581 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
1995 was used for additional analysis of the deduced amino acid sequence.
2.4. RNA and protein blot analysis RNA was separated by formaldehyde gel electro-
phoresis Maniatis et al., 1982 and transferred by pass- ive diffusion to HyBond-N
+
Amersham nylon mem- brane according to the manufacturer’s instructions.
Random-primed,
32
P-labeled DNA probes were incu- bated overnight with the blots at 65
° C in 50 mM PIPES
pH 6.5, 100 mM NaCl, 50 mM Na phosphate buffer pH 7.0, 1 mM EDTA and 5 SDS, followed by 65
° C
and room temperature washes in 1 ×
SSC with 5 SDS Virca et al., 1990. Kodak X-OMAT film was exposed
to hybridized filters and the resulting autoradiographs were scanned with a Molecular Dynamics Scanning
Densitometer ImageQuant program: Molecular Dynam- ics: Sunnyvale, CA.
Tissues were lysed in TEP buffer [10 mM Tris–HCl pH 9.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 6 M urea, 0.2 Triton X-100, 0.2 sodium deoxycholate] by sonication at 4
° C Sonifier Cell Dis-
rupter Model W140; Branson Sonic Power Co., Dan- bury, CT for 20 s at a setting of 5 and protein content
determined according to Bradford 1976 using bovine serum albumen as the standard. Proteins were then sep-
arated
by SDS–PAGE
Laemmli, 1970,
electro- transferred to polyvinylidene difluoride membranes, fol-
lowed by blocking for 1 h at room temperature in TBST [10 mM Tris–HCl pH 7.5 with 0.9 NaCl and 0.1
Tween 20] with 1 non-fat milk. Blots were then incu- bated at 4
° C for 14 to 16 h with an anti-hsphsc 70
monoclonal antibody Sigma: clone BRM-22 dissolved in TBST 12,000. Blots were then washed 3
× 10 min
in TBST, incubated with an alkaline phosphatase-conju- gated anti-mouse IgG 12,000: Sigma for 1–1.5 h at
room temperature, washed as above and signals developed using nitroblue tetrazolium and 5-bromo-4-
chloro-3-indolyl phosphate, as described previously Rybczynski et al., 1996. Blots were scanned while wet
using a Molecular Dynamic Computing Densitometer and the ImageQuant program.
2.5. Protein dephosphorylation Prothoracic gland extracts were treated with lambda
protein phosphatase New England Biolabs, which removes phosphates from proteins phosphorylated at ser-
ine, threonine or tyrosine residues Zhuo et al., 1993, to determine if any apparent hsphsc 70 isoforms
detected with immunoblotting were phosphorylated. Pro- thoracic glands were sonicated, as described above,
directly in phosphatase buffer [50 mM Tris–HCl pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01 Brij
35, 2 mM MnCl
2
with added protease inhibitors 0.2 mM phenylmethylsulfonyl fluoride, 1
µ gml aprotinin,
0.75 µ
gml pepstatin, 0.5 µ
gml leupeptin. Following sonication, samples were centrifuged for 3 min at 15,800
g at 4 °
C and the supernatant was incubated for 90 min at 25
° C with 400 units of lambda protein phosphatase
per 50 µ
l of supernatant 1 prothoracic gland equivalent per 10
µ l. Samples were then flash frozen and stored at
280 °
C until PAGE analysis.
3. Results
3.1. Isolation and sequence analysis of a hsc 70 cDNA clone from M. sexta
A Trichoplusia ni hsc 70 cDNA clone Schelling and Jones, 1996 was used to screen at low stringency i.e.
3 ×
SSC at 50 °
C 120,000 independent clones from a M. sexta prothoracic gland cDNA library. After two
rounds of rescreening, 20 putative hsphsc 70 clones were chosen, phagemids containing the putative positive
cDNAs excised and recovered, and the cDNA inserts subjected to reprobing, and restriction site mapping. Six
apparent clone types resulted but partial sequencing of these clones showed that they were identical to one
another. They were also highly homologous with the hsc 70-4 coding sequence of D. melanogaster Perkins et al.,
1990 and with the probe hsc sequence from T. ni Schelling and Jones, 1996. One of these clones phsc5
contained an insert of 2,124 base pairs, the largest among those characterized. Complete sequencing of phsc5
revealed an open reading frame of 652 amino acids Fig. 1, coding for a protein with a deduced molecular weight
of 71,431. The 85 bases preceding the putative initiation methionine contain no other initiation codon and the
CAAA immediately upstream of this presumptive trans- lation initiation site matches the consensus sequence for
an insect D. melanogaster initiation site Caverner and Ray, 1991. A single conventional polyadenylation sig-
nal AATAA occurs 54 bases after the stop codon TAA, and 13 bases separate this AATAA from the
polyA
+ tail sequence of this clone Fig. 1.
Comparisons of the translated sequence from clone phsc5 with D. melanogaster, T. ni and vertebrate hsp 70
and hsc 70 sequences are presented in Fig. 2 and Table 1. These analyses indicated that the sequence from M.
sexta is nearly identical to the hsc gene product of T. ni, which was used at low stringency to screen our library.
Like the T. ni sequence, the M. sexta hsc 70 deduced amino acid sequence shows the highest identity with the
Drosophila hsc 70-4 gene product. Overall, the differ- ences between the moth sequences and the Drosophila
sequence appear to be randomly spaced throughout their lengths, with a slight increase in frequency in the last
100 residues.
The deduced amino acid sequence from M. sexta hsc
582 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
Fig. 1. The nucleotide sequence and deduced amino acid sequence of Manduca sexta hsc 70 based on the sequence of the cDNA clone ptub-10.
The consensus polyadenylation signal AATAA is indicated by double underlining.
583 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
Fig. 2. Comparison of the deduced hsc 70 amino acid sequence from M. sexta Msexta with the hsc 70 from T. ni and hsc 70-4 from D.
melanogaster Dmhsc4. Double dots indicate identity with the sequence from M. sexta. and dashes indicate gaps required to align the sequences.
70 contains multiple potential sites for phosphorylation by cAMPcGMP-dependent kinases, protein kinase C
and casein kinase II [Prosite analysis Bairoch et al., 1995] and phosphorylation of proteins of the hsp 70
family has been reported from vertebrates tissues e.g., Egerton et al., 1996; Cvoro et al., 1999 However, treat-
ment of prothoracic gland extracts with lambda phospha- tase, which removes phosphates from proteins phos-
phorylated at serine, threonine or tyrosine residues Zhuo et al., 1993, failed to reveal any gel mobility shifts or
584 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
Table 1 A comparison of the deduced amino acid sequence of the M. sexta
cognate heat shock protein with hsp 70 and hsc 70 sequences from Trichoplusia ni, Drosophila melanogaster and humans
a
Identity with M. AAs
sexta hsc Trichoplusia hsc
98.2 653
Drosophila hsc 4 90.5
652 Drosophila hsc 1
82.1 654
Drosophila hsp 1 74.5
651 Drosophila hsp 2
74.3 649
Drosophila hsc 3 63.7
614 Drosophila hsc 5
50.5 604
Human hsc 71 86.7
652 Human hsp 70-1
82.5 652
a
The percent identity with the M. sexta sequence 652 AAs is given in the middle column while the right column indicates the num-
ber of amino acids in the compared sequence.
other changes using immunoblot detection. In the same samples, dephosphorylation-dependent band mobility
shifts were readily detected for the ecdysone receptor components EcR and USP, as described previously by
Song and Gilbert 1998. Prosite analysis also revealed the presence of a presumed ATPGTP binding site
AEAYLGKT, at residues 131–138, that has been termed the “P-loop”: Saraste et al., 1990 and which is
to be expected in a hspc 70 protein.
3.2. The expression of M. sexta hsc 70 in prothoracic glands and other tissues of M. sexta
Northern and immunoblot blot analyses were used to assess the abundance of hsc 70s in prothoracic glands
relative to other tissues. Fig. 3 shows that the Manduca hsc 70-4 cDNA hybridizes strongly with an mRNA of
about 2.4 kb in all tissues probed. In Drosophila, hsc 70-4 mRNA is 2.3 kb Perkins et al., 1990, and the
Trichoplusia hsc 70-4 homologue mRNA is about 2.0 kb Schelling and Jones, 1996.
The expression of hspc 70 proteins in these same tissues was also assessed using an antibody that reacts
with many cognate and stress-inducible hspc 70 pro- teins. These data show that several hsc 70 proteins
appear to be expressed in most if not all non-stressed tissues Fig. 3. All tissues surveyed express an hsc of
70 kDa and most also express a presumptive “hsc 70” of 72 kDa. Several tissues show additional immunoreac-
tivity at 66 and 62 kDa e.g., fat body and gut while Malphigian tubules contain a 71 kDa putative hspc 70
family member. The identification of these immunoreac- tive proteins as member of the hspc 70 is tentative and
we can not rule out the possibility that the lower molecu- lar weight proteins are proteolytic fragments of one or
more of the larger proteins. Heat shock of prothoracic glands in vitro 42
° C for 1–2 h followed by Northern
Fig. 3. The tissue distribution of hsc 70 mRNA as determined by
Northern blot hybridization 5 µ
g total RNAlane: lower panel and hsc 70 protein as determined by immunoblot analysis 5
µ g
proteinlane: upper panel. The bottom panel shows the amount of ribosomal RNA loaded per lane ethidium staining, as a control for
loading variation. All tissues were from V
4
larvae. Abbreviations: PG, prothoracic gland; M, Malphigian tubules; G, thoracic ganglia; Br,
brain; SG, salivary gland; FB, fat body; Ep, epidermis; Gut, midgut; rRNA, ribosomal RNA.
analysis or immunoblotting revealed that the mRNA and hsc 70 protein changes after such treatment were insig-
nificant to modest; per gland, heat shock increased hsc 70 mRNA only 13
± 20 and hsc 70 protein
increased 68 ±
18 relative to 25 °
C controls. Previous work demonstrated that the levels of hsc 70
protein in the prothoracic gland change during late larval and early pupal development in a manner that suggests
a role for hsc 70s in ecdysteroidogenesis andor cell growth Rybczynski and Gilbert, 1995a. Changes in the
hsc 70 mRNA concentration in the prothoracic gland during the fifth larval instar and during early pupal-adult
development were assessed using Northern blot analysis Fig. 4A. Hsc levels were expressed per
µ g protein or
RNA because the prothoracic gland cells undergo a large change in cell size during the developmental period stud-
ied, e.g.,
10-fold change
in protein
content Rybczynski and Gilbert, 1995b, making per gland
comparisons difficult to interpret. Hsc 70 mRNA and protein levels appear well correlated during the fifth lar-
val instar but diverge somewhat in early pupal-adult development. The maximum hsc 70 mRNA level, per
µ g RNA, was found on V
5
while the maximum hsc 70 protein per
µ g protein occurred on P
. Both hsc 70 mRNA and protein showed a minimum on V
6
, just prior to the late fifth instar peak of ecdysteroidogenesis that
occurs on V
7
. Developmental profiles of hsc 70 mRNA
585 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
Fig. 4. Hsc 70 mRNA and protein levels during the fifth larval instar
and early pupal-adult development. Data are derived from densito- metric scans of immunoblots or autoradiographs of Northern blots
probed with an anti-hsc 70 monoclonal antibody or with the radiolab- eled M. sexta hsc 70 cDNA, respectively. Levels are expressed as a
percentage of the maximum concentration measured during this devel- opmental period. A hsc 70 mRNA and hsc 70 protein protein data
from Rybczynski and Gilbert, 1995a in the prothoracic gland. B hsc 70 mRNA in the brain and fat body. Tissues from at least five animals
were pooled for each time-point. Two separate series of prothoracic glands were collected for Northern blots and for the immunoblots.
Note that the actual concentration of hsc 70 mRNA per
µ g RNA com-
prising the peaks is tissue-specific see also Fig. 3. The onset of larval wandering is denoted by the arrowhead.
levels were also determined for two non-steroidogenic organs, brain and fat body Fig. 4B. Neither organ
shows a pattern of hsc 70 mRNA expression matching that seen in the prothoracic gland. In brain, hsc 70
mRNA peaks occur just after the molt from fourth to fifth larval instar V
1
and just after the larval-pupal molt P
and P
1
. In fat body, hsc 70 mRNA levels are highest just before, and shortly after, the larval-pupal molt. Earl-
ier work indicated also that hsc 70 protein levels in the prothoracic gland were not correlated with levels meas-
ured in brain and fat body Rybczynski and Gilbert, 1995a.
3.3. Hormonal regulation of hsc 70 mRNA in the prothoracic gland
The possibility that the developmentally-specific changes in hsc 70 mRNA described above were hor-
monally controlled was explored in a series of in vitro and in vivo experiments using partially purified PTTH,
20E and hydroprene, a slowly metabolized JH analogue. Two developmental periods were investigated, the first
being the three days centered on the commitment peak V
3
in our colony. At this time a PTTH-stimulated small increase in circulating ecdysteroids occurs in the relative
absence of juvenile hormone and results in the “commit- ment” of larvae to develop into pupae at the next molt
Riddiford, 1976. The second period chosen was the first day of pupal-adult development, a period charac-
terized by extensive remodeling of the organism to pro- duce an adult moth.
Short-term 2 h exposure in vitro of prothoracic glands to PTTH resulted in a decrease in hsc 70 mRNA
levels on V
3
but no changes were noted with V
4
or P glands Fig. 5A. However, a longer exposure 14 h to
dibutyryl cAMP, which mimics the effects of PTTH Rybczynski and Gilbert, 1994, resulted in decreased
prothoracic gland hsc 70 mRNA levels at all three stages studied Fig. 5B. Dibutyryl cAMP was used to conserve
PTTH because of the larger incubation volumes employed 5 ml vs 25
µ l during these longer incu-
bations. Small incubation volumes may yield results reflecting the influence of ecdysteroids produced by the
gland during the incubation rather than the direct effect of PTTH.
When V
2
prothoracic glands were exposed for 20 h in vitro to 150 nM 20E, a level normally seen at the V
3
commitment peak Grieneisen et al., 1993, hsc 70 mRNA levels decreased by more than 60 Fig. 5C.
Exposure of P prothoracic glands to 1
µ M 20E, a level
present normally at stages P
3
to P
4
Warren and Gilbert, 1986, also resulted in a large drop in prothoracic gland
hsc 70 mRNA levels 40: Fig. 5C. The purpose of these 20E experiments was to determine the steroid’s
possible effects on hsc 70 mRNA levels but the effect of 20E on hsc 70 protein was also addressed briefly. V
5
prothoracic glands incubated for 10 to 24 h with 10 µ
M 20 E, a physiological concentration on V
7
, respond by changing expression of the ecdysone receptor proteins
EcR and USP, an event that is associated with down- regulation of ecdysteroid synthesis Song and Gilbert,
1998. Hsc 70 protein levels in glands so treated decline considerably 44
± 3 of control in line with the hypo-
thesized negative feedback of 20E on ecdysteroid syn- thesis Song and Gilbert, 1998.
Application of hydroprene just prior to the commit-
586 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
Fig. 5. The effects of PTTH, dibutyryl cAMP and 20E on prothoracic
gland hsc 70 mRNA levels in vitro. A hsc 70 mRNA levels after 2 h in vitro incubation with PTTH 0.25 brain equivalents B hsc 70
mRNA levels after 14 h in vitro incubation with 5 mM dibutyryl cAMP. PTTH stimulates cAMP production and dibutyryl cAMP mim-
ics the effect of PTTH on prothoracic gland protein synthesis and ecdy- steroid synthesis. C hsc 70 mRNA levels after 20 h incubation with
20E in vitro. V
2
and P prothoracic glands were incubated with 150
nM 20E and 1 µ
M, respectively. In these experiments, one prothoracic gland from an animal received the experimental treatment while the
other gland served as a control.
ment peak early V
3
retards development Rountree and Bollenbacher, 1986 and a topical application of 1.5
µ g
hydroprene delayed larvae by about two days in reaching the aphagic, wandering phase data not shown. No dif-
ference was found between control and treated protho- racic gland hsc 70 mRNA levels when animals were
sampled 24 h after the topical application Fig. 6 but a large increase seen 48 h later in control animals was
clearly inhibited by hydroprene application. Incubation of prothoracic glands in vitro 2–24 h with a similar
concentration of hydroprene did not result in detectable changes in hsc 70 mRNA levels data not shown.
4. Discussion
