Biomedical subjects

L M Riddiford

Publications and source records attributed to L M Riddiford.

At least 37 records · Page 2Linked to original sources

PubMed1997

Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta.

To determine which ecdysteroids may be biologically active in the larval epidermis of the tobacco hornworm, Manduca sexta, we studied the action of several known ecdysteroids and metabolites on the expression of the genes encoding the ecdysone receptor (EcR), Manduca hormone receptor 3 (MHR3), dopa decarboxylase (DDC), and a larval cuticle protein (LCP-14). Both Day 2 fourth- and Day 2 fifth-instar larval epidermis contained significant 3 beta-reductase activity which metabolized 3-dehydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E) to ecdysone (E) and 20-hydroxyecdysone (20E), respectively, but had only very low amounts of ecdysone oxidase activity (E to 3DE) and no detectable ecdysone 20-monooxygenase activity (E to 20E). When the expression of the various genes was studied in the epidermis in vitro, 20E and 3D20E had similar effects, whereas E, 3DE, 26-hydroxyecdysone and 20,26-dihydroxyecdysone were ineffective. Exposure of Day 2 fifth-instar epidermis to 500 ng/ml of either 20E or 3D20E for 24 hr caused a rapid, biphasic increase in EcR-B1 mRNA. By contrast, EcR-A mRNA showed a less rapid initial increase followed by a slow steady rise and was less responsive to 3D20E. Ecdysone in a 1:1 mixture with 20E effectively halved the concentration of 20E needed to induce EcR-B1 mRNA but showed no synergism in the induction of EcR-A mRNA. The induction of MHR3 mRNA and of DDC mRNA in Day 2 fourth-instar epidermis as well as the suppression of DDC and LCP-14 gene expression by 3D20E was indistinguishable from that of 20E. Therefore, for Manduca larval epidermis, only 20E and 3D20E are biologically active ecdysteroids. Since the 3D20E can be converted to 20E by the epidermis, its effects are likely mediated by 20E.

Animals↗

PubMed1997

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta.

The embryonic cell line, GV1, from Manduca sexta was transiently transfected with DNA constructs of the Drosophila hsp70 promoter fused to either a beta-galactosidase (pXH70ZT) or a chloramphenicol acetyl transferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell density, DNA:lipofectin ratio, and time of incubation were varied to determine the optimal conditions: 2 x 10(5) cells/ml, 1:3, and 5 h. Under these conditions, the transfection efficiency was about 40%. Heat inducibility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than that of pXH70ZT, containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in these cells. A 2 kb fragment of the proximal promoter region of the MHR3 gene containing a putative ecdysone response element was shown to be responsive to 20-hydroxyecdysone after its transfection into these cells.

Animals↗

PubMed1997

Disruption of a behavioral sequence by targeted death of peptidergic neurons in Drosophila.

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

Animals↗

PubMed1997

Structure and spatial expression of the Manduca sexta MSCP14.6 cuticle gene.

The genomic and cDNA sequences of the MSCP14.6 gene section, which encodes a Manduca sexta cuticle protein, have been determined. The genomic sequence presented here includes 1428 N of 5' flanking DNA, 1587 N of transcribed DNA, and 465 N of 3' flanking DNA. The intron/exon boundaries mapped by comparison of these sequences were confirmed by primer extension and SI nuclease mapping. Prior work showed that the RNA produced by this gene is regulated both temporally and spatially during larval, pupal, and adult development, and also showed that both 20-hydroxyecdysone (20E) and juvenile hormone (JH) are important in controlling the amount of RNA present in the epidermis (Riddiford et al., 1986). In situ hybridizations show that MSCP14.6 is expressed throughout the epidermis in larvae and is spatially restricted in pharate pupal and pharate adult stages. The results presented here delimiting the transcription unit and 5' flanking DNA of the MSCP14.6 gene will facilitate further research to determine the DNA elements necessary for regulation of gene expression.

Amino Acid Sequence↗

PubMed1997

Regulation of the ecdysone receptor, USP, E75 and MHR3 mRNAs by 20-hydroxyecdysone in the GV1 cell line of the tobacco hornworm, Manduca sexta.

The responsiveness of several nuclear transcription factor genes to 20-hydroxyecdysone (20E) was characterized in an embryonic cell line, GV1, from Manduca sexta. The mRNA for the Manduca ecdysone receptor (MsEcR) was present in the GV1 cells and transiently increased 2.3-fold by 5 h after the addition of 2 micrograms/ml (4 x 10(-6) M) 20-hydroxyecdysone (20E). In contrast, Manduca ultraspiracle (MsUSP) mRNA level in the GV1 cells decreased slowly to half of its initial level by 12 h when exposed to the same concentration of 20E. The mRNAs for two putative transcription factors, MsE75 and MHR3, were induced in the GV1 cells by 20E, with that for E75 appearing within 1 h whereas that for MHR3 within 2 h. The ED50S for induction of MsE75 and MHR3 gene expression in GV1 cells were 1.2 x 10(-6) and 2.5 x 10(-6) M 20E, respectively.

Animals↗

PubMed1997

Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta.

cDNAs were isolated from Manduca sexta that encode two isoforms of an ultraspiracle (USP) homologue MsUSP-1 and MsUSP-2 with different N-terminal A/B regions. The MsUSP-1 cDNA predicts a protein with 97% and 45% amino acid identities in the DNA- and ligand-binding domains respectively to the Drosophila USP and 89% overall identity with Bombyx mori CF1 (an USP homologue). Northern blot hybridizations with probes specific to MsUSP-1 and MsUSP-2 showed transcripts of an approximately equal size (4.5 kb), but with diverse developmental profiles in Manduca epidermis during the two final larval instars and the onset of the adult moult. The MsUSP-1 mRNA was expressed during the intermoult periods, with higher levels around the time of the larval ecdyses and at the onset of wandering behaviour. In contrast, the MsUSP-2 mRNA was up-regulated at times of high ecdysteroid titre during the larval moults, when the MsUSP-1 mRNA disappeared. Together, these conversely regulated isoform mRNAs contribute to the constitutive expression profile of total MsUSP mRNA.

Amino Acid Sequence↗

PubMed1997

A cluster of cuticle protein genes of Drosophila melanogaster at 65A: sequence, structure and evolution.

A 36-kb genomic DNA segment of the Drosophila melanogaster genome containing 12 clustered cuticle genes has been mapped and partially sequenced. The cluster maps at 65A 5-6 on the left arm of the third chromosome, in agreement with the previously determined location of a putative cluster encompassing the genes for the third instar larval cuticle proteins LCP5, LCP6 and LCP8. This cluster is the largest cuticle gene cluster discovered to date and shows a number of surprising features that explain in part the genetic complexity of the LCP5, LCP6 and LCP8 loci. The genes encoding LCP5 and LCP8 are multiple copy genes and the presence of extensive similarity in their coding regions gives the first evidence for gene conversion in cuticle genes. In addition, five genes in the cluster are intronless. Four of these five have arisen by retroposition. The other genes in the cluster have a single intron located at an unusual location for insect cuticle genes.

Animals↗

PubMed1996

Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta.

Ecdysteroids acting through multiple isoforms of the ecdysone receptor (EcR) initiate molting and metamorphosis of insects. Two isoforms of EcR, A (this paper) and B1 (Fujiwara et al., Insect Biochem. Mol. Biol. 25, 845-856, 1995), were isolated from the tobacco hornworm, Manduca sexta, and shown to be similar to the corresponding Drosophila EcR isoforms. The developmental profiles of both EcR-A and EcR-B1 (determined by both analysis of isoform-specific mRNAs and use of monoclonal antibodies that detect either EcR-B1 or all forms), however, were different in Manduca epidermis, which produces sequentially the larval, the pupal, and the adult cuticles. EcR-B1 predominated through the larval, pupal, and early developing adult stages with an upregulation early in each molt. By contrast, EcR-A was present only at the onset of new cuticle synthesis during the larval molt, but in the pupal and adult molts was upregulated slightly later than EcR-B1 during the commitment period and was present during the predifferentiative phase. Both isoforms appeared in the larval wing discs after pupal commitment and persisted through pupal differentiation. The mRNAs for both isoforms were directly induced in larval epidermis in vitro by 20-hydroxyecdysone, but EcR-B1 mRNA accumulated more rapidly, peaking at 3 hr. In the presence of a protein synthesis inhibitor, the accumulation of EcR-B1 mRNA was slower and its subsequent decline was prevented, but the accumulation of EcR-A mRNA was unaffected. Thus, in this polymorphic epidermis both isoforms appear in every molt, with EcR-B1 present during the commitment and predifferentiative phases and then at the onset of cuticle synthesis EcR-A prevails. Additionally, EcR-A is apparently associated with the switching and predifferentiative events necessary for a new synthetic program.

Amino Acid Sequence↗

PubMed1996

Juvenile hormone: the status of its "status quo" action.

Juvenile hormone (JH) allows larval molting in response to ecdysteroids but prevents the switching of gene expression necessary for metamorphosis. I first review our efforts to isolate the nuclear receptor for JH in the larval epidermis of Manduca sexta using photoaffinity analogs and our recent findings that the molecule isolated does not bind JH I with high affinity. The reported apparent high affinity binding of JH I by the recombinant 29 kDa protein (rJP29) was artifactual due to the presence of contaminating esterases. Purified rJP29 bound little detectable JH I, but its binding of the photoaffinity analog was prevented by JH I as well as other isoprenoids, indicating a low affinity for these compounds. Our recent studies focus on the effects of JH on the early molecular events induced by 20-hydroxyecdysone (20E). Culture of day 2 5th larval epidermis with 10(-6)M 20E for 24 h caused first pupal commitment, then the onset of the predifferentiative events necessary for pupation. Biphasic increases in the mRNAs of the two isoforms of the ecdysone receptor (EcR-A and EcR-B1) and of E75A, an ecdysteroid-induced transcription factor, coincided with these two phases. The mRNAs for Ultraspiracle (USP) and the metamorphosis-specific Broad-Complex (BR-C) increased only during the second phase. The presence of JH had no effect on the initial increases in EcR mRNAs but caused an increased accumulation of E75A mRNA. This JH also prevented the later changes in EcR, USP, and BR-C mRNAs. Thus, JH influences only certain of the early actions of 20E which then result in its preservation of the "status quo."

Animals↗

PubMed1995

Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone.

Manduca dopa decarboxylase (DDC) cDNA was isolated, sequenced, and found to be most closely related to Drosophila DDC (72% amino acid identity). Culture of Day 2 fourth instar larval epidermis with 20-hydroxyecdysone (20E) showed that 20E was necessary to determine the later expression of the gene, but its removal was required for this expression to occur. Experiments with the protein synthesis inhibitors, cycloheximide and anisomycin, and the mRNA synthesis inhibitor, alpha-amanitin, showed that 20E induced a protein(s) which suppressed transcription of the DDC gene. Gel mobility shift assays using epidermal extracts and various fragments of the first 1.1 kb of the 5' flanking region of the DDC gene showed only one DNA fragment 87 to 167 bp upstream of the 5' initiation site that bound a nuclear protein(s) with the expected developmental specificity. The protein was abundant at the time of high ecdysteroid titer when no DDC mRNA was present, but low both before the rise (no DDC mRNA) and after the decline of ecdysteroid titer (maximal DDC mRNA). Thus, this protein(s) is a candidate for an ecdysteroid-induced transcription factor which acts to suppress DDC transcription. DNase I footprinting assays confirmed by use of a specific oligonucleotide showed that this protein(s) bound to the sequence 5'-GGCTTATGCGCTGCA-3'.

Amino Acid Sequence↗

PubMed1995

Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings.

Using the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA clone, we isolated three genomic clones for EcR from the tobacco hornworm, Manduca sexta. Subsequent isolation and sequencing of several cDNAs yielded a homolog of the B1 isoform with 50, 95 and 70% amino acid identities with DmEcR in the N-terminal A/B, the DNA binding and the ligand binding domains respectively. Unlike Drosophila, an intron occurs between the exons encoding the two zinc fingers of Manduca EcR (MsEcR). A 6.0 kb mRNA encoding MsEcR was found in both larval wing discs and prothoracic glands and in pupal wings. During the final larval instar, the mRNA was maximal in the wing discs at one day after wandering (W1), whereas in the prothoracic gland EcR mRNA increased rapidly to high levels on day 2 and remained high thereafter. During the onset of adult development, two peaks of EcR mRNA were observed in wings from day 3 to 5 and on day 8 after pupal ecdysis. These two peaks correlated with the time of increasing titers of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The EcR mRNA peaks always preceded the large ecdysteroid peak, suggesting that the transcription of the EcR gene is induced by a low concentration of ecdysteroid in vivo.

Amino Acid Sequence↗

PubMed1994

The two duplicated insecticyanin genes, ins-a and ins-b are differentially expressed in the tobacco hornworm, Manduca sexta.

Two gene-specific probes were generated from the unique sequences in the 3' non-coding regions of the two insecticyanin genes, ins-a and ins-b to study the developmental expression of these genes in Manduca sexta. Both genes were initially transcribed in the freshly hatched first instar larvae and then expressed in the epidermis and to a lesser degree in the fat body during every larval feeding stage. In the epidermis of the 4th and 5th instar larvae, both mRNAs appeared shortly before ecdysis and accumulated to maximal levels within a day. As the larval epidermis became pupally committed on day 3 of the 5th (final) instar, INS-a mRNA quickly decreased, whereas INS-b mRNA showed a second peak of accumulation. In the fat body, both genes showed a similar expression pattern within the 4th instar to that of the epidermis except that levels were lower and ins-b mRNA dominated. In the final instar, only ins-b mRNA was present in significant amount. These findings not only reveal that the two duplicated insecticyanin genes have diverged in their expression pattern but also demonstrate, for the first time, that fat body also expresses insecticyanin genes.

Animals↗

PubMed1994

Isolation, characterization and developmental expression of the ecdysteroid-induced E75 gene of the wax moth Galleria mellonella.

Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone did not encode the N-terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75A and GmE75B, which differed in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA was induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.

Amino Acid Sequence↗

PubMed1994

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect.

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels then increased and remained at intermediate levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion.

Animals↗

PubMed1994

Evidence of a stimulatory effect of cyclic AMP on corpus allatum activity in Manduca sexta.

Injection of dibutyryl-cAMP prevents cuticular melanization of black Manduca sexta larvae, whose pigmentation is related to a defect in the control of the corpus allatum. The cAMP analog has no effect in allatectomized black larvae. Significant stimulation of corpus allatum activity was obtained in vitro with compounds which elicit or mimic elevated intracellular cAMP levels (dibutyryl-, 8-bromo-, N6 benzoyl-, and 8-thiomethyl-cAMP, 3-isobutyl-1-methylxanthine), but not with dibutyryl-cGMP. Relatively inactive glands, such as those on day 4 of last larval stadium or from black mutant larvae, were more sensitive to these compounds than glands actively synthesizing JH/JH acid. JH acid synthesis by corpora allata taken after pupal commitment in the last larval stadium (days 6 and 8) was not stimulated by either dibutyryl-cAMP or 3-isobutyl-1-methylxanthine, but day 8 glands appeared to be inhibited by dibutyryl--cAMP. The results indicate that a cAMP second messenger system is involved in the transduction of signals which stimulate JH/JH acid synthesis by Manduca corpora allata prior to pupal commitment and suggest that it may be involved in the inhibition of JH acid synthesis after commitment. They also imply that the proposed hemolymph factor to which the black mutant corpora allata are differentially sensitive interfaces with the cAMP system.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Larva lights: a decade of photoaffinity labeling with juvenile hormone analogues.

The introduction of photoaffinity labeling into the mode of action of insect hormones and pheromones started 12 yr ago with the photoaffinity labeling of juvenile hormone binding proteins (JHBPs) from cockroaches in the laboratory of the late John K. Koeppe. Applying this technique to Manduca sexta led ultimately to a three-laboratory collaborative project that has begun to dissect the molecular basis for JH transport, metabolism, and nuclear binding and gene activation in Lepidoptera. This review provides (1) a history of the first experiments; (2) an idea of the breadth of the technique in the arthropod classes Insecta, Crustacea, and Arachnida; and (3) evidence for the depth of the technique in unearthing key details about three different types of the molecular action of JH in M. sexta.

Affinity Labels↗

PubMed1994

Isolation and developmental expression of the ecdysteroid-induced GHR3 gene of the wax moth Galleria mellonella.

Three degenerate primers were designed to match the most conserved regions within the DNA-binding domains of several selected members of the steroid hormone receptor family. Use of these primers in the polymerase chain reaction with cDNA from Galleria mellonella prepupae detected a 177 bp fragment that had 87% identity to the Manduca sexta gene MHR3 and 75% to the Drosophila melanogaster DHR3 gene, and therefore was named "GHR3". Screening of a Galleria penultimate instar cDNA library with this fragment yielded a cDNA clone that contained a 557 codon open reading frame, predicting a 62.3 kDa protein. The deduced amino acid sequence of GHR3 showed 92% overall identity with the MHR3 protein and 97 and 70% identity with DHR3 in the putative DNA- and ligand-binding domains, respectively. Hybridization of whole body RNA revealed high GHR3 mRNA levels during both the larval and pupal molts, coincident with the molt-inducing ecdysteroid pulses, and low or undetectable levels during the first half of the last instar. During the larval-pupal transformation, no GHR3 mRNA was found at the beginning of the stemmatal pigment retraction at the onset of the ecdysteroid rise; maximal levels were observed 4 h later, coincident with the peak ecdysteroid titer (over 2.3 micrograms 20E equivalents/ml hemolymph). Two mRNAs (4.6 and 3.6 kb) were detected when the ecdysteroid titer was high. Injection of 2 micrograms/gm 20E into isolated final instar larval abdomens induced the appearance of the 4.6 kb mRNA within 1.5 h; the mRNA level then reached maximum by 3 h and declined by 6 h. No 3.6 kb mRNA was detectable during that time. A 10-fold lower 20E dose caused only trace induction by 3 h.

Amino Acid Sequence↗

PubMed1993

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster.

Eclosion hormone (EH) is a neuropeptide that triggers the performance of ecdysis behaviors at the end of a molt. We have isolated the EH gene from Drosophila melanogaster, and localized the gene to the right arm of chromosome 3 at band position 90B1-2. The 97-amino-acid translation product contains a signal peptide followed by a 73-amino-acid prohormone. The N-terminus of the prohormone has diverged from lepidopteran EH both in its length and amino acid composition, and contains a potential endoproteolytic cleavage site. The deduced sequence of Drosophila EH is 58% identical (36 of 62 amino acids) to that of Manduca EH. The EH gene is expressed as a 0.8-kb transcript in a single pair of brain neurons which extend their processes the entire length of the central nervous system and also to the corpora cardiaca portion of the ring gland. These cells show massive depletion of immunoreactive EH at ecdysis.

Amino Acid Sequence↗