The Effect of 20-Hydroxyecdysone on Expression of Genes Coding for Low Molecular Weight Silk Proteins of Galleria mellonella KRYSTYNA GRZELAK,* BARBARA KLUDKIEWICZ,* ZOFIA LASSOTA*
Received 27 November 1991; revtsed and accepted 24 September 1992
The low molecular weight GaUeria silk proteins of 24 and 29-30 kDa are coded by the 1100 nucleotides long mRNAs of the posterior silk gland (PSG) cells. The synthesis of these proteins starts during the first 2 days of the last instar when the endogenous ecdysteroid titre is very low. The synthesis of 24 kDa protein precedes the synthesis of 30 kDa protein. PSGs from day 1 last instar larvae are sensitive to exogenous 20-hydroxyecdysone (20-HE) in vitro at 0.5 pg/ml concentration causing an increase of 1100 nucleotides long mRNAs. The electrophoretic analysis of the in vitro labelling proteins indicates that 20-HE may induce the synthesis of 30 kDa protein. Juvenile hormone II in vitro suppresses the stimulatory effect of 20-HE. PSGs from day 3 last instar larvae are insensitive to exogenous 20-HE in vitro.
The control of expression of silk protein genes is not yet understood. In B o m b y x mori, ecdysteroids and juvenile hormones affect transcription of the silk protein genes and stability of their mRNAs (DaiUie, 1979; Couble et al., 1983; Tripoulos and Samols, 1986).
Larvae of Galleria mellonella produce in the posterior silk glands (PSG) 24 and 29-30 kDa silk proteins which appear to be analogous to the low molecular weight silk proteins of B. mori (Tokutake, 1980; Shimura et al., 1982; Couble et al., 1983, 1985; Prudhomme et al., 1985) but they do not bear any significant nucleot~de sequence homology (Grzelak et al., 1988). These proteins appear to be covalently linked by disulfide bonds to the fibroin. Galleria PSG polyadenylated RNA contains an abun- dant class of about 1100 nucleotide long RNAs which seem to include messengers for 24 and 29-30 kDa pro- teins (Grzelak et al., 1988).
In the present paper, we describe studies on the role of 20-hydroxyecdysone (20-HE) in activation of low molecular weight silk protein genes in PSG of Galleria in vitro. The interference of juvenile hormone II (JH II) with the stimulatory effect of 20-HE is also described.
MATERIALS AND METHODS lnsects
Wax moth, G. mellonella (Lepidoptera: Pyralidae) was reared on a standard diet at 29°C as reported by Sehnal
*Institute of Blochenustry and Biophysics, Polish Academy of Sciences, Rakowlecka 36, Warszawa, Poland.
(1966). Last instar larvae, 20--24h after their ecdysis were regarded as day 1, 24 h later as day 2 and so on. Silk glands were dissected under Ringers from larvae of different stages of the last instar and were used immedi- ately or were frozen in liquid nitrogen.
R N A extraction
Total RNA was extracted from fresh as well as from frozen PSG by the LiCl-urea method of Auffray and Rougeon (1980). Poly (A) RNA was isolated on oligo- dT-cellulose according to Aviv and Leder (1972). Low salt released poly(A)-containing RNA was ethanol pre- cipitated and submitted to a second cycle of oligo-dT- cellulose chromatography after heating at 65°C in low salt buffer in order to remove contaminating ribosomal RNA. Further fractionation of Galleria PSG mRNA was carried out on a linear 5-20% sucrose gradient according to Couble et al. (1981).
Synthesis o f complementary D N A
DNA complementary to 10S--16S fraction of PSG mRNA from day 5 larvae was synthesized according to Maniatis et al. (1982) as previously described (Grzelak et al., 1988). Specific activity of 3Zp-labelled cDNA was from 1.08 x 106 to 1.5 x 107 cpm/pg.
Northern analysis
"Nor thern" blots were performed according to Mani- atis et al. (1982). The formaldehyde-formamide de- natured total PSG RNA (20°°25/zg) was electrophoresed in 1.5% agarose gel and transferred onto nitrocellulose
211
212 K R Y S T Y N A G R Z E L A K et al
filters. Hybridizations were made with 32p-labelled cDNA as previously described (Grzelak et al., 1988). Filters were washed stepwise with two changes of 2 x SSC containing 0.1% SDS for 30 min each at room temperature and then three times in 1, 0.3, and 0.1 x SSC containing 0.1% SDS for 30mln each at 55°C with gentle agitation. Filters were exposed to X-ray films at -70°C using intensifying screen. Each experiment was repeated at least once with RNA extracted from a different batch of PSG.
Dot blot hybridization
Total PSG RNA was denatured at 65°C for 10 min in 10raM Tris buffer, pH 8.0 containing 0.5% SDS and was hand-spotted on Hybond-N membrane (Amersham). Each sample was serially diluted to spot 4, 2, 1 and 0.5/~g of RNA. The hybridization conditions and washing were as described above for Northern blots.
Purification of 1100 nucleotide PSG mRNAs
Urea-denatured PSG mRNA was electrophoresed in 1.5% agarose and the position of the 1100 nucleotide PSG mRNAs was located with reference to size markers. The class of 1100 nucleotide mRNA was eluted electro- phoretically and ethanol precipitated.
Cultivation of silk glands & vitro and hormone treatments
The PSG were dissected from water anaesthetized larvae and rinsed in cold Grace's medium. Five pairs of PSG were transferred to 0.5 ml fresh medium in siliclad- treated glass vials according to Sehnal et al. (1987). The silk glands were incubated at 29°C for 3 h (normal atmosphere, no shaking).
20-hydroxyecdysone (Sigma, 20-HE) and juvenile hormone II (Sigma, JH II) were dissolved in Grace's medium and acetone respectively. Experimental samples received 20-HE (0.5/~g/ml) or 20-HE and JH II (1 #g/ml) simultaneously in the volume of 2.5#1. Un- treated or acetone-treated silk glands were used as controls.
In vitro labelling and analysis of silk proteins
PSG were incubated with [14C]leucine (211 mCi/mmol) or [14C]amino acid mixture (50 mg Ci/mg atom C) in vitro as described above. 1/zCi of [~4C]leucine in 5 #1 or 2.5/zCi of [J4C]amino acid mixture in 50~1 per 0.5ml medium was used. mac-labelled proteins were extracted from PSG by 8 M urea, 2% SDS and 5% fl-mercap- toethanol, then heated at 100°C for 3rain and elec- trophoresed in 10% polyacrylamide SDS gels according to Laemmli (1970). Gels were treated for fluorography according to Bonner and Laskey (1974), then autoradio- graphed and quantified by an LKB laser densitometer.
5/~1 of 14C-labelled PSG protein extract was spotted on Whatman 3mm filter paper, precipitated with trichloroacetic acid (TCA) and counted in LKB 1209 scintillation counter. Equal counts were then run on gel.
Analysis of cell-free translation products
Wheat germ S-23 extract was prepared by the method of Morch et al. (1986). The translation mixture (25 #1) was as described previously (Grzelak et al., 1988). 2.4-6.0/~g of RNA was tested per assay. The translation product was denatured and electrophoresed in 15% polyacrylamide SDS gel. Gels were treated as described previously.
RESULTS
Developmental pattern of low molecular weight silk pro- teins m early stages of the last larval instar
The 24 and 30 kDa silk proteins of Gallerta were previously shown to differ in amino acid composition (Grzelak et al., 1988). The 1100 nucleotides long RNA class of PSG polyadenylated RNA from day 5 last instar larvae translated in wheat germ cell-free system with [14C]leucine yielded both 24 and 30 kDa polypeptides (Fig. 1). This result confirms our earlier suggestion (Grzelak et al., 1988) that this size class poly (A)RNA contains mRNAs for both 24 and 30 kDa silk proteins. However, in isolated PSG from day 1 larvae during incubation with [~4C]leucine [Fig. 2(1)] as well as [t4C]amino acid mixture (data not shown) the label could be found only in 24 kDa protein and in an umdentified 14 kDa protein while 30 kDa protein was not detectable. In PSG from day 2 and day 3 larvae both 24 and 30 kDa
- - 6 9
24 kD -
- 4 6
- 3 0
- 1 4 3
F I G U R E I. TranslaUon of 1100 nucleotldes long Gallerla PSG m R N A s m a wheat germ cell-free system Autora&ogram corresponds to the cell-free [~4C]leucme-labelled proteins translated from agarose purified i 100 nucleotide long m R N A s originated from day 5 last mstar larvae Proteins were denatured m SDS and fl-mercaptoethanol prior to electrophorests m a 15% polyacrylamtde gel Size of marker proteins (lysozyme, carbonic anhydrase, ovalbumm and bovine serum albumin,
respectively) are expressed m kdodaltons
ECDYSONE-DEPENDENT GENE EXPRESSION 213
6 9 -
4 6 -
3 0 -
14.,3 - -
""=- 30 kO
W .,e- 24 kD
a b c
o
b
6 9 - -
4 6 - -
3 0 -
14.3 -
I II
-L
b c
FIGURE 2. The low molecular weight proteins in PSG of Gallerta larvae at early stages of the last instar (I) 14C-labelled silk proteins in PSG of day 1 (a), day 2 (b) and day 3 (c) last mstar larvae of Gallerla. PSGs were incubated m vttro with [14C]leuclne for 3 h at 29°C, Proteins extracted from PSG as described in Materials and Methods, were analysed m 10% polyacrylamlde gel, Equal amounts of radioactive material (9000 cpm) were loaded into each slot of the gel Autoradiograms were analysed by densltometnc scanning. The fragment of the scans in wluch the low molecular weight proteins regions occur is displayed in (I). (II) [~4C]leucine-labelled proteins translated with PSG mRNAs of day I (a) day 2 (b) and day 3 (c) in a wheat germ cell-free system Equal amounts of radioactive material (2000 cpm) were loaded into each slot of the gel. Proteins were
denatured and separated in the 15% polyacrylamlde gel Marker proteins are as in Fig 1
,,t- 30 kD
4'=-24 kD
proteins were labelled, the ratio of 24:30 kDa bands was 4:1 during this period. When mRNAs from PSG of day 1, day 2 and day 3 Galleria larvae were translated in wheat germ cell-free translation system with [~4C]leucine [Fig. 2 (II)], 30 kDa proteins were not de- tectable in day 1 preparations suggesting that the gene for 30 kDa silk protein is not expressed during the first day of the last larval instar.
Effect of 20-HE on expression of genes coding for low molecular weight silk proteins
Since Sehnal et al. (1981) showed that ecdysteroids titres are low in day 1 and day 2 last instar larvae and increase on day 3 we studied the effect of exogenous 20-HE on the synthesis of mRNAs for low molecular weight proteins in isolated PSG in tissue culture and the data are presented in Fig. 3. Northern blot analysis of total RNA from PSG of day 1 larvae shows that the level of 1100 nucleotide transcripts is increased by 20-HE [Fig. 3(A)]. These RNAs were more abundant in hor-
mone treated (lane 2) than in control silk glands (lane 1). This is consistent with dot hybridization [Fig. 3(B)]: the dot blots from the hormone-treated PSG from day 1 last instar larvae (row 2) were more intense than those in untreated controls (row 1). However, the effect of 20-HE on 1100 nucleotide PSG poly (A) RNA in day 2 larvae (rows 3 and 4) is less than that in day 1 larvae and in PSG from day 3 larvae 20-HE does not affect the level of transcripts (see rows 5 and 6). The effect of 20-HE on silk gland gene expression was confirmed when control and hormone treated RNA was probed with PSG- specific clone, designated PG-1 (Zurovec et aL, 1992) containing transcripts for small silk proteins [Fig. 3(B) rows 7 and 8]. The effect of exogenous 20-HE on the incorporation of [14C]leucine into proteins of PSG cul- tured in vitro is shown in Fig. 3(C). In hormone-treated PSG from day 1 larvae the label was found in both 24 and 30 kDa proteins while no labelled 30 kDa protein was seen in untreated control silk glands. The scans confirm the presence of labelled 30 kDa protein in
214 KRYSTYNA GRZELAK et al
-~rr r 1
1 2
- ~ - 2 8 S
- 2 3 S
- 1 8 S
- 1 6 S
+ 20-HE
3
4
0 5 1 2 4 ~g
" ~ T r 1
{' +20-HE
"~rn" 2
+ 20-HE
"~TTT 3
÷ 20 - HE
,oOo "~rl'l" I
+20- HE
69- -
46- -
30--
14 3 --
"~TTl" 1
+20-HE
30 kD
" - 2 4 kD
( A ) (B ) (C) FIGURE 3 Effect of 20-hydroxyecdysone on the level of low molecular weight proteins mRNAs [(A) and (B)] as well as on the m vttro synthesm of silk proteins (C) m PSG of Gallerta larvae at early stages of the last instar. PSGs were dissected from day 1 [(A) lanes 1 and 2, (B) rows 1, 2, 7, 8, and (C) lanes 1 and 2], day 2 [(B) rows 3 and 4] and day 3 [(B) rows 5 and 6] last mstar larvae and incubated m vitro without [(A) lane 1, (B) rows 1, 3, 5, 7 and (C) lane 1] or with 20-HE [(A) lane 2, (B) rows 2, 4, 6, 8] for 3 h at 29°C Total RNA was either blotted on mtrocellulose (A) or hand-spotted on Hybond-N membrane (B) and hybn&zed with Gallena [32p]PSG cDNA or with PG-1 [(B) rows 7 and 8] clone (Zurovec et al, 1992) ~4C-labelled silk proteins (C) were extracted and analysed on the gel as described m Matenals and Methods Equal amounts of ra&oactwe material (6000 cpm) were loaded into each slot of the gel Hela cell 28 S and 18 S rRNA and E coh 23 S and 16 S rRNA were used as molecular weight standards (A) Size of marker proteins (C) as m Fig 1 Note the hybn&zatlon mgnal (arrow) over
1100 nucleotlde mRNA species m (A) lanes 1 and 2
h o r m o n e t rea ted P S G of day 1 larvae and the mcreased intensi ty o f 24 and 30 k D a pro te in bands in h o r m o n e t rea ted P S G o f day 2 larvae (Table 1). Thus exogenous 20-HE induces the express ion o f 30 k D a pro te in gene and increases the level o f synthesis o f bo th 24 and 30 k D a proteins.
Influence o f juvenile hormone on the effect o f 20 -H E on expression o f genes coding the low molecular weight silk protems
The results on in te rac t ion o f JH II with the effect o f 20-HE on the synthesis o f low molecu la r weight pro te ins in P S G cul tured m vitro are presented m Fig. 4. N o r t h e r n blot analysis o f to ta l R N A from P S G of day 1 larvae shows that the band o f 1100 nucleot ide R N A s in prep- a ra t ions t rea ted s imul taneous ly with bo th 20-HE and JH II is the same as that in the con t ro l ones [Fig. 4(A)]. This
lS consis tent with do t hybr id iza t ion da t a [Fig. 4(B)]. The intensi ty o f the a u t o r a d i o g r a p h s remained unchanged in the unt rea ted (row 1), acetone t rea ted (row 2) and JH- t r ea t ed silk g lands (row 5) as well as in the 20-HE plus JH I I - t rea ted silk g lands (row 3). Thus juveni le ho rmone in vitro suppresses the s t imula to ry effect o f the exogenous 20-HE in silk g lands f rom day 1 last ins tar larvae.
DISCUSSION
Previously we have demons t r a t ed (Grze lak et al., 1988) that the low molecu la r weight pro te ins o f Galleria silk and their messengers are p roduced m the pos te r ior pa r t of the silk gland. The d a t a presented in this paper reveal tha t the expression o f indiv idual genes coding for these pro te ins does not s tar t s imul taneously . So dur ing
ECDYSONE-DEPENDENT GENE EXPRESSION 215
TABLE 1. 20-HE reduced changes m the synthesis of low molecular weight sdk pro- terns of Gallena larvae m early stages of the
last mstar
Absorption units*
Treatment 30 kDa 24 kDa
Day 1 Control - - 0 499 20-HE 0.178 0.971
Day 2 Control 0 244 0 890 20-HE 0 358 1.246
*Numbers in the table represent absorption units obtained by dens]tometnc scanning of the autoradlogram presented m Fig 2 and of the autora&ogram of [~4C]leuclne labelled proteins of hormone-treated PSG of day 2 larvae (not shown)
the first day of the last larval instar mRNA for 24 kDa protein is synthesized and translated while the gene coding for 30 kDa protein is silent (Fig. 2). On day 2 the genes for both 24 and 30 kDa protems are expressed and the process continues on days 3 and 5 of the last instar (Figs 1 and 2).
According to Sehnal et al. (1981) and Sehnal and Michalik (1984) the first 2 days after last larval ecdysis represent the development of Galleria silk gland in the preparatory phase characterized by low silk production and low level of ecdysteroids (about 20 ng ecdysteroid per 1 g of larval body weight). Only on day 3 does the level of ecdysteroid increase (to 65 ng per g) initiating the onset of metamorphosis.
In the present study we demonstrate that in the silk gland from day 1 larvae the treatment in vitro with 2 x 10 -6 M 20-HE induces the expression of the gene for 30 kDa protein [Fig. 3(C)]. The effect is clearly visible after 3 h of exposure pointing to the direct action of hormone. In our in vitro system the incorporation of radioactive precursors into RNA and proteins remains linear during at least 3 h (Sehnal et al., 1987). The a p p e a r a n c e o f e cdys t e ro ld - i nduc ib l e p ro t e in s m vitro was
n o t e d in K c - H cells o f Drosophi la wi th in 1 h (Savak is
et al., 1980) a n d m la rva l ep ide rmis o f Tenebrto wi th in
2 h (Oue l le t t e and C a v e n e y , 1990) a f te r the e x p o s u r e to
h o r m o n e . T h e sensi t iv i ty o f Galleria g lands to e x o g e n o u s
2 0 - H E po in t s to the p resence o f f unc t i ona l r ecep to r s fo r
the h o r m o n e in this t issue. D e a k et al. (1988) r e p o r t e d
tha t in Drosophi la l a rvae the level o f 2 0 - H E recep to r s
increases wi th in 2 h a f te r inges t ion o f the h o r m o n e . T h e
I 2
20-HE
+ JHII
--28S
0.5
- 2 3 S
- 1 8 S 2 0
- 1 6 S
3 0
4
I 2 4 /zg
",,- 20 -HE + J HII
.4- + JHII
(A) (B)
FIGURE 4 Effect of 20-hydroxyecdysone and juvende hormone II on the level of low molecular weight proteins mRNAs m PSG of day 1 last mstar Gallena larvae PSG were incubated ,n vttro m the presence of 20-HE and JH II [(A) lane 2 and (B) row 3] Untreated [(B) row 1], acetone-treated PSG [(A) lane 1 and (B) rows 2 and 4] and JH-treated [(B) row 5] were used as controls Gallerta PSG total RNA was either blotted on mtrocellulose (A) or hand-spotted on Hybond-N membrane (B) and hybn&zed with Gallena [~2p]PSGcDNA or with PG-1 [(B) rows 4 and 5] clone (Zurovec et al., 1992) Size of markers
(A) as m Fig 3 Note the hybn&zatlon signal (arrow) over 1100 nucleot]de mRNA species m (A) lanes 1 and 2.
216 KRYSTYNA GRZELAK et al.
ecdysteroid-dependent induction of the gene for 30 kDa protein in the gland tissue in vitro is repressed in the presence of 4 x 10-6M exogenous juvenile hormone (Fig. 4). The endogenous JH II content of the Galleria larval body amounts to 0.3 and 0.15pmol per 1 g of body weight on days 1 and 2 of the last larval instar respectively (Rembold and Sehnal, 1987). Thus in vivo the expression of the gene for 30 kDa protein is probably negatively regulated by the endogenous juvenile hor- mone on day 1 and the gene is expressed on day 2 when 20-HE is still present and the level of JH II decreases below the critical level.
Beginning with day 3 of the last instar the JH II vanishes and the effect of 20-HE dominates enabling the efficient synthesis of the low molecular weight silk proteins.
The antagonism of juvenile hormone and ecdysteroid was observed in wvo in fat body of Galleria (Grzelak and Kumaran, 1985), in vivo and in vitro m the epidermis of Manduca sexta (Rlddiford, 1986) and in Drosophila Kc cells (Cherbas et al., 1989). According to Michahk and Sehnal (1989) juvenile hormone in vitro does not affect total RNA synthesis in Galler~a PSG from first 2 days of last instar. However, the results of these authors are hardly comparable with ours: they used silk glands from hgated larvae (probably lacking hormones and recep- tors) and the effects were studied 20-24 h after hormone application. Juvenile hormone, on the other hand, in vivo reduced total RNA synthesis in Galleria PSG acting selectively on the synthesis and/or turnover of only certain classes of RNA when applied topically during the preparatory phase (Michalik and Sehnal, 1989). This observation confirms the effects we have observed in vitro.
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Acknowledgements--The authors thank Professor A K Kumaran for helpful comments on the manuscript We acknowledge the gifts of the PG-1 probe by Dr Zurovec of Institute of Entomology Czechoslovak Academy of Sciences, (~eske Budejovlce, Czechoslovakia
