- Email: [email protected]
Characterization of four pupal wing cuticular protein genes of the silkmoth Antheraea polyphemus
Insect Biochem. Molec. BioL Vol. 24, No. 3, pp. 291 299, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/94 $6.00 + 0.00
Pergamon
Characterization of Four Pupal Wing Cuticular Protein Genes of the Silkmoth
Antheraea polyphemus M. N. KUMAR,* S. SRIDHARA*t Received 5 October 1992; revised and accepted 24 June 1993
Three different clones have been isolated from a genomic library of the silkmoth Antheraea polyphemus by employing a subtractive hybridization technique. The clones with inserts of 13--16 kb of DNA each, code for mRNAs expressed in the wing epidermis during JH induced second pupal cuticle deposition. While two of the clones code for a single mRNA each, the third one codes for two mRNAs. All the four mRNAs code for distinct polypeptides that can be precipitated with antibodies raised against pupal cuticular proteins. These genes are activated at the same period of pupal development and their transcripts follow similar patterns of accumulation. Although these genes are expressed in a tissue and time specific manner attesting to their pupal wing epidermal specificity, three of them are expressed in the adult wing epidermis also, but not at the larval stage. While DNAs from other silkmoths and insects hybridize to these genes, only one of the A. polyphemus genes hybridizes to RNA from second pupal wings of two other silkmoths tested. Antheraea polyphemus protein genes
Silkmoths Cuticle Pupal development Wingepidermis mRNA
INTRODUCTION The nature of the cuticle secreted during the post embryonic development of the insects, whether larval, pupal or adult, depends upon at least two hormones: molting hormone (MH or 20-hydroxyecdysone or 20-HE) and juvenile hormone (JH) (Sridhara et al., 1978). The pupal stage of holometabolous insects is a fascinating period of development in that the hardened outer cuticle of a pupa (Lepidoptera) or a puparium (Diptera) functions as an outer case in which the transformation of the larva or maggot into the volant adult moth or fly takes place. Several studies concerning the larval-pupal transformation in Drosophila and Manduca demonstrate that the switch from larval to pupal expression requires subtle changes in 20-HE levels, which (directly or indirectly) are eventually responsible both for the repression of the larval cuticle genes and activation of the pupal cuticle genes (Doctor et al., 1985; Wolfgang and Riddiford, 1986; Wolfgang et al., 1986). The effects of 20-HE during these changes can be *Department of Biochemistryand Molecular Biology,Texas Tech University Health SciencesCenter, 3601 Fourth Street, Lubbock, TX 79430, U.S.A. tAuthor for correspondence. Abbreviations used: kb, kilo base(s); bp, base pair(s); SDS--PAGE, sodium dodecyl sulfate polyacrylamidegel electrophoresis; SSC, sodium chloride+ sodiumcitrate; PBS, phosphate bufferedsaline.
Cuticle
nullified or modified by JH (Riddiford, 1985; Hiruma et al., 1991). Drosophila imaginal discs which can be mass isolated and which respond to hormone regimens in defined culture conditions have been utilized to study hormonal regulation of larval-pupal metamorphosis, especially pupal cuticle formation (Fristrom, 1981; Fristrom et al., 1991). In Lepidoptera, however, such discrete groups of cells do not exist and changes occur sequentially in 'polymorphic cells' derived directly from the already differentiated larval cells (Riddiford, 1984). Studies of the hormonal regulation of pupal cuticle protein genes of lepidopterans are hindered by the difficulty to isolate such cells in large quantities and by the asynchronous acquisition of competency for larval-pupal molt by the different larval cells during normal larval-pupal metamorphosis. However, the epidermal cells of diapausing silkmoth pupae (Antheraea polyphemus, Hyalophora cecropia, etc.) circumvent some of these problems and provide good opportunities to study the molecular mechanisms involved in this regulation of metamorphosis by the two hormones. Transfer of the diapausing pupa (characterized by a low level of metabolism) from low temperature (4°C) to room temperature (23-25°C) results in the initiation of adult development as a consequence of a small rise in the titer of the molting hormone. Normally the pupal wing epidermis (and other epidermal cells) does not encounter JH and goes on to
291
292
M.N. KUMAR and S. SRIDHARA
develop and differentiate into adult wing structures in about 15-18 days. However, if JH is provided experimentally along with 20-HE, development towards the adult is suppressed and the program for the pupal cuticle is re-expressed. Consequently, a second pupal wing that is made up of proteins different from those of the adult wing is synthesized in about 6 days (Willis and Hollowell, 1976; Sridhara et al., 1980; Willis and Cox, 1984; Sridhara, 1985b). The ability of JH to set in motion the program for the second pupal development is limited to about the first 24h while the deposition of the pupal cuticle begins at about 4 days (Ruh et al., 1980; Sridhara, 1985b). At the time of transfer of diapausing pupae from the cold to the incubator and/or administration of the hormone, the tissue is neither engaged in cuticle synthesis nor has it made any cuticle during the several months it has been in diapause (Willis, 1981). This special situation whereby epidermal cells can be directed to take alternate developmental pathways has been utilized to study the cuticular proteins and related macromolecular changes in the silkmoth H. cecropia (Willis et al., 1981; Willis and Cox, 1984; Willis, 1987; Binger and Willis, 1990). The wing epidermal cells of silkmoths A. polyphemus and A. pernyi have been used in this laboratory to follow changes in RNAs and proteins during pupal and adult development. Some of the results pertinent to this report are: (i) new mRNAs which are absent at day 1 and that belong to the moderately abundant class appear during days 4-6 of second pupal development: (ii) these new messages can be translated into polypeptides whose mobilities in 2-D gels resemble those of pupal cuticular proteins: (iii) some of these messages are different from those making the adult wing cuticle and (iv) the 2-D patterns of the pupal and adult cuticular protein patterns are quite different although some of the proteins may be common to the two stages (Sridhara et al., 1980; Sridhara, 1985a, b, 1993). These results have provided opportunities to isolate genes that are expressed by the wing epidermis in a tissue and time specific manner. This report presents results on the characterization of four genes that are expressed in the pupal wing epidermis at the time of cuticular deposition. EXPERIMENTAL P R O C E D U R E S
Diapausing pupae of the silkmoths A. polyphemus, A. pernyi, Actius luna, and H. cecropia were obtained commercially and stored at 4°C. 20-HE and the juvenile hormone (t, t, c-JH1) were purchased from Calbiochem. The 20-HE was dissolved in water while JH was dissolved in olive oil. Development of the animals towards second pupae was initiated by removing the pupae from the cold, placing them in an incubator programmed for 16h light-8 h dark cycle and maintained at 23-25°C. The hormones (2/~g/g pupa of each hormone) were injected through an intersegmental membrane after 2 h of warming. Under these conditions the pupae develop second pupal cuticles by day 6. Pupae injected 20-HE
alone synthesize adult wing cuticle by day 15. The wing tissue (fore and hind wings from both sides) from developing pupae or adults was dissected and either processed immediately for extraction of RNA or stored at -80°C as a powder prepared in liquid nitrogen. Fat bodies from day 6 second pupae were also collected for RNA extraction. Tissues or corresponding RNA obtained during successive days of second pupal development are referred to as J l, J2, etc., while that obtained at day 15 of adult development is referred to as El5. RNA was also extracted from five to six dorsal and ventral segments of abdominal integuments of feeding fifth instar larvae (~ 10-12 g wt). Isolation of total RNA and poly (A +) RNA, preparation of cDNA, hybridization with mRNA, in vitro translation of mRNA and analysis of translation products, were carried out essentially as described earlier (Sridhara, 1985a, b, 1994). The recombinant DNA techniques followed standard procedures (Maniatis et al., 1982; Davis et al., 1986). The antibodies were obtained by immunizing rabbits with pupal cuticular proteins solubilized from cleaned second pupal wing cuticle following standard techniques (Johnstone and Thorpe, 1982). Both immune and non immune serum were processed by ammonium sulfate precipitation followed by hydroxylapatite chromatography. The antibodies were dialysed and stored in PBS containing 20% glycerol. Probe generation and screening of the genomic library A cDNA probe that is specific to the time period when pupal cuticle was being actively deposited was prepared as described earlier (Sridhara, 1985a, b, 1993) while employing ~ 3 2 p dCTP instead of 3H-dCTP. Briefly, about 108DPM of 32p cDNA was synthesized using poly (A +) mRNA derived from wing tissue on day 6 of second pupal development (J6), oligo dT and AMV reverse transcriptase. This cDNA was hybridized to saturation over 8 days with poly (A +) RNA derived from wing tissue 1 day after hormone administration (Jl). The unhybridized single stranded cDNA was recovered from a hydroxyapatite column. Elimination of the RNA with alkali and recovery of the cDNA from a Sephadex-50 column provided the desired probe. 2 x 105 recombinant phages of a A. polyphemus genomic library prepared in the vector EMBL-4 were screened with the probe. Four nitrocellulose lifts were made from each plate. Differential screening was carried out whereby two filters were hybridized to the probe while the other two were hybridized to a similar amount of 32p-labeled J1 cDNA. Hybridization and washing conditions are described below. Plaques that hybridized uniquely to the J6 specific cDNA probe were collected. Further selection and purification were carried out by similar differential screening with Jl and J6 cDNAs. Hybridization and washing Library screening and the other hybridizations (Northerns, dot blots, Southerns, etc.) were carried out
SILKMOTH PUPAL CUTICLE PROTEIN GENES in a solution of formamide 50%, SSC 5 x , phosphate buffer 50mM, Denhart solution 5 x , and sonicated denatured calf thymus D N A 100/tg/ml. Prehybridization was done overnight and hybridization for 24-48 h, both at 42°C. Washings were done successively in 2 × 2SSC at room temperature, 2 × 2SSC at 65°C, 2 × I SSC at 65°C, and 2 × 0.2SSC at 65°C. Occasionally the washing was stopped at 2 × 2SSC at 65°C (indicated at appropriate places).
Membranes and probes The majority of studies were carried out with nitrocellulose (BA85, Schliecher & Schuell) and probes prepared by nick translation with a - 3 2 P dCTP. Southern blots of genomic DNA were prepared with the BioRad Zeta Probe membrane and the probes were generated by the random primer labeling procedure (Feinberg and Vogelstein, 1983).
Hybrid selection and translation RNAs specific to the clones were isolated by hybridization of total poly (A ÷) RNA to denatured plasmid DNAs fixed on 5 m m dia Zeta Probe membrane discs for 48 h at 42°C. RNAs corresponding to individual clones were translated in a wheat germ cell free system in the presence of five 3H-amino acids (Sridhara, 1985a, b). The entire translation product was boiled with the sample buffer and analysed by SDS-PAGE. Alternately, the translation mixture was diluted with immune precipitation buffer, cleared with 25/~1 of
293
Protein A conjugated to agarose (Repligen Corp.) and mixed overnight with antibodies produced against pupal cuticular proteins. The mixture was treated with another 25/~1 aliquot of Protein A agarose for 3 h in the cold. The agarose was washed, the bound material eluted by boiling with sample buffer and analyzed by SDS-PAGE. The gels were stained, destained, and prepared for fluorography with Enhance (NEN). The dried gels were exposed to X-ray film for varying periods of time. RESULTS
Isolation of pupal specific genomic clones Earlier studies demonstrated that new mRNAs that probably coded for cuticular proteins, appeared in the wing epidermis during 4~6 days of JH induced second pupal development (Sridhara, 1985b, 1993). This information and the techniques developed were utilized here to obtain a pupal specific probe to screen a recombinant genomic library prepared in the laboratory. The specificity of selection was enhanced by screening alternate filters with J1 cDNA and the specific probe simultaneously. This screening procedure yielded 20 clones after three rounds of selection and purification. Three clones, each showing a different level of hybridization with J6 cDNA, were plaque purified and their DNAs were analyzed. These clones are designated as 2ApPcpl4, 15, and 112 (Lambda Antheraea polyphemus pupal cuticular protein clone).
Clone i 4 t~l W
n'~
rr
II
. tO) '~-
l II
I
"~
~
~
(/3 1,1
I
0
o WU
II
I
IS
~
o.~=
~
"-- W ~
II I Clone
"~
~
.
rv"
,., ~
.
"--~
o
.
t~J
I
~
f'r"
~ "rV
I II
~
iv"
tJ.IC,I taJX
~
I
re"
o
U t.t.l
II I
iv"
oo
U U t.~ taJ
I
II
clone I12
L~
.--
mm
¢~
U
f"
mmm
1 2kb
I
FIGURE 1. Restrictionmaps of the three genomicclones isolated by differentialscreening. The phage DNAs were subjected to digestion with single and multiple restriction enzymes and the maps arrived at by standard procedures. The fragment(s) hybridizing to the J6 cDNA are marked below each map (~). The DNA fragmentssubclonedfor further analysis are marked above (~-+).
294
M.N. KUMAR and S. SRIDHARA
Analyses of the clones The D N A insert in each clone had a distinct pattern by restriction analysis (Fig. 1). Southern blots of each of the three DNAs were probed with individually labeled phage DNAs. In each case the insert(s) hybridized with its own probe (Fig. 2). These results and the different restriction maps demonstrated that the insert in each clone was a different fragment of the genomic DNA. Dot blot analysis confirmed that the phage DNAs hybridized with J6 cDNAs. We probed strips of a Northern blot of J6 poly (A +) RNA with nick translated phage DNAs. It was seen that the first two clones identified one m R N A each while the third clone identified two mRNAs (Fig. 3). The m R N A signal for the clone 2 14 was quite faint in this blot, but was stronger in other blots (see later). The D N A segments corresponding to the specific mRNAs were identified by probing various Southern blots with J6 cDNAs. It was determined that the first two 2 clones contained one D N A segment each that hybridized to the cDNA while the third contained two such fragments (marked in Fig. 1). The combined results allowed us to conclude that each 2 clone contains 13-16kb of genomic D N A which includes smaller fragments that code for mRNAs expressed at day 6 of ~14 ~15
pupal development. These fragments, marked in Fig. l, were sub cloned into pUCI8 or Bluescript plasmids. The derived plasmids are pApPcpl4, 15, and l I2-PI, P2 (abbreviated as p14, p15, pll2-P1 and pl12-P2 respectively in the text).
Characterization of the mRNAs corresponding to the &serts Northern blot analysis with labeled plasmid DNAs confirmed that the plasmids detected one m R N A each. The sizes of mRNAs corresponding to clones P14 and P15 were 2.1 kb and 1.3 kb respectively. The two subcloned fragments of 2112 coded for one m R N A each of sizes 1.1 and 2.4 kb. The mRNAs corresponding to each of the plasmid clones were isolated by hybrid selection from J6 poly (A +) RNA and subsequently translated in a wheat germ cell free system, mRNAs derived from each plasmid yielded a single major translation product (Fig. 4). The translation products were recovered as immune-precipitates confirming that these clones code for cuticular proteins. No radioactive signals were seen in the fluorograms of the precipitates with non immune antibodies and translation products of hybrid selected RNAs from J1 poly (A +) RNA by the same plasmid
~14 ~15
k112
1'
kb
2
23.1
9.4
A
B
C
D
FIGURE 2. Determinationof the relationshipsbetween the three clones. Southern blots of EcoR1 digests of the phage DNAs were probed with nick translated 2 DNAs. A and C: labeled 2 14 DNA. B and D: labeled 215 DNA. Three hybridizingbands in all the blots correspond to the DNAs of undigested phage (-) and two arms (---~).
SILKMOTH PUPAL CUTICLE PROTEIN GENES k14
k15
kl12
kb 9.5 7.5
4.4
2.4
1.4
FIGURE 3. Identification of mRNAs corresponding to the phage DNAs in the pupal wing epidermis. Northern blots of 5 pg of J6 poly (A ÷) RNA were probed with nick translated phage DNAs. The sizes of the mRNAs were calculated based on the mobility of BRL RNA standards. The mRNA signal correspondingto clone 14 was quite faint in this study.
DNAs (data not included). Included in Fig. 4 are the translation products of the total J4 and J6 mRNAs separated on a different gel. Each poly (A ÷) RNA preparation yielded a large number of translation products and individual bands that may correspond to the hybrid selected translation products are marked by solid dots. On the basis of the sizes of the mRNAs and the corresponding translation products, it can be deduced that each of the mRNAs will contain untranslated regions.
Expression patterns of the clones The time of activation and/or expression of these genes during second pupal development was determined by probing Northern blots of RNAs prepared from wing tissue every 24 h after hormone administration. The m R N A corresponding to each of the four genes appeared on day 4 of second pupal development (Fig. 5). A comparison of the intensity of hybridization shows
295
that while the mRNAs corresponding to clones p14 and p 15 appear to increase slightly from day 4 to day 6, the two mRNAs corresponding to pll2-P1 and pl12-P2 do not change significantly. Since other hybridization experiments indicated the presence of mRNAs hybridizing to some of these clones in the developing adult wing epidermis, their expression at the time of synthesis of different cuticles was followed. Northern blots prepared with RNAs derived from fifth instar larval abdominal epidermis (L), day 6 second pupal wing epidermis (P), day 15 pharate adult wing epidermis (A) and day 6 second pupal fat body (Pf) were probed with labeled plasmid DNAs. Results (Fig. 6) showed that (i) mRNAs of similar size corresponding to clones p 14 and p 15 were expressed in the adult wing epidermis albeit at a lower level, (ii) an m R N A of similar size corresponding to clone p112-P2 was expressed in the adult wing, (iii) the expression of p112-P1 was restricted to the second pupal epidermis, and (iv) none of the cloned genes were expressed either in the larval abdominal epidermis or in the second pupal fat body.
Representation of these genes in the A. polyphemus genome and expression in other silkmoths Strips of a Southern blot of EcoRl digested A. polyphemus genomic D N A were individually probed with labeled plasmid DNAs. Each probe hybridized to the fragment(s) (Fig. 7) predicted from the restriction maps. Measurement of radioactivity hybridized to DNA on slot blots prepared with increasing quantities of known amounts of inserts and genomic D N A next to each other indicated that the genes are probably single copy (data not included) assuming 1 pg as the genomic content (Efstratiadis et al., 1976). Dot blot analysis of other insect DNAs showed cross hybridization with all the plasmid DNA probes while calf thymus and rat liver DNAs used as controls were negative. Northern blots of mRNAs isolated from second pupal wing tissue of two other silkmoths isolated 6 days after hormone administration were probed with labeled plasmid DNAs. m R N A of the same size as that of A. polyphemus was detected in two other silkmoths by the p15 D N A (Fig. 8). The corresponding m R N A was absent in the cecropia wing epidermis on day 1 (C1) after hormone administration used as a control. DISCUSSION A differential subtractive hybridization technique was utilized to derive a probe putatively specific for the pupal cuticular protein genes. Utilizing this probe it was possible to isolate genomic clones that contained silkmoth D N A fragments coding for mRNAs expressed in the pupal wing epidermis at the time of cuticle deposition. The genes for the larval cuticular proteins (Lcp) of Drosophila melanogaster and Manduca sexta are present in short segments of D N A and appear to belong to a multigene family (Snyder et al., 1981, 1982; Rebers and Riddiford, 1988; Horodyski and Riddiford, 1989).
M. N. KUMAR and S. SRIDHARA
296 kDa
14A 14B
15A
15B
P1A
PaB
P2A
J6
PzB
97--
J4
kDa --
97
--
66
m
45
45--
B31
34-29--
21-m21
iiiiiiiiii
18.4 - -
14.3 - -
14
FIGURE 4. Characterization of the polypeptides corresponding to the cloned genes. Hybrid selected RNAs were translated in a wheat germ cell free system. The translation products were then analyzed by SDS-PAGE and fluorography. Duplicate translation mixtures were subjected to immune precipitation with antibodies prepared against pupal cuticular proteins. A: Translation product directly loaded on the gel. B: Immunoprecipitated translation product. J4 and J6 correspond to translation products of poly (A ÷) RNA (from second pupal wings isolated on days 4 and 6 of development respectively) analyzed under similar conditions. The solid dots identify the translation products that may correspond to the individual cloned genes. The mobilities of protein size standards in the two cases were different. J1
J2
J3
J4
J6
J1
J2
J3
J4
J6
2.1kb - i! i!iii~!i!~ii~!!!!!ii~iii~!i¸¸i~!ii~
i~
--
ei
1.3kb
~~ ~~! ii~iiI~~i/ ? !!ii~ii ~i¸i iiiill !i
14
J1
J2
J3
15
J4
J6
Jl
J2
J3
J,t
J6
2.4kb
1.1kb
112-P 1
112-P 2
FIGURE 5. Time of expression of the genes during pupal development. Northern blots containing 15/~g each of RNA isolated from wing epidermis every 24 h (J1-J6) of second pupal development were probed with labeled plasmid DNAs.
SILKMOTH PUPAL CUTICLE PROTEIN GENES Hence, it was expected the A. polyphemus pupal cuticular protein genes might also be clustered. It is clear the ones isolated here are not. Recent characterization of three pupal cuticular protein genes of Drosophila (Apple and Fristrom, 1991) as well as an earlier characterization of another one (Henikoff et al., 1986) show that clustering is not a requirement for proper hormonal and developmental regulation. The closest location is seen in clone 2ApPcp112 in which two genes expressing mRNAs that code for polypeptides of differing sizes, are situated within 4 kb of each other. Analysis of more clones will permit a determination of whether other pupal and/or larval cuticle protein genes are situated close to each other as found in Drosophila (Apple and Fristrom, 1991). Each gene codes for a single mRNA and must therefore utilize a unique initiation site. The absence of hybridizing mRNAs in day 6 pupal fat body and in day 1 wing epidermis confirms the tissue and time specificity of expression. The ability of antibodies against pupal cuticular proteins to bind the in vitro translation products from hybrid selected RNAs support the possibility that these genes code for cuticular proteins. The need for long exposure times, due to the use of 3H amino acids and probably the presence of minute amounts of individual mRNAs (compare the intensity of individual bands to the total number and intensity of the other L
P
A
Pf
297
bands in J6 lane of Fig. 4) may account for the somewhat blurred background and faint specific bands on the fluorograms. This is especially true after immuneprecipitation. This approach of hybrid selection and translation rather than obtaining large amounts of translation products by other approaches was followed to test the ability of each clone to select related mRNAs from the total poly (A + ) RNA. Since a large number of pupal cuticular polypeptides with close pIs and molecular weights were present in 2-D gels (Sridhara, 1985b) it was expected that there might exist other related mRNAs as in multigene families of larval Lcps and Pcps of M. sexta and D. melanogaster (Riddiford et al., 1990; Apple and Fristrom, 1991). However, it is clear that each clone selects a single mRNA coding for a single polypeptide. All the four genes are activated after day 3 during second pupal development and the transcripts continue to accumulate until day 6. The presence mRNAs corresponding to the genes at day 4 of second pupal development was predictable based on the similarity of the newly appearing mRNAs on days 4 and 6 as determined by c D N A - m R N A hybridizations (Sridhara, 1985b, 1993) and analysis of their translation products (Sridhara, 1985b and Fig. 4). The observation that three of the clones hybridized to mRNAs of the same size expressed by the developing adult wing epidermis was unexpected. While L
P
A
Pf
2.1kb m 1.3kb
14
L
P
A Pf
15
L
P
A
Pf
2.4kb
1.1kb
112-P1
112-P2
FIGURE 6. Tissue and stage specificityof expression. Northern blots prepared with 15 #g of RNA isolated from fifth instar larval abdominal epidermis (L), wing epidermis at day 6 of second pupal development (P), wing epidermis at day 15 of adult development (A) and fat body at day 6 of second pupal development (Pf) were probed with random primer labeled plasmid DNAs.
298
M.N. KUMAR and S. SRIDHARA
c D N A - m R N A hybridizations showed that some of the m R N A s coding for the pupal and adult cuticles were different, 2-D analysis of the cuticular proteins showed that some proteins might be common between the two stages (Sridhara et al., 1980; Sridhara, 1985b). Although same amounts of the different RNAs were used to prepare Northern blots, the lack of an internal control limits our ability to attribute significance to the variation in hybridization intensities of the bands in different tissues and developmental periods. Furthermore, the differences in the intensity of hybridization of the corresponding m R N A s between the second pupal and adult wing epidermis cannot be related to the synthesis of the adult cuticle without more detailed analysis of their expression during adult development. It is known that flexible and hard cuticles of larval, pupal and adult stages share common proteins (Willis, 1987, 1989). Similarly, a set of proteins called cutins appear to be common to the larval and pupal cuticles of Manduca and Drosophila (Rebers and Riddiford, 1988; Apple and Fristrom, 1991). The proteins that are common to the pupal and adult cuticles, but not present in the larval cuticle, like the products of three of the genes isolated here may form a third group. However, to formulate such a group, it will be necessary to produce adequate amounts of the protein products corresponding to each of these genes and carry out rigorous analysis and comparisons with the pupal and adult cuticular proteins. The gene p112-P1 appears to be pupal specific. Since this gene is situated within 4 kb ofp112-P2, a gene that is expressed at both the pupal and adult stages, the clone 2ApPcp112 will offer opportunities to study the mechanisms by which genes are differentially activated and repressed.
kb
kb 3.9
3.7,,11~ 3.2,,1~
C1
C6
L6
P6
1.3kb
FIGURE 8. Detection of mRNA corresponding to p15 in other silkmoths. A Northern blot prepared with 15 #g RNA each isolated from the second pupal wing tissue of three silkmoths 6 days after hormone administration was probed with labeled p15 DNA insert. Washing was stopped at 2SSC at 65°C. A. polyphernus (P6), A. luna (L6) and H. cecropia (C6). RNA from wing tissue of H. cecropia after 24 h of hormone administration (C1) was used as control.
The ability of the cloned D N A s to hybridize to the genomic D N A s of other insects was predictable based on the similarities among the larval cuticular genes of Drosophila and Manduca (Rebers and Riddiford, 1988). It was expected that the isolated genes might be expressed in the wing tissue of other silkmoths during pupal cuticle deposition, since their m R N A s could drive A. polyphemus pupal specific c D N A to hybrids and their translation products were also similar (Sridhara, 1993). In view of the presence of hybridizing m R N A corresponding to only one of the clones (p15) in the pupal wing epidermis of two other silkmoths, it will be necessary to follow the expression of these genes in the epidermis at different time periods of each developmental stage.
~1- 2.1 REFERENCES
14
15
P1
P2
FIGURE 7. Representation of the genes in the silkmoth genome. A Southern blot of EcoR1 digested A. polyphemus genomicDNA was cut into strips and probed with labeled plasmid probes. Each strip contained about 10#g of DNA.
Apple R. T. and Fristrom J. W. (1991) 20-Hydroxyecdysone is required for, and negatively regulates transcription of Drosophila pupal cuticle protein genes. Dev. Biol. 146, 569 582. Binger L. E. and Willis J. H. (1990) In vitro translation of epidermal RNA from different anatomical regions and metamorphic stages of Hyalophora cecropia. Insect Biochem. 20, 573-583. Cox D. L. and Willis J. H. (1987) Analysis of cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis. Insect Biochem. 17, 457 468.
SILKMOTH PUPAL CUTICLE PROTEIN GENES Davis L. G., Dibner M. D. and Baltey J. F. (1986) Basic Methods in Molecular Biology, Elsevier, New York. Doctor J., Fristrom D. and Fristrom J. W. (1985) The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxy ecdysone. J. cell. Biol. 101, 189-200. Efstratiadis A., Crain W. R., Britten R. J., Davidson E. H. and Kafotos F. C. (1976) DNA sequence organization in the lepidopteran Antheraea pernyi. Proc. natn Acad Sci. U.S.A. 73, 2289~293. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analyt. Biochem. 132, 6-13. Fristrom J. W. (1981) Drosophila imaginal discs as a model system for the study of metamorphosis. In Metamorphosis: A Problem in Developmental Biology (Edited by Gilbert L. I. and E. Frieden), 2nd Edn, pp. 217-260. Plenum Press, New York. Fristrom J. W., Fristrom D. K., Apple R. T., Birr C., Fechtel K. and Wolfgang W. (1991) Hormone induced differentiation of the imaginal disc epidermis: pupal cuticle formation in Drosophila. In The Physiology of Insect Epidermis (Edited by Retnakaran A. and Binnington K., pp. 57-78. Pergamon Press, Oxford. Henikoff S., Keene M. A., Fechtel K. and Fristrom J. W. (1986) Gene within a gene: nested Drosophila genes encode unrelated proteins on opposite strands. Cell 44, 33-42. Hiruma K., Hardie J. and Riddiford L. M. (1991) Hormonal regulation of epidermal metamorphosis in vitro: control of expression of a larval-specific cuticle gene. Dev. Biol. 144, 369 378. Horodyski F. M. and Riddiford L. M. (1989) Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm. Dev. Biol. 132, 292 303. Johnstone A. and Thorpe R. (1982) Immunochemistry in Practice. Blackwell, Oxford. Katula K., Gilbert L. I. and Sridhara S. (1981) mRNA populations during wing development in the silkmoth Antheraea polyphemus. Molec. cell. Endocr. 26, 293-313. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York. Nowock J., Sridhara S. and Gilbert L. I. (1978) Ecdysone-initiated developmental changes in wing epidermis RNA polymerase activity during adult development of the oak silkmoth Antheraea pernyi. Molec. cell. Endocr. 11, 325 341. Rebers J. F. and Riddiford L. M. (1988) Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J. Molec. Biol. 203, 411 423. Riddiford L. M. (1984) Hormonal control of sequential gene expression in insect epidermis. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones (Edited by Hoffmann J. and Porchet M.), pp. 265 272. Springer, Berlin. Riddiford L. M. (1985) Hormone action at the cellular level. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and L. I. Gilbert), Vol. 8, pp. 37-84. Pergamon Press, Oxford. Riddiford L. M., Palli S. R. and Hiruma K. (1990) Hormonal control of sequential gene expression in Manduca epidermis. In Progress in
299
Comparative Endocrinology (Edited by Epple A., Scares C. G. and Stetson M. H.), pp. 226 231. Wiley, New York. Ruh M. H., Bryzski R. G., Connors N. A. and Dwyer K. A. (1980) DNA synthesis and cell division during second pupal development in the silkmoth Antheraea polyphemus. Insect Biochem. 10, 607-616. Snyder M., Hirsh J. and Davidson N. (1981) The cuticle genes of Drosophila: a developmentally regulated gene cluster. Cell 25, 165-177. Snyder M., Hunkapiller M., Yuen D., Silvert D., Fristrom J. and Davidson N. (1982) Cuticle protein genes of Drosophila: structure, organization, and evolution of four clustered genes. Cell 29, 1027-1040.
Sridhara S. (1985a) Sequence complexities of mRNA populations during adult wing development in the silkmoth Antheraea polyphemus. Insect Biochem. 15, 103-I 10. Sridhara S. (1985b) Evidence that pupal and adult cuticular proteins are coded by different genes in the silkmoth Antheraea polyphemus. Insect Biochem. 15, 333-339. Sridhara S. (1993) mRNAs of wing epidermis of the oak silkmoth Antheraea polyphemus and other silkmoths during pupal development. Insect Biochem. Molec. Biol. 23, 631~:r41. Sridhara S., Katula K. S. and Gilbert L. I. (1980) Hormonal control of RNA synthesis in wing epidermis. In lnsect Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 439 462. Academic Press, New York. Sridhara S., Nowock J. and Gilbert L. I. (1978) In International Review of Biochemistry: Biochemistry and Mode of Action of Hormones H (Edited by Rickenberg H. V.), Vol. 20, pp. 133-188. University Park Press, Baltimore. Willis J. H. (1981) Juvenile hormone: the status of 'status quo'. Am. Zool. 21, 763-773. Willis J. H. (1987) Cuticular proteins: the neglected component. Archs Insect. Biochem. Physiol. 6, 203-215. Willis J. H. (1989) Partial amino acid sequences of cuticular proteins from Hyalophora cecropia. Insect Biochem. 19, 41-46. Willis J. H. and Cox D. L. (1984) Defining the anti-metamorphic action of juvenile hormone. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones(Edited by Hoffmann J. and Porchet M.), pp. 466-474. Springer, Berlin. Willis J. H. and Hollowell M. P. (1976) The interaction of juvenile hormone and ecdysone: antagonistic, synergistic, or permissive. In The Juvenile Hormones (Edited by Gilbert L. I.), pp. 270-287. Plenum Press, New York. Wolfgang W. J. and Riddiford L. M. (1986) Larval cuticular morphogenesis in the tobacco hornworm Manduca sexta, and its hormonal regulation. Dev. Biol. 113, 305-316. Wolfgang W. J., Fristrom D. and Fristrom J. W. (1986) The pupal cuticle of Drosophila: differential ultrastructural immunolocalization of cuticle proteins. J. cell. Biol. 102, 306-311.
Acknowledgements--This work was partially supported by seed grants from Texas Tech University Health Sciences Center. Our thanks are due to Ms Agnes Moine who constructed the genomic library and helped with the screening and Mr Harvey Olney for preparation of the figures. Our sincere thanks to a reviewer who pointed out an error in the restriction map of one of the clones and for valuable suggestions to improve the manuscript.
Pergamon
Characterization of Four Pupal Wing Cuticular Protein Genes of the Silkmoth
Antheraea polyphemus M. N. KUMAR,* S. SRIDHARA*t Received 5 October 1992; revised and accepted 24 June 1993
Three different clones have been isolated from a genomic library of the silkmoth Antheraea polyphemus by employing a subtractive hybridization technique. The clones with inserts of 13--16 kb of DNA each, code for mRNAs expressed in the wing epidermis during JH induced second pupal cuticle deposition. While two of the clones code for a single mRNA each, the third one codes for two mRNAs. All the four mRNAs code for distinct polypeptides that can be precipitated with antibodies raised against pupal cuticular proteins. These genes are activated at the same period of pupal development and their transcripts follow similar patterns of accumulation. Although these genes are expressed in a tissue and time specific manner attesting to their pupal wing epidermal specificity, three of them are expressed in the adult wing epidermis also, but not at the larval stage. While DNAs from other silkmoths and insects hybridize to these genes, only one of the A. polyphemus genes hybridizes to RNA from second pupal wings of two other silkmoths tested. Antheraea polyphemus protein genes
Silkmoths Cuticle Pupal development Wingepidermis mRNA
INTRODUCTION The nature of the cuticle secreted during the post embryonic development of the insects, whether larval, pupal or adult, depends upon at least two hormones: molting hormone (MH or 20-hydroxyecdysone or 20-HE) and juvenile hormone (JH) (Sridhara et al., 1978). The pupal stage of holometabolous insects is a fascinating period of development in that the hardened outer cuticle of a pupa (Lepidoptera) or a puparium (Diptera) functions as an outer case in which the transformation of the larva or maggot into the volant adult moth or fly takes place. Several studies concerning the larval-pupal transformation in Drosophila and Manduca demonstrate that the switch from larval to pupal expression requires subtle changes in 20-HE levels, which (directly or indirectly) are eventually responsible both for the repression of the larval cuticle genes and activation of the pupal cuticle genes (Doctor et al., 1985; Wolfgang and Riddiford, 1986; Wolfgang et al., 1986). The effects of 20-HE during these changes can be *Department of Biochemistryand Molecular Biology,Texas Tech University Health SciencesCenter, 3601 Fourth Street, Lubbock, TX 79430, U.S.A. tAuthor for correspondence. Abbreviations used: kb, kilo base(s); bp, base pair(s); SDS--PAGE, sodium dodecyl sulfate polyacrylamidegel electrophoresis; SSC, sodium chloride+ sodiumcitrate; PBS, phosphate bufferedsaline.
Cuticle
nullified or modified by JH (Riddiford, 1985; Hiruma et al., 1991). Drosophila imaginal discs which can be mass isolated and which respond to hormone regimens in defined culture conditions have been utilized to study hormonal regulation of larval-pupal metamorphosis, especially pupal cuticle formation (Fristrom, 1981; Fristrom et al., 1991). In Lepidoptera, however, such discrete groups of cells do not exist and changes occur sequentially in 'polymorphic cells' derived directly from the already differentiated larval cells (Riddiford, 1984). Studies of the hormonal regulation of pupal cuticle protein genes of lepidopterans are hindered by the difficulty to isolate such cells in large quantities and by the asynchronous acquisition of competency for larval-pupal molt by the different larval cells during normal larval-pupal metamorphosis. However, the epidermal cells of diapausing silkmoth pupae (Antheraea polyphemus, Hyalophora cecropia, etc.) circumvent some of these problems and provide good opportunities to study the molecular mechanisms involved in this regulation of metamorphosis by the two hormones. Transfer of the diapausing pupa (characterized by a low level of metabolism) from low temperature (4°C) to room temperature (23-25°C) results in the initiation of adult development as a consequence of a small rise in the titer of the molting hormone. Normally the pupal wing epidermis (and other epidermal cells) does not encounter JH and goes on to
291
292
M.N. KUMAR and S. SRIDHARA
develop and differentiate into adult wing structures in about 15-18 days. However, if JH is provided experimentally along with 20-HE, development towards the adult is suppressed and the program for the pupal cuticle is re-expressed. Consequently, a second pupal wing that is made up of proteins different from those of the adult wing is synthesized in about 6 days (Willis and Hollowell, 1976; Sridhara et al., 1980; Willis and Cox, 1984; Sridhara, 1985b). The ability of JH to set in motion the program for the second pupal development is limited to about the first 24h while the deposition of the pupal cuticle begins at about 4 days (Ruh et al., 1980; Sridhara, 1985b). At the time of transfer of diapausing pupae from the cold to the incubator and/or administration of the hormone, the tissue is neither engaged in cuticle synthesis nor has it made any cuticle during the several months it has been in diapause (Willis, 1981). This special situation whereby epidermal cells can be directed to take alternate developmental pathways has been utilized to study the cuticular proteins and related macromolecular changes in the silkmoth H. cecropia (Willis et al., 1981; Willis and Cox, 1984; Willis, 1987; Binger and Willis, 1990). The wing epidermal cells of silkmoths A. polyphemus and A. pernyi have been used in this laboratory to follow changes in RNAs and proteins during pupal and adult development. Some of the results pertinent to this report are: (i) new mRNAs which are absent at day 1 and that belong to the moderately abundant class appear during days 4-6 of second pupal development: (ii) these new messages can be translated into polypeptides whose mobilities in 2-D gels resemble those of pupal cuticular proteins: (iii) some of these messages are different from those making the adult wing cuticle and (iv) the 2-D patterns of the pupal and adult cuticular protein patterns are quite different although some of the proteins may be common to the two stages (Sridhara et al., 1980; Sridhara, 1985a, b, 1993). These results have provided opportunities to isolate genes that are expressed by the wing epidermis in a tissue and time specific manner. This report presents results on the characterization of four genes that are expressed in the pupal wing epidermis at the time of cuticular deposition. EXPERIMENTAL P R O C E D U R E S
Diapausing pupae of the silkmoths A. polyphemus, A. pernyi, Actius luna, and H. cecropia were obtained commercially and stored at 4°C. 20-HE and the juvenile hormone (t, t, c-JH1) were purchased from Calbiochem. The 20-HE was dissolved in water while JH was dissolved in olive oil. Development of the animals towards second pupae was initiated by removing the pupae from the cold, placing them in an incubator programmed for 16h light-8 h dark cycle and maintained at 23-25°C. The hormones (2/~g/g pupa of each hormone) were injected through an intersegmental membrane after 2 h of warming. Under these conditions the pupae develop second pupal cuticles by day 6. Pupae injected 20-HE
alone synthesize adult wing cuticle by day 15. The wing tissue (fore and hind wings from both sides) from developing pupae or adults was dissected and either processed immediately for extraction of RNA or stored at -80°C as a powder prepared in liquid nitrogen. Fat bodies from day 6 second pupae were also collected for RNA extraction. Tissues or corresponding RNA obtained during successive days of second pupal development are referred to as J l, J2, etc., while that obtained at day 15 of adult development is referred to as El5. RNA was also extracted from five to six dorsal and ventral segments of abdominal integuments of feeding fifth instar larvae (~ 10-12 g wt). Isolation of total RNA and poly (A +) RNA, preparation of cDNA, hybridization with mRNA, in vitro translation of mRNA and analysis of translation products, were carried out essentially as described earlier (Sridhara, 1985a, b, 1994). The recombinant DNA techniques followed standard procedures (Maniatis et al., 1982; Davis et al., 1986). The antibodies were obtained by immunizing rabbits with pupal cuticular proteins solubilized from cleaned second pupal wing cuticle following standard techniques (Johnstone and Thorpe, 1982). Both immune and non immune serum were processed by ammonium sulfate precipitation followed by hydroxylapatite chromatography. The antibodies were dialysed and stored in PBS containing 20% glycerol. Probe generation and screening of the genomic library A cDNA probe that is specific to the time period when pupal cuticle was being actively deposited was prepared as described earlier (Sridhara, 1985a, b, 1993) while employing ~ 3 2 p dCTP instead of 3H-dCTP. Briefly, about 108DPM of 32p cDNA was synthesized using poly (A +) mRNA derived from wing tissue on day 6 of second pupal development (J6), oligo dT and AMV reverse transcriptase. This cDNA was hybridized to saturation over 8 days with poly (A +) RNA derived from wing tissue 1 day after hormone administration (Jl). The unhybridized single stranded cDNA was recovered from a hydroxyapatite column. Elimination of the RNA with alkali and recovery of the cDNA from a Sephadex-50 column provided the desired probe. 2 x 105 recombinant phages of a A. polyphemus genomic library prepared in the vector EMBL-4 were screened with the probe. Four nitrocellulose lifts were made from each plate. Differential screening was carried out whereby two filters were hybridized to the probe while the other two were hybridized to a similar amount of 32p-labeled J1 cDNA. Hybridization and washing conditions are described below. Plaques that hybridized uniquely to the J6 specific cDNA probe were collected. Further selection and purification were carried out by similar differential screening with Jl and J6 cDNAs. Hybridization and washing Library screening and the other hybridizations (Northerns, dot blots, Southerns, etc.) were carried out
SILKMOTH PUPAL CUTICLE PROTEIN GENES in a solution of formamide 50%, SSC 5 x , phosphate buffer 50mM, Denhart solution 5 x , and sonicated denatured calf thymus D N A 100/tg/ml. Prehybridization was done overnight and hybridization for 24-48 h, both at 42°C. Washings were done successively in 2 × 2SSC at room temperature, 2 × 2SSC at 65°C, 2 × I SSC at 65°C, and 2 × 0.2SSC at 65°C. Occasionally the washing was stopped at 2 × 2SSC at 65°C (indicated at appropriate places).
Membranes and probes The majority of studies were carried out with nitrocellulose (BA85, Schliecher & Schuell) and probes prepared by nick translation with a - 3 2 P dCTP. Southern blots of genomic DNA were prepared with the BioRad Zeta Probe membrane and the probes were generated by the random primer labeling procedure (Feinberg and Vogelstein, 1983).
Hybrid selection and translation RNAs specific to the clones were isolated by hybridization of total poly (A ÷) RNA to denatured plasmid DNAs fixed on 5 m m dia Zeta Probe membrane discs for 48 h at 42°C. RNAs corresponding to individual clones were translated in a wheat germ cell free system in the presence of five 3H-amino acids (Sridhara, 1985a, b). The entire translation product was boiled with the sample buffer and analysed by SDS-PAGE. Alternately, the translation mixture was diluted with immune precipitation buffer, cleared with 25/~1 of
293
Protein A conjugated to agarose (Repligen Corp.) and mixed overnight with antibodies produced against pupal cuticular proteins. The mixture was treated with another 25/~1 aliquot of Protein A agarose for 3 h in the cold. The agarose was washed, the bound material eluted by boiling with sample buffer and analyzed by SDS-PAGE. The gels were stained, destained, and prepared for fluorography with Enhance (NEN). The dried gels were exposed to X-ray film for varying periods of time. RESULTS
Isolation of pupal specific genomic clones Earlier studies demonstrated that new mRNAs that probably coded for cuticular proteins, appeared in the wing epidermis during 4~6 days of JH induced second pupal development (Sridhara, 1985b, 1993). This information and the techniques developed were utilized here to obtain a pupal specific probe to screen a recombinant genomic library prepared in the laboratory. The specificity of selection was enhanced by screening alternate filters with J1 cDNA and the specific probe simultaneously. This screening procedure yielded 20 clones after three rounds of selection and purification. Three clones, each showing a different level of hybridization with J6 cDNA, were plaque purified and their DNAs were analyzed. These clones are designated as 2ApPcpl4, 15, and 112 (Lambda Antheraea polyphemus pupal cuticular protein clone).
Clone i 4 t~l W
n'~
rr
II
. tO) '~-
l II
I
"~
~
~
(/3 1,1
I
0
o WU
II
I
IS
~
o.~=
~
"-- W ~
II I Clone
"~
~
.
rv"
,., ~
.
"--~
o
.
t~J
I
~
f'r"
~ "rV
I II
~
iv"
tJ.IC,I taJX
~
I
re"
o
U t.t.l
II I
iv"
oo
U U t.~ taJ
I
II
clone I12
L~
.--
mm
¢~
U
f"
mmm
1 2kb
I
FIGURE 1. Restrictionmaps of the three genomicclones isolated by differentialscreening. The phage DNAs were subjected to digestion with single and multiple restriction enzymes and the maps arrived at by standard procedures. The fragment(s) hybridizing to the J6 cDNA are marked below each map (~). The DNA fragmentssubclonedfor further analysis are marked above (~-+).
294
M.N. KUMAR and S. SRIDHARA
Analyses of the clones The D N A insert in each clone had a distinct pattern by restriction analysis (Fig. 1). Southern blots of each of the three DNAs were probed with individually labeled phage DNAs. In each case the insert(s) hybridized with its own probe (Fig. 2). These results and the different restriction maps demonstrated that the insert in each clone was a different fragment of the genomic DNA. Dot blot analysis confirmed that the phage DNAs hybridized with J6 cDNAs. We probed strips of a Northern blot of J6 poly (A +) RNA with nick translated phage DNAs. It was seen that the first two clones identified one m R N A each while the third clone identified two mRNAs (Fig. 3). The m R N A signal for the clone 2 14 was quite faint in this blot, but was stronger in other blots (see later). The D N A segments corresponding to the specific mRNAs were identified by probing various Southern blots with J6 cDNAs. It was determined that the first two 2 clones contained one D N A segment each that hybridized to the cDNA while the third contained two such fragments (marked in Fig. 1). The combined results allowed us to conclude that each 2 clone contains 13-16kb of genomic D N A which includes smaller fragments that code for mRNAs expressed at day 6 of ~14 ~15
pupal development. These fragments, marked in Fig. l, were sub cloned into pUCI8 or Bluescript plasmids. The derived plasmids are pApPcpl4, 15, and l I2-PI, P2 (abbreviated as p14, p15, pll2-P1 and pl12-P2 respectively in the text).
Characterization of the mRNAs corresponding to the &serts Northern blot analysis with labeled plasmid DNAs confirmed that the plasmids detected one m R N A each. The sizes of mRNAs corresponding to clones P14 and P15 were 2.1 kb and 1.3 kb respectively. The two subcloned fragments of 2112 coded for one m R N A each of sizes 1.1 and 2.4 kb. The mRNAs corresponding to each of the plasmid clones were isolated by hybrid selection from J6 poly (A +) RNA and subsequently translated in a wheat germ cell free system, mRNAs derived from each plasmid yielded a single major translation product (Fig. 4). The translation products were recovered as immune-precipitates confirming that these clones code for cuticular proteins. No radioactive signals were seen in the fluorograms of the precipitates with non immune antibodies and translation products of hybrid selected RNAs from J1 poly (A +) RNA by the same plasmid
~14 ~15
k112
1'
kb
2
23.1
9.4
A
B
C
D
FIGURE 2. Determinationof the relationshipsbetween the three clones. Southern blots of EcoR1 digests of the phage DNAs were probed with nick translated 2 DNAs. A and C: labeled 2 14 DNA. B and D: labeled 215 DNA. Three hybridizingbands in all the blots correspond to the DNAs of undigested phage (-) and two arms (---~).
SILKMOTH PUPAL CUTICLE PROTEIN GENES k14
k15
kl12
kb 9.5 7.5
4.4
2.4
1.4
FIGURE 3. Identification of mRNAs corresponding to the phage DNAs in the pupal wing epidermis. Northern blots of 5 pg of J6 poly (A ÷) RNA were probed with nick translated phage DNAs. The sizes of the mRNAs were calculated based on the mobility of BRL RNA standards. The mRNA signal correspondingto clone 14 was quite faint in this study.
DNAs (data not included). Included in Fig. 4 are the translation products of the total J4 and J6 mRNAs separated on a different gel. Each poly (A ÷) RNA preparation yielded a large number of translation products and individual bands that may correspond to the hybrid selected translation products are marked by solid dots. On the basis of the sizes of the mRNAs and the corresponding translation products, it can be deduced that each of the mRNAs will contain untranslated regions.
Expression patterns of the clones The time of activation and/or expression of these genes during second pupal development was determined by probing Northern blots of RNAs prepared from wing tissue every 24 h after hormone administration. The m R N A corresponding to each of the four genes appeared on day 4 of second pupal development (Fig. 5). A comparison of the intensity of hybridization shows
295
that while the mRNAs corresponding to clones p14 and p 15 appear to increase slightly from day 4 to day 6, the two mRNAs corresponding to pll2-P1 and pl12-P2 do not change significantly. Since other hybridization experiments indicated the presence of mRNAs hybridizing to some of these clones in the developing adult wing epidermis, their expression at the time of synthesis of different cuticles was followed. Northern blots prepared with RNAs derived from fifth instar larval abdominal epidermis (L), day 6 second pupal wing epidermis (P), day 15 pharate adult wing epidermis (A) and day 6 second pupal fat body (Pf) were probed with labeled plasmid DNAs. Results (Fig. 6) showed that (i) mRNAs of similar size corresponding to clones p 14 and p 15 were expressed in the adult wing epidermis albeit at a lower level, (ii) an m R N A of similar size corresponding to clone p112-P2 was expressed in the adult wing, (iii) the expression of p112-P1 was restricted to the second pupal epidermis, and (iv) none of the cloned genes were expressed either in the larval abdominal epidermis or in the second pupal fat body.
Representation of these genes in the A. polyphemus genome and expression in other silkmoths Strips of a Southern blot of EcoRl digested A. polyphemus genomic D N A were individually probed with labeled plasmid DNAs. Each probe hybridized to the fragment(s) (Fig. 7) predicted from the restriction maps. Measurement of radioactivity hybridized to DNA on slot blots prepared with increasing quantities of known amounts of inserts and genomic D N A next to each other indicated that the genes are probably single copy (data not included) assuming 1 pg as the genomic content (Efstratiadis et al., 1976). Dot blot analysis of other insect DNAs showed cross hybridization with all the plasmid DNA probes while calf thymus and rat liver DNAs used as controls were negative. Northern blots of mRNAs isolated from second pupal wing tissue of two other silkmoths isolated 6 days after hormone administration were probed with labeled plasmid DNAs. m R N A of the same size as that of A. polyphemus was detected in two other silkmoths by the p15 D N A (Fig. 8). The corresponding m R N A was absent in the cecropia wing epidermis on day 1 (C1) after hormone administration used as a control. DISCUSSION A differential subtractive hybridization technique was utilized to derive a probe putatively specific for the pupal cuticular protein genes. Utilizing this probe it was possible to isolate genomic clones that contained silkmoth D N A fragments coding for mRNAs expressed in the pupal wing epidermis at the time of cuticle deposition. The genes for the larval cuticular proteins (Lcp) of Drosophila melanogaster and Manduca sexta are present in short segments of D N A and appear to belong to a multigene family (Snyder et al., 1981, 1982; Rebers and Riddiford, 1988; Horodyski and Riddiford, 1989).
M. N. KUMAR and S. SRIDHARA
296 kDa
14A 14B
15A
15B
P1A
PaB
P2A
J6
PzB
97--
J4
kDa --
97
--
66
m
45
45--
B31
34-29--
21-m21
iiiiiiiiii
18.4 - -
14.3 - -
14
FIGURE 4. Characterization of the polypeptides corresponding to the cloned genes. Hybrid selected RNAs were translated in a wheat germ cell free system. The translation products were then analyzed by SDS-PAGE and fluorography. Duplicate translation mixtures were subjected to immune precipitation with antibodies prepared against pupal cuticular proteins. A: Translation product directly loaded on the gel. B: Immunoprecipitated translation product. J4 and J6 correspond to translation products of poly (A ÷) RNA (from second pupal wings isolated on days 4 and 6 of development respectively) analyzed under similar conditions. The solid dots identify the translation products that may correspond to the individual cloned genes. The mobilities of protein size standards in the two cases were different. J1
J2
J3
J4
J6
J1
J2
J3
J4
J6
2.1kb - i! i!iii~!i!~ii~!!!!!ii~iii~!i¸¸i~!ii~
i~
--
ei
1.3kb
~~ ~~! ii~iiI~~i/ ? !!ii~ii ~i¸i iiiill !i
14
J1
J2
J3
15
J4
J6
Jl
J2
J3
J,t
J6
2.4kb
1.1kb
112-P 1
112-P 2
FIGURE 5. Time of expression of the genes during pupal development. Northern blots containing 15/~g each of RNA isolated from wing epidermis every 24 h (J1-J6) of second pupal development were probed with labeled plasmid DNAs.
SILKMOTH PUPAL CUTICLE PROTEIN GENES Hence, it was expected the A. polyphemus pupal cuticular protein genes might also be clustered. It is clear the ones isolated here are not. Recent characterization of three pupal cuticular protein genes of Drosophila (Apple and Fristrom, 1991) as well as an earlier characterization of another one (Henikoff et al., 1986) show that clustering is not a requirement for proper hormonal and developmental regulation. The closest location is seen in clone 2ApPcp112 in which two genes expressing mRNAs that code for polypeptides of differing sizes, are situated within 4 kb of each other. Analysis of more clones will permit a determination of whether other pupal and/or larval cuticle protein genes are situated close to each other as found in Drosophila (Apple and Fristrom, 1991). Each gene codes for a single mRNA and must therefore utilize a unique initiation site. The absence of hybridizing mRNAs in day 6 pupal fat body and in day 1 wing epidermis confirms the tissue and time specificity of expression. The ability of antibodies against pupal cuticular proteins to bind the in vitro translation products from hybrid selected RNAs support the possibility that these genes code for cuticular proteins. The need for long exposure times, due to the use of 3H amino acids and probably the presence of minute amounts of individual mRNAs (compare the intensity of individual bands to the total number and intensity of the other L
P
A
Pf
297
bands in J6 lane of Fig. 4) may account for the somewhat blurred background and faint specific bands on the fluorograms. This is especially true after immuneprecipitation. This approach of hybrid selection and translation rather than obtaining large amounts of translation products by other approaches was followed to test the ability of each clone to select related mRNAs from the total poly (A + ) RNA. Since a large number of pupal cuticular polypeptides with close pIs and molecular weights were present in 2-D gels (Sridhara, 1985b) it was expected that there might exist other related mRNAs as in multigene families of larval Lcps and Pcps of M. sexta and D. melanogaster (Riddiford et al., 1990; Apple and Fristrom, 1991). However, it is clear that each clone selects a single mRNA coding for a single polypeptide. All the four genes are activated after day 3 during second pupal development and the transcripts continue to accumulate until day 6. The presence mRNAs corresponding to the genes at day 4 of second pupal development was predictable based on the similarity of the newly appearing mRNAs on days 4 and 6 as determined by c D N A - m R N A hybridizations (Sridhara, 1985b, 1993) and analysis of their translation products (Sridhara, 1985b and Fig. 4). The observation that three of the clones hybridized to mRNAs of the same size expressed by the developing adult wing epidermis was unexpected. While L
P
A
Pf
2.1kb m 1.3kb
14
L
P
A Pf
15
L
P
A
Pf
2.4kb
1.1kb
112-P1
112-P2
FIGURE 6. Tissue and stage specificityof expression. Northern blots prepared with 15 #g of RNA isolated from fifth instar larval abdominal epidermis (L), wing epidermis at day 6 of second pupal development (P), wing epidermis at day 15 of adult development (A) and fat body at day 6 of second pupal development (Pf) were probed with random primer labeled plasmid DNAs.
298
M.N. KUMAR and S. SRIDHARA
c D N A - m R N A hybridizations showed that some of the m R N A s coding for the pupal and adult cuticles were different, 2-D analysis of the cuticular proteins showed that some proteins might be common between the two stages (Sridhara et al., 1980; Sridhara, 1985b). Although same amounts of the different RNAs were used to prepare Northern blots, the lack of an internal control limits our ability to attribute significance to the variation in hybridization intensities of the bands in different tissues and developmental periods. Furthermore, the differences in the intensity of hybridization of the corresponding m R N A s between the second pupal and adult wing epidermis cannot be related to the synthesis of the adult cuticle without more detailed analysis of their expression during adult development. It is known that flexible and hard cuticles of larval, pupal and adult stages share common proteins (Willis, 1987, 1989). Similarly, a set of proteins called cutins appear to be common to the larval and pupal cuticles of Manduca and Drosophila (Rebers and Riddiford, 1988; Apple and Fristrom, 1991). The proteins that are common to the pupal and adult cuticles, but not present in the larval cuticle, like the products of three of the genes isolated here may form a third group. However, to formulate such a group, it will be necessary to produce adequate amounts of the protein products corresponding to each of these genes and carry out rigorous analysis and comparisons with the pupal and adult cuticular proteins. The gene p112-P1 appears to be pupal specific. Since this gene is situated within 4 kb ofp112-P2, a gene that is expressed at both the pupal and adult stages, the clone 2ApPcp112 will offer opportunities to study the mechanisms by which genes are differentially activated and repressed.
kb
kb 3.9
3.7,,11~ 3.2,,1~
C1
C6
L6
P6
1.3kb
FIGURE 8. Detection of mRNA corresponding to p15 in other silkmoths. A Northern blot prepared with 15 #g RNA each isolated from the second pupal wing tissue of three silkmoths 6 days after hormone administration was probed with labeled p15 DNA insert. Washing was stopped at 2SSC at 65°C. A. polyphernus (P6), A. luna (L6) and H. cecropia (C6). RNA from wing tissue of H. cecropia after 24 h of hormone administration (C1) was used as control.
The ability of the cloned D N A s to hybridize to the genomic D N A s of other insects was predictable based on the similarities among the larval cuticular genes of Drosophila and Manduca (Rebers and Riddiford, 1988). It was expected that the isolated genes might be expressed in the wing tissue of other silkmoths during pupal cuticle deposition, since their m R N A s could drive A. polyphemus pupal specific c D N A to hybrids and their translation products were also similar (Sridhara, 1993). In view of the presence of hybridizing m R N A corresponding to only one of the clones (p15) in the pupal wing epidermis of two other silkmoths, it will be necessary to follow the expression of these genes in the epidermis at different time periods of each developmental stage.
~1- 2.1 REFERENCES
14
15
P1
P2
FIGURE 7. Representation of the genes in the silkmoth genome. A Southern blot of EcoR1 digested A. polyphemus genomicDNA was cut into strips and probed with labeled plasmid probes. Each strip contained about 10#g of DNA.
Apple R. T. and Fristrom J. W. (1991) 20-Hydroxyecdysone is required for, and negatively regulates transcription of Drosophila pupal cuticle protein genes. Dev. Biol. 146, 569 582. Binger L. E. and Willis J. H. (1990) In vitro translation of epidermal RNA from different anatomical regions and metamorphic stages of Hyalophora cecropia. Insect Biochem. 20, 573-583. Cox D. L. and Willis J. H. (1987) Analysis of cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis. Insect Biochem. 17, 457 468.
SILKMOTH PUPAL CUTICLE PROTEIN GENES Davis L. G., Dibner M. D. and Baltey J. F. (1986) Basic Methods in Molecular Biology, Elsevier, New York. Doctor J., Fristrom D. and Fristrom J. W. (1985) The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxy ecdysone. J. cell. Biol. 101, 189-200. Efstratiadis A., Crain W. R., Britten R. J., Davidson E. H. and Kafotos F. C. (1976) DNA sequence organization in the lepidopteran Antheraea pernyi. Proc. natn Acad Sci. U.S.A. 73, 2289~293. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analyt. Biochem. 132, 6-13. Fristrom J. W. (1981) Drosophila imaginal discs as a model system for the study of metamorphosis. In Metamorphosis: A Problem in Developmental Biology (Edited by Gilbert L. I. and E. Frieden), 2nd Edn, pp. 217-260. Plenum Press, New York. Fristrom J. W., Fristrom D. K., Apple R. T., Birr C., Fechtel K. and Wolfgang W. (1991) Hormone induced differentiation of the imaginal disc epidermis: pupal cuticle formation in Drosophila. In The Physiology of Insect Epidermis (Edited by Retnakaran A. and Binnington K., pp. 57-78. Pergamon Press, Oxford. Henikoff S., Keene M. A., Fechtel K. and Fristrom J. W. (1986) Gene within a gene: nested Drosophila genes encode unrelated proteins on opposite strands. Cell 44, 33-42. Hiruma K., Hardie J. and Riddiford L. M. (1991) Hormonal regulation of epidermal metamorphosis in vitro: control of expression of a larval-specific cuticle gene. Dev. Biol. 144, 369 378. Horodyski F. M. and Riddiford L. M. (1989) Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm. Dev. Biol. 132, 292 303. Johnstone A. and Thorpe R. (1982) Immunochemistry in Practice. Blackwell, Oxford. Katula K., Gilbert L. I. and Sridhara S. (1981) mRNA populations during wing development in the silkmoth Antheraea polyphemus. Molec. cell. Endocr. 26, 293-313. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York. Nowock J., Sridhara S. and Gilbert L. I. (1978) Ecdysone-initiated developmental changes in wing epidermis RNA polymerase activity during adult development of the oak silkmoth Antheraea pernyi. Molec. cell. Endocr. 11, 325 341. Rebers J. F. and Riddiford L. M. (1988) Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J. Molec. Biol. 203, 411 423. Riddiford L. M. (1984) Hormonal control of sequential gene expression in insect epidermis. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones (Edited by Hoffmann J. and Porchet M.), pp. 265 272. Springer, Berlin. Riddiford L. M. (1985) Hormone action at the cellular level. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and L. I. Gilbert), Vol. 8, pp. 37-84. Pergamon Press, Oxford. Riddiford L. M., Palli S. R. and Hiruma K. (1990) Hormonal control of sequential gene expression in Manduca epidermis. In Progress in
299
Comparative Endocrinology (Edited by Epple A., Scares C. G. and Stetson M. H.), pp. 226 231. Wiley, New York. Ruh M. H., Bryzski R. G., Connors N. A. and Dwyer K. A. (1980) DNA synthesis and cell division during second pupal development in the silkmoth Antheraea polyphemus. Insect Biochem. 10, 607-616. Snyder M., Hirsh J. and Davidson N. (1981) The cuticle genes of Drosophila: a developmentally regulated gene cluster. Cell 25, 165-177. Snyder M., Hunkapiller M., Yuen D., Silvert D., Fristrom J. and Davidson N. (1982) Cuticle protein genes of Drosophila: structure, organization, and evolution of four clustered genes. Cell 29, 1027-1040.
Sridhara S. (1985a) Sequence complexities of mRNA populations during adult wing development in the silkmoth Antheraea polyphemus. Insect Biochem. 15, 103-I 10. Sridhara S. (1985b) Evidence that pupal and adult cuticular proteins are coded by different genes in the silkmoth Antheraea polyphemus. Insect Biochem. 15, 333-339. Sridhara S. (1993) mRNAs of wing epidermis of the oak silkmoth Antheraea polyphemus and other silkmoths during pupal development. Insect Biochem. Molec. Biol. 23, 631~:r41. Sridhara S., Katula K. S. and Gilbert L. I. (1980) Hormonal control of RNA synthesis in wing epidermis. In lnsect Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 439 462. Academic Press, New York. Sridhara S., Nowock J. and Gilbert L. I. (1978) In International Review of Biochemistry: Biochemistry and Mode of Action of Hormones H (Edited by Rickenberg H. V.), Vol. 20, pp. 133-188. University Park Press, Baltimore. Willis J. H. (1981) Juvenile hormone: the status of 'status quo'. Am. Zool. 21, 763-773. Willis J. H. (1987) Cuticular proteins: the neglected component. Archs Insect. Biochem. Physiol. 6, 203-215. Willis J. H. (1989) Partial amino acid sequences of cuticular proteins from Hyalophora cecropia. Insect Biochem. 19, 41-46. Willis J. H. and Cox D. L. (1984) Defining the anti-metamorphic action of juvenile hormone. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones(Edited by Hoffmann J. and Porchet M.), pp. 466-474. Springer, Berlin. Willis J. H. and Hollowell M. P. (1976) The interaction of juvenile hormone and ecdysone: antagonistic, synergistic, or permissive. In The Juvenile Hormones (Edited by Gilbert L. I.), pp. 270-287. Plenum Press, New York. Wolfgang W. J. and Riddiford L. M. (1986) Larval cuticular morphogenesis in the tobacco hornworm Manduca sexta, and its hormonal regulation. Dev. Biol. 113, 305-316. Wolfgang W. J., Fristrom D. and Fristrom J. W. (1986) The pupal cuticle of Drosophila: differential ultrastructural immunolocalization of cuticle proteins. J. cell. Biol. 102, 306-311.
Acknowledgements--This work was partially supported by seed grants from Texas Tech University Health Sciences Center. Our thanks are due to Ms Agnes Moine who constructed the genomic library and helped with the screening and Mr Harvey Olney for preparation of the figures. Our sincere thanks to a reviewer who pointed out an error in the restriction map of one of the clones and for valuable suggestions to improve the manuscript.