580 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
The function of hsc 70 in PTTH-stimulated ecdystero- idogenesis has proven difficult to determine. Hsc 70 pro-
teins participate in a variety of cellular processes in addition to their roles in protein folding and import into
organelles see Mayer and Bukau, 1998; Fink, 1999. For instance, hsc 70s are also involved in clathrin
uncoating Chappell et al., 1986 and in vertebrate ster- oid hormone receptor assembly and function see Pratt
and Toft, 1997. Hsc 70s are also abundant proteins such that PTTH-stimulated changes in abundance must be
measured against a very high background. For these reasons, a molecular approach was chosen, with the hope
that determining the hsc 70 isoform distribution in the prothoracic gland and other tissues during development
and under in vitro PTTH stimulation might afford further insight into the relationship between ecdysteroid syn-
thesis and hsc 70 synthesis. The data indicate that the chief hsc 70 expressed in the prothoracic gland is a Man-
duca homologue of the hsc 70-4 gene product of Droso- phila Perkins et al., 1990. The translation of this hsc
70 undergoes short-term up-regulation by PTTH in the gland but longer exposure to PTTH or PTTH analogues
and to 20-hydroxyecdysone 20E result in down-regu- lation of both protein and mRNA levels, suggesting a
role in the negative feedback control of ecdysteroid receptor complex assembly in the prothoracic gland see
Gilbert et al., 1997.
2. Methods and materials
2.1. Animal rearing, prothoracic gland dissections and in vitro manipulations
M. sexta were reared and staged as described pre- viously Rybczynski and Gilbert, 1995a. The 24 h fol-
lowing the molt to the fifth instar is designated as fifth instar day 1 V
1
, while the 24 h following the pupal molt is designated as pupal day 0 P
. Subsequent 24 h periods are numbered successively, e.g., V
2
, P
1
etc. Following dissection under insect Ringers Weevers,
1966, glands to be treated with PTTH or other agents were transferred to spot test plate wells containing Gra-
ce’s medium and pre-incubated at 25 °
C for 30–45 min. The medium was then removed and replaced rapidly by
25 µ
l incubations2 h. For overnight incubations 14– 20 h, the glands were transferred to small Petri dishes
containing 5 ml Grace’s medium. All incubations were performed under high humidity conditions at 25
° C. For
in vitro experiments, one of the two prothoracic glands present in each animal received the experimental treat-
ment while the other gland served as a control. Control glands were incubated for the same time, in the same
volume, and also experienced the same solvents or buf- fers, as the experimental glands. The juvenile hormone
JH analogue hydroprene gift of ZoeconNovartis was dissolved in acetone 1.5
µ g15
µ l and applied to the
dorsal surface of day 3 fifth instar V
3
larvae weighing 6.0 to 6.5 g. This dose of hydroprene consistently
resulted in a 24 h delay in pupal commitment Rountree and Bollenbacher, 1986; Rybczynski and Gilbert, unpub-
lished observations.
2.2. Prothoracicotropic hormone PTTH PTTH extracts were prepared from P
1
brains of M. sexta homogenized in methionine-free Grace’s medium
at 4 °
C 10 µ
lbrain followed by boiling and centrifug- ation Bollenbacher et al., 1979; Rybczynski and Gilbert,
1995a. The resultant supernatant contains big PTTH 25,000 kDa and small PTTH ,10,000 kDa
Bollenbacher et al., 1984 which were separated by three cycles of ultrafiltration YM10 filter: Amicon,
Beverly, MA after which the big PTTH concentrate was brought up to the starting volume with methionine-free
Grace’s medium estimated 1,000-fold dilution of small PTTH concentration in the big PTTH fraction. Methion-
ine-free Grace’s medium was used to make experiments consistent with earlier studies in which
35
S-methionine labeled protein synthesis was studied e.g., Rybczynski
and Gilbert, 1994; Rybczynski and Gilbert, 1995a and PTTH aliquots were stored at 280
° C and discarded after
two freeze-thaw cycles. 2.3. RNA isolation and construction and screening of
a prothoracic gland cDNA library Tissues were extirpated under insect Ringer at 25
° C,
frozen rapidly in pre-chilled 270 °
C microfuge tubes and stored at 280
° C. Total RNA was isolated by either
the hot phenol method or the SDS–urea method Jowett, 1986.
An hsc 70 cDNA clone isolated from the moth Tricho- plusia ni and containing the entire coding sequence [gift
of Dr Grace Jones, University of Kentucky, Lexington, KY Schelling and Jones, 1996] was used to screen an
unamplified prothoracic gland library as described pre- viously Rybczynski and Gilbert, 1998. After two
rounds of screening, Bluescript phagemids containing the putative positive cDNAs were excised and recovered
according to the manufacturer’s instructions Stratagene, La Jolla, CA. DNA was sequenced at the University of
North Carolina at Chapel Hill Automated Sequencing facility on a Model 373A DNA Sequencer Applied
Biosystems, Foster City, CA using the Taq DyeDeox- y
TM
Terminator Cycle
Sequencing Kit
Applied Biosystems. Overlapping sequences were obtained from
restriction fragment and nested deletion subclones Erase-a-Base System: Promega, Madison, WI. DNA
and deduced amino acid sequence analyses were perfor- med using the Genetics Computer Group program
Madison, WI. The Prosite program Bairoch et al.,
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
