich GD, Hammock BD, Riddiford LM (1994): A nuclear juvenile
Turner and Wilson
hormone binding protein from larvae of Manduca sexfa: A putative receptor for the metamorphic action of JH. Proc Natl Acad Sci USA 91:6191-6195.
Pardue ML (1986): In situ hybridization to DNA of chromosomes and nuclei. In Roberts DB
(ed): Drosophila: A Practical Approach. Oxford: IRL Press, pp 111-137.
Postlethwait JH, Handler AM (1979):The roles of juvenile hormone and 20-hydroxyecdysone
during vitellogenesis in isolated abdomens of Drosopkila melanogastev. J Insect Physiol
Postlethwait JH, Jones J (1978): Endocrine control of larval fat body histolysis in normal and
mutant Drosopkila melanogaster. J Exp Zoo1 203:207-214.
Postlethwait JH, Weiser K (1973): Vitellogenesis induced by juvenile hormone in the female
sterile mutant apterous-four in Drosopkila melanogaster. Nature New Biol244:284-285.
Postlethwait JH, Handler AM, Gray PW (1976): A genetic approach to the study of juvenile
hormone control of vitellogenesis in Drosopkila melanogaster. In Gilbert LI (ed): The Juvenile
Hormones. New York: Plenum Press, pp 449-469.
Pratt GE, Jennings RC, Hamnett AF, Brooks GT (1980): Lethal metabolism of precocene-1 to a
reactive epoxide by locust corpora allata. Nature 284:320-323.
Richard DS, Applebaum SW, Sliter TJ, Baker FC, Schooley DA, Reuter CC, Henrich VC, Gilbert LI (1989):Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosopkila melanogaster: A putative juvenile hormone in the higher Diptera. Proc Natl Acad Sci
USA 86:1421-1425.
Richard DS, Arnim AE, Gilbert LI (1993): A reappraisal of the hormonal regulation of larval
fat body histolysis in female Drosopkila rnelanogaster. Experientia 49:150-156.
Riddiford LM, Ashburner M (1991): Effects of juvenile hormone mimics on larval development and metamorphosis of Dvosopkila melanogaster. Gen Comp Endocrinol82:172-183.
Saunders DS, Richard DS, Applebaum SW, Ma M, Gilbert LI (1990): Photoperiodic diapause
in Drosopkila melanogaster involves a block to the juvenile hormone regulation of ovarian
maturation. Gen Comp Endocrinol79:174-184.
Searles LL, Greenleaf AL, Kemp WE, Voelker RA (1986): Sites of P element insertion and
structures of P element deletions in the 5’ region of Drosopkila melanogaster RpII215. Mol
Cell Biol 6:3312-3319.
Shemshedini L, Wilson TG (1988): A high affinity, high molecular weight juvenile hormone
binding protein in the hemolymph of Drosopkila melanogaster. Insect Biochem 18:681-689.
Shemshedini L, Wilson TG (1990): Resistance to juvenile hormone and an insect growth regulator in Drosopkila is associated with an altered cytosolic juvenile hormone binding protein.
Proc Natl Acad Sci USA 87:2072-2076.
Shemshedini L, Lanoue M, Wilson TG (1990): Evidence for a juvenile hormone receptor involved in protein synthesis in Drosopkila melanogaster. J Biol Chem 265:1913-1918.
Sliter TJ, Sedlak BJ, Baker FC, Schooley PA (1987): Juvenile hormone in Drosopkila melanogaster. Identification and titer determination during development. Insect Biochem 17:161-165.
Staal GB (1975): Insect growth regulators with juvenile hormone activity. Ann Rev Entomol
Methoprene-tolerant From Drosophila
Stevens B, Alvarez CM, Bohman R, OConnor JD (1980): An ecdysteroid-induced alteration in
the cell cycle of cultured Drosopkila cells. Cell 22:675-682.
Trowel1 SC, Hines ER, Herlt AJ, Rickards RW (1994): Characterisation of a juvenile hormone
binding lipophorin from the blowfly Lucilia cuprina. Comp Biochem Physiol B.
Turner C (1992): Isolation of a region from the Drosopkila genome containing the Metkoprenetolerant (Met) gene. PhD dissertation, University of Vermont, Burlington.
Voelker RA, Wisely GB, Huang S-M, Gyurkovics H (1985):Genetic and molecular variation in
the Xp11225 region of Drosopkila melanogaster. Mol Gen Genet 201:437-445.
Wilson TG (1980): Correlation of phenotypes of the apterous mutant in Drosopkila melanogaster.
Dev Genet 1:195-204.
Wilson TG (1981): A mosaic analysis of the apterous mutation in Drosopkila melanogaster. Dev
Wilson TG (1982): A correlation between juvenile hormone deficiency and vitellogenic oocyte
degeneration in Drosopkila melanogaster. Wilhelm Roux Arch Entwicklungsmech Org
Wilson TG (1993):Involvement of transposable genetic elements in initiating insecticide resistance. J Econ Entomol86:645-651.
Wilson TG, Fabian J (1986): A Drosopkila melanoguster mutant resistant to a chemical analog of
juvenile hormone. Dev Biol118:190-201.
Wilson TG, Fabian J (1987): Selection of methoprene-resistant mutants of Drosopkila melanogaster. In Law J (ed): Molecular Entomology. UCLA Symposia on Molecular and Cellular
Biology, New Series, 49:179-188.
Wilson TG, Turner C (1992): Molecular analysis of Metkoprene-tolerant, a gene in Drosopkila
involved in resistance to juvenile hormone analog insect growth regulators. In Mullin C,
Scott J (eds): Molecular Mechanisms of Insecticide Resistance: Diversity Among Insects.
American Chemical Society Symposium Series 505:99-112.
Wilson TG, Landers MH, Happ GM (1983): Precocene I and I1 inhibition of vitellogenic oocyte development in Drosopkila melanogaster. J Insect Physiol29:249-254.
Zhang P, Knowles BA, Goldstein LS, Hawley RS (1990): A kinesin-like protein required for
distributive chromosome segregation in Drosopkila. Cell 62:1053-1062.
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molecular, tolerantia, melanogaster, analysis, genes, drosophila, regions, methorprene
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