Alternative splicing of transcripts expressed by the Manduca sexta ...

Peptides 22 (2001) 263–269 

Alternative splicing of transcripts expressed by the Manduca sexta 

allatotropin (Mas-AT) gene is regulated in a tissue-specific manner 

Frank M. Horodyski* a,b,c , Seema R. Bhatt d , and Kyeong-Yeoll Lee a,b 

a Department of Biomedical Sciences 

b College of Osteopathic Medicine 

c Molecular and Cellular Biology Program 

d Department of Biological Sciences, Ohio University, Athens, OH 45701 

Received 15 January 2000; accepted 15 July 2000 

Abstract 

The Manduca allatotropin (Mas-AT) gene is expressed as at least three mRNA isoforms that differ from each other by alternative 

splicing. The location at which the alternative exons are included in the mature mRNAs occur within the open reading frame, so that three 

different propeptides are predicted as translation products. In the pharate adult insect, the major mRNA isoform expressed in the brain and 

frontal ganglion differs from that expressed in the nerve cord. Examination of the deduced translations of the alternative exons reveals the 

presence of three additional Mas-AT-like sequences that are flanked by basic amino acid residues. Therefore, the Mas-AT-like sequences 

present within the gene may be derived from a duplication of an ancestral Mas-AT-like sequence followed by divergence. © 2001 Elsevier 

Science Inc. All rights reserved. 

Keywords: Allatotropin; Manduca sexta; Alternative splicing; Nervous system; Neuropeptides 

1. Introduction 

Manduca allatotropin (Mas-AT) is an amidated tridecapeptide 

that was isolated from heads of pharate adult 

insects [14]. Purification of the peptide was based on an in 

vitro bioassay, measuring the ability to stimulate juvenile 

hormone (JH) synthesis by the adult female corpora allata 

(CA) [11,19], an endocrine organ that is the source of JH 

[30]. It was demonstrated that Mas-AT is active only on the 

adult CA; it has no effect on the larval or pupal CA [14]. 

Therefore, despite the presence of JH in both larvae and 

adults and the importance of the precise regulation of its 

titers during both life stages [20,31], JH levels in larvae are 

apparently controlled by mechanisms distinct from the stimulation 

of its synthesis by Mas-AT. In larvae, JH levels may 

be controlled by the action of stimulatory or inhibitory 

peptides on the CA. These factors may include allatotropins 

that have not yet been characterized, allatostatin (Mas-AST) 

which inhibits JH synthesis by larval CA in vitro [16], an 

Taken from a paper presented at the Winter Neuropeptide Conference 

2000, Invertebrate Division, Hua Hin, Thailand, January 10–15, 2000. 

* Corresponding author. Tel.: 740-593-0851; fax: 740-597-2778. 

E-mail address: horodysk@ohiou.edu 

allatostatin with sequence similarity to the cockroach allatostatins 

(lepidostatin) [7,9], or allatinhibin which stably 

inactivates the CA and was identified by its physiological 

action in the intact insect [3]. In addition to regulating the 

level of JH synthesis, its rate of degradation may be controlled 

by changing titers of enzymes that inactivate JH [13] 

or by the presence of binding proteins that alter its interactions 

with degradative enzymes [12,26]. 

Although Mas-AT apparently does not regulate JH synthesis 

levels in larvae [14], the mRNA encoding the propeptide 

from which Mas-AT is derived was detected at high 

levels in two cells of the frontal ganglion and two cells of 

the terminal abdominal ganglion; and at lower levels in 

10–12 pairs of cells in the brain and 4 pairs of cells in the 

subesophogeal ganglion [4,25]. 

Similar cells in larvae contain material that reacted to a 

polyclonal antiserum to Mas-AT [4,25,32]. In addition to 

these cells, the larval midgut endocrine cells contain 

Mas-AT immunoreactivity [33]. The role of Mas-AT in 

larval insects was examined by studying its effect on active 

ion transport across the midgut epithelium [17]. Mas-AT, at 

concentrations from 10–100 nM, was shown to inhibit active 

ion transport in vitro in a dose-dependent manner. In 

another lepidopteran insect, Helicoverpa armigera, Mas-AT 

0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. 

PII: S0196-9781(00)00378-8

264 F.M. Horodyski et al. / Peptides 22 (2001) 263–269 

stimulated peristaltic contractions of the foregut; and it was 

proposed that this effect was mediated by the two Mas-AT 

immunoreactive cells in the frontal ganglion [8]. 

The cells that express the Mas-AT gene and contain 

Mas-AT-like immunoreactivity after metamorphosis are 

similar to those observed in larvae, but there are some 

significant differences [4]. Two pairs of cells in the pupal 

pterothoracic ganglion and 1–3 pairs of cells in the unfused 

abdominal ganglia contain Mas-AT mRNA and immunoreactivity 

[4,29]. The number of reactive cells in the abdominal 

ganglia increases during adult development, so that by 

the pharate adult stage, 3 pairs of cells are consistently seen 

[4]. In addition, the terminal abdominal ganglion of pupae 

and pharate adults contains one pair of cells in the most 

anterior neuromere and a cluster of cells in the posterior 

region that contain Mas-AT mRNA and immunoreactivity 

that are not reactive in the larval stage. An additional function, 

cardioexcitatory activity, has been described for 

Mas-AT in the adult [29]. Therefore, Mas-AT is a multifunctional 

peptide, whose cellular targets and biological 

activities change during development. 

The Mas-AT gene is transcribed as at least three mR- 

NAs, which differ from one another by alternative splicing 

[25]. Exons that are unique to a specific Mas-AT mRNA are 

located within the protein-coding region, so that three different 

propeptides can be derived from the mRNAs. The 

deduced translation product of each mRNA contains a sequence 

identical to that of Mas-AT that is flanked by basic 

residues characteristically found at cleavage sites. Additional 

peptides are predicted from regions of the precursor 

flanking the Mas-AT sequence. Some of these putative 

peptides are found in each deduced propeptide, whereas 

others are unique so that they are derived specifically from 

one Mas-AT mRNA. We show that in the pharate adult 

nervous system, the alternative splicing of Mas-AT transcripts 

is regulated in a tissue-specific manner. Furthermore, 

the deduced translation within the alternative exons contains 

one sequence within each exon that resembles the sequence 

of Mas-AT and is also flanked by basic residues. This raises 

the possibility that additional Mas-AT-like peptides may be 

derived from the alternatively spliced Mas-AT mRNAs. 

2. Materials and methods 

2.1. Animals 

Larvae of the tobacco hornworm, Manduca sexta, were 

reared individually on an artificial diet (Bioserv) at 26°C 

under a 17-hr light: 7-hr dark photoperiod as described [2]. 

At the start of the wandering stage, the larvae were transferred 

to wooden blocks until pupation. Freshly ecdysed 

pupae were transferred to cardboard boxes until 1 day before 

adult emergence. Pupae were staged according to the 

number of days postpupal ecdysis, with day 0 designated as 

the day of pupal ecdysis. Pharate adults were selected when 

the pupal cuticle above the wing and the abdomen is soft 

[22]. 

2.2. RNA extraction 

Tissues were dissected and frozen in liquid nitrogen and 

stored at 80°C until use (for the RT-PCR experiment); or 

stored overnight at 4°C in RNAlater (Ambion) followed by 

removal of the solution and storage of the tissue at 80°C 

(for the Northern blot). RNA was extracted using Trizol 

reagent (Gibco-BRL), and mRNA was purified using the 

Micro-Oligo(dT) Spin Column Kit (5 prime-3 prime). 

2.3. Reverse transcriptase-polymerase chain reaction (RT- 

PCR) 

Reverse transcriptase reactions were performed with the 

entire yield of total RNA isolated from tissue dissected from 

a single pharate adult animal, and carried out using the 

conditions previously described using murine leukemia virus 

(MLV) reverse transcriptase (Gibco-BRL) [15]. PCR 

[21] was performed in 1 Taq polymerase buffer (Stratagene) 

supplemented to a final concentration of 3.5 mM 

MgCl 2 ,1M of each primer (synthesized by DNA International), 

and 200 M dNTPs in a volume of 50 l. The 

mixtures were overlaid with 80 l mineral oil and amplified 

in a thermal cycler (Perkin Elmer 480) for 35 cycles (94°C, 

1 min; 55°C, 1 min; 72°C 1 min) preceded by a4min 

denaturation step at 94°C, and followed by a 5 min final 

extension at 72°C. The exon-specific PCR primers 5- 

GCAACAGCGACCCACGCG-3 and 5-CTTTCAGATTT 

AAACCACGAC-3 were previously described and were 

designed based on the sequence of the Mas-AT gene within 

exons II and VII [25]. PCR products were electrophoresed 

on a 3% NuSieve-GTG agarose (FMC) gel and transferred 

to Hybond-N (Amersham). 

Three exon-specific probes were designed to distinguish 

between RT-PCR products derived from the three Mas-AT 

isoforms. Probe A was labeled using the insert of the 

Mas-AT cDNA clone, pF6-1 [25]. The cDNA clone was 

isolated from a pupal brain cDNA library and is derived 

from exons I, II, VI, and VII. These exons are common to 

all known Mas-AT mRNAs, and thus should detect mRNAs 

I, II, and III. pF6-1 DNA was digested with Eco RI and the 

digestion products were electrophoresed on a 0.8% low 

melting point agarose gel in Tris-acetate buffer. The 1.1 kb 

insert was excised and 3 volumes of H 2 O were added before 

using the DNA as a template for random hexamer primer 

labeling. 

Probe B was designed to specifically hybridize to DNA 

derived from exon IV, and thus should detect only mRNA 

II. The plasmid, p408-5, was constructed by subcloning the 

677 bp RT-PCR amplification product from day 3, 5th instar 

larval brain obtained by using the exon-specific primers 

described above [25]. Then, to eliminate common sequences 

to all Mas-AT mRNA isoforms, p408-5 DNA was

F.M. Horodyski et al. / Peptides 22 (2001) 263–269 

265 

amplified using the primers 5-CGAACTTACAACGTC 

CC-3 and 5-TTCGTTGTAGTTCTCTTCA-3, which 

were designed to sequences at the boundaries of exon IV. 

The 103 bp PCR product was then purified on a 3% 

NuSieve-GTG agarose gel in Tris-acetate buffer as described 

above. 

Probe C was designed to specifically hybridize to DNA 

derived from exons III and V, and thus should detect only 

mRNA III. The plasmid, p409-1, was constructed by subcloning 

the 797 bp RT-PCR amplification product from day 

2, 5th instar larval nerve cord obtained by using the exonspecific 

primers described above [25]. Then, to eliminate 

common sequences to all Mas-AT mRNA isoforms, p409-1 

was amplified using the primers 5-CGACGTAGATC 

ACCAGGCA-3 and 5-TACCGTCCATCGATTTGT-3, 

which are designed to sequences at the upstream boundary 

of exon III and the downstream boundary of exon V, respectively. 

The 221 bp PCR product was then purified on a 

3% NuSieve-GTG agarose gel in Tris-acetate buffer as 

described above. 

The probes were labeled with 32 P by random hexamer 

priming [10] and purified from unincorporated nucleotides 

using a NucTrap Probe Purification Column (Stratagene) 

and hybridized using the identical high stringency conditions 

used for the Northern blot hybridization. 

2.4. Sequence analysis 

Sequences were analyzed using the DNASIS v.2.1 (Hitachi 

Software Engineering Co., Ltd.), and the BLASTP 

algorithm [1] to search the NCBI database by internet access. 

3. Results 

The Mas-AT gene contains at least 7 exons, 4 of which 

are common to all known Mas-AT mRNAs [25]. The 

Mas-AT cDNA clone, isolated from a pupal brain cDNA 

library, consists of sequences that include only the common 

exons. When a probe was constructed from this cDNA clone 

and hybridized to a genomic Southern blot, a single hybridizing 

region was observed indicating that Mas-AT is a 

single copy gene, so any mRNAs detected using this probe 

must be derived from this gene. To determine the size of the 

Mas-AT mRNA, the identical probe was used to hybridize 

to a Northern blot of poly A mRNA extracted from the 

pupal nerve cord. A single 1.2 kb mRNA hybridized to this 

probe (Fig. 1). The size of the mRNA is slightly larger than 

that of the cDNA clone (1,064 bp), and is nearly full-length. 

Since it was previously shown that at least three Mas-AT 

mRNAs differed from each other by alternative splicing 

[25], we wanted to determine whether different Mas-AT 

mRNAs are present in different tissues isolated from the 

same animal. The pharate adult stage was chosen, since the 

cellular distribution of Mas-AT mRNA in the brain and 

Fig. 1. Expression of the Mas-AT gene. Northern blot of Manduca sexta 

day 2 pupal poly A RNA hybridized to the 32 P-labelled insert of the 

Mas-AT cDNA clone. The size of the hybridizing RNA is shown. 

frontal ganglion was not yet described, and this experiment 

would provide evidence for the presence of Mas-AT mRNA 

in these tissues. The brain, frontal ganglion, and nerve cord 

was extracted from a single pharate adult insect, and RNA 

was prepared from these tissues. Mas-AT mRNA was detected 

by reverse transcriptase-polymerase chain reaction 

(RT-PCR) using primers designed to portions of common 

exons (exons II and VII) flanking the alternative exons (Fig. 

2a). Using these primers, each mRNA is predicted to yield 

a specific-sized product that can be detected by ethidium 

bromide staining of the gel containing the RT-PCR products 

and by hybridization of a Southern blot of that gel with 

probes derived from specific regions of the Mas-AT gene 

between these primers. Analysis of the Southern blot ensures 

that the RT-PCR products observed are derived from 

the (single-copy) Mas-AT gene. A negative control was 

included in parallel in which the RT-PCR was conducted 

with no added RNA, and a positive control was included in 

which a PCR reaction was carried out with the Mas-AT 

cDNA clone as a template. 

The RT-PCR products were electrophoresed on three 

separate gels, and a Southern blot of each gel was hybridized 

to a different probe (Fig. 2b). One probe (A) was 

derived from the cDNA clone, and contains sequences only 

from the common exons. Therefore, this probe was expected 

to detect each of the three alternatively spliced 

Mas-AT mRNAs. The remaining two probes are composed 

exclusively of sequences present in the alternative exons 

known to be represented in each of the two longer mRNAs, 

respectively. Therefore, probe B is designed to specifically 

detect RT-PCR products derived from RNA II, the only 

known Mas-AT mRNA that contains sequences from exon


Fig. 2. Expression of the three alternatively spliced Mas-AT mRNAs in tissues of a pharate adult Manduca sexta. a. Design of the RT-PCR experiment to 

detect Mas-AT mRNAs. A schematic representation of the Mas-AT gene shows the positions of the known exons. Common exons are shown in darker 

shading. The region that contains exon I (predicted from the cDNA sequence) has not yet been identified, and is at least 3 kb upstream of exon II. Shown 

below the restriction map is a representation of the structure of each Mas-AT mRNA [adapted from Taylor et al. [25]]. Downstream and upstream primers 

for PCR are located within exons II and VII, respectively. Relative locations of the RT-PCR products derived from each mRNA are blackened, and the sizes 

of the expected RT-PCR products are designated. b. Products of RT-PCR reactions from brain (B), frontal ganglion (F), and nerve cord (N) were hybridized 

to 32 P-labelled probes specific to the designated exons (shown at right). A negative control in which no RNA was added to the RT-PCR reaction (), and 

a positive control in which the Mas-AT cDNA clone was included as a template in the PCR reaction () are included. The sizes of the hybridizing products 

are shown. The Mas-AT exons from which the probe is derived, and the Mas-AT mRNA isoforms detected by each probe are shown. 

IV. Similarly, probe C is designed to specifically detect 

RT-PCR products derived from RNA III, the only known 

Mas-AT mRNA that contains sequences from exons III and 

V. The positive control is expected to hybridize only to 

probe A, since the Mas-AT cDNA lacks sequences from 

exons III, IV and V. The negative control is not expected to 

hybridize to any of the three probes, and is included to 

eliminate the possibility that the RT-PCR products are derived 

from a contaminant. 

The pharate adult brain contains predominantly Mas-AT 

mRNA isoform I, since the major product hybridizing to a 

probe consisting of common exons (probe A) is 578 bp, 

characteristic of mRNA I amplification (Fig. 2b, lane B, 

probe A). In this hybridization to brain RT-PCR products, a 

less intense band of 677 bp band is also detected, which is 

the size expected from mRNA II amplification. To confirm 

that the 677 bp product is derived from mRNA II, the 

identical RT-PCR products were hybridized to probe B, 

which is specific to exon IV and is predicted to detect only 

mRNA II. Hybridization of the brain RT-PCR products to 

probe B detected exclusively the 677 bp product, so a low 

level of mRNA II is also present in the brain (Fig. 2b, lane 

B, probe B). Although hybridization of the brain RT-PCR 

products to probe A did not reveal a 797 bp product characteristic 

of the presence of mRNA III, we tested whether 

mRNA III was present in the brain by hybridization to probe 

C. Indeed, a hybridizing 797 bp product was detected, so 

mRNA III is also present in the brain, although at very low 

levels compared with mRNAs I and II, since this product 

was not detectable with probe A (Fig. 2b, lane B, probe C).


267 

Table 1 

Summary of Mas-AT mRNA isoforms expressed in pharate adult 

Manduca sexta 

Brain 

Frontal Ganglion 

Nerve Cord 

The pharate adult nerve cord contains predominantly 

Mas-AT mRNA isoform II, since the major product hybridizing 

to the common exon probe is 677 bp (Fig. 2b, lane N, 

probe A); and a product of identical size hybridized to probe 

B, designed to detect only mRNA II (Fig. 2b, lane N, probe 

B). A less intense product of 578 bp was detectable with 

probe A, indicating that mRNA I is also present in the nerve 

cord, but at very low levels compared with mRNA II (Fig. 

2b, lane N, probe A). No hybridization of nerve cord RT- 

PCR products was detected to probe C, so mRNA III is 

absent from the nerve cord (Fig. 2b, lane N, probe C). The 

pharate adult frontal ganglion contains exclusively mRNA 

I, since the only RT-PCR product that hybridized to any of 

the probes was the 578 bp product that hybridizes to probe 

A (Fig. 2b, lane F, probe A). RNAs II and III are apparently 

absent in the pharate adult frontal ganglion (Fig. 2b, lane F, 

probes B and C). A summary of the Mas-AT mRNA isoforms 

present in each pharate adult tissue is presented in 

Table 1. 

4. Discussion 

Mas-AT mRNAs 

I, (II, III) 

I 

II, (I) 

Like many neuropeptides, Mas-AT appears to possess 

multiple biological activities [8,14,17,29]. These data are 

mostly based on available in vitro assays, and because of 

this, the actual role of Mas-AT at any developmental time is 

difficult to ascertain. Whether the peptide actually possesses 

these activities in the intact animal depends in part on 

whether the peptide is present in high enough concentrations 

at the target tissue at the developmental times in which 

it is active in vitro. This concentration depends on the 

affinity of the peptide ligand for its receptor protein in the 

membrane of the target cell. In the case of Mas-AT, this 

information is still unknown. An ELISA has been developed 

using a polyclonal antiserum to Mas-AT, and it was 

shown that the peptide was present in the brain, nerve cord, 

and the retrocerebral complex [28]. However, it is not 

known whether the peptide is released into the hemolymph 

where it can act as a hormone on distant targets, or whether 

it primarily acts locally upon release. Localization of cells 

that express the Mas-AT gene and contain the peptide, and 

characterization of the axonal structure of these cells can 

provide clues as to where Mas-AT can be released. For 

example, the two Mas-AT cells in the larval frontal ganglion 

project their axons down the recurrent nerve to the foregutmidgut 

boundary [4,6,8]. Release of Mas-AT from these 

cells may account for the increase in peristaltic contractions 

observed [8]. 

Three propeptides are predicted translation products of 

the three Mas-AT mRNAs, and Mas-AT is the only peptide 

whose sequence is within the propeptides that has been 

identified in the insect. It is flanked by a single Arg and a 

Lys-Arg sequence, which are characteristic sites of prohormone 

processing [23]. With the exception of Mas-AT, the 

other predicted peptides have no similarities to known peptides 

in the database. However, a close examination of the 

predicted translated sequences from each of the alternative 

exons (III, IV and V) reveals the presence of a short sequence, 

from 16–17 residues, flanked by a single Arg and a 

Lys-Arg, like Mas-AT (Fig. 3). The C-terminal residue of 

each sequence is Gly, which functions as a substrate for 

C-terminal amidation [5]. Comparison of these three sequences 

with that of Mas-AT demonstrates that 7, 6, and 2 

residues are identical to that in Mas-AT at the same position 

(Fig. 4). These sequences are designated Mas-AT-like I, 

Mas-AT-like II, and Mas-AT-like III; respectively. Mas- 

AT-like I coding sequence is within exon V, Mas-AT-like II 

coding sequence is within exon III, and Mas-AT-like III 

Fig. 3. Localization of deduced Mas-AT-like peptides within the predicted propeptides. The structures of the propeptides derived from the three Mas-AT 

mRNAs all contain a predicted signal peptide (blackened), a 15-residue peptide, Mas-AT (AT), and a 79 or 80 residue C-terminal peptide that may be further 

processed. Propeptides derived from RNAs II and III contain additional predicted peptides including Mas-AT-like peptides I, II, and III; whose relative 

localization is shown.


Fig. 4. Alignment of the Mas-AT sequence with those of the predicted Mas-AT-related peptides. Residues of the Mas-AT-like peptides identical to Mas-AT 

are shown in bold. 

coding sequence is within exon IV (Fig. 3). One residue 

(Phe-2, numbered from the N-terminus of Mas-AT) is conserved 

in all 4 sequences, and five residues (Gly-1, Lys-3, 

Ala-10, Arg-11, and Phe-13) are conserved in 3 of the 4 

sequences (Fig. 4). 

Although Mas-AT-like III shares only 2 of 13 identities 

with Mas-AT, it shares an additional 4 identities to either 

Mas-AT-like I or Mas-AT-like II. The deduced sequences 

flanking the Mas-AT-like residues share no identity with the 

deduced sequence flanking Mas-AT. From this information, 

we can speculate that Mas-AT and the Mas-AT-like sequences 

are derived from a single ancestral sequence, which 

underwent duplication to 4 copies and subsequent divergence. 

Since the similarity is exclusively within the Mas- 

AT-like sequences, there may have been evolutionary pressure 

to maintain this common sequence motif. 

Recently, a Mas-AT-like sequence was predicted from a 

DNA sequence of an Aedes aegypti cDNA clone that shares 

identity in 10 of 13 residues [27]. The predicted structure of 

the Aedes propeptide is similar to that of Manduca, and the 

greatest identity was observed in the Mas-AT-like region. A 

lower level of similarity was observed in the C-terminal 

peptide, so the greatest evolutionary pressure on the propeptide 

sequence was to maintain the Mas-AT-like sequence. 

Based on the identical sequence of several cDNA clones 

isolated from Aedes, alternative splicing of the Mas-AT-like 

transcript was not detected. It is evident from this study, and 

from the isolation of Mas-AT-like peptides from Locusta 

migratoria (Lom-AG-myotropin) [18], and Leptinotarsa 

decemlineata [24] that an Mas-AT-like sequence is present 

in a wide variety of insect orders. Some of the functions of 

these peptides may be conserved among many insect species, 

or may be specific to a particular order. 

Unlike Mas-AT, there is no evidence that the cleavage 

sites flanking the Mas-AT-like peptides are used, since these 

peptides have not been isolated or detected using specific 

antisera. It is difficult to speculate as to the functions of 

these sequences until evidence for their existence has been 

obtained. However, it is possible to test these synthetic 

peptides for activity in the bioassays in which Mas-AT is 

active. 

We have clearly demonstrated that the Mas-AT transcripts 

are alternatively spliced in a tissue-specific manner 

in the pharate adult (Fig. 2b). The biological significance for 

this finding is not known, but it is intriguing that the different 

Mas-AT mRNAs may yield multiple Mas-AT-like 

peptides (Fig. 4). The brain and the nerve cord contain 

primarily one isoform, but low levels of other isoforms were 

also detected (Fig. 2b). In situ hybridization studies using 

exon-specific probes are necessary to determine whether 

different cells in these tissues contain different isoforms, or 

whether a single cell may contain more than one isoform. 

We have preliminary evidence that the Mas-AT mRNA 

isoform present in the larval nerve cord differs from the 

prominent isoform found in the pharate adult. Additional 

cells express the Mas-AT gene in the nerve cord after 

metamorphosis [4]. It will be interesting to determine 

whether the presumed switch in Mas-AT isoform is due to 

a change in the splicing pattern within a single cell, or is due 

to the onset of expression of the new isoform in the additional 

Mas-AT cells. 

Acknowledgments 

We are grateful to Drs. Tejal Bhatt and Phil Taylor for 

helpful discussions and to Paul Wiehl for excellent technical 

assistance. This work was supported by a grant from the 

National Science Foundation (IBN-9307051 and 

IBN-9807907) to FMH. SRB was supported by a Research 

Experience for Undergraduates (REU) Supplement to a 

grant from the National Science Foundation (IBN- 

9307051). 

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Alternative splicing of transcripts expressed by the Manduca sexta ...

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