Vitellogenin in locusts (Locusta migratoria): Translation of vitellogenin mRNA in Xenopus oocytes and analysis of the polypeptide products

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 266-276, 1980

Vitellogenin

in Locusts (Locusta migratoria): Translation of Vitellogenin mRNA in Xenopus Oocytes and Analysis of the Polypeptide Products THOMAS T. CHEN

Department

of Biology,

McMaster

Hamilton,

University,

Accepted November

Ontario

L8S hK1, Canada

20, 1979

Cytoplasmic poly(A)+-RNA, prepared from fat bodies of reproductively active locust females, directed the synthesis of two large polypeptides in Xenopus oocytes. Occasionally two smaller polypeptides (X, and X,) were also detectable. The two larger polypeptides were immunologically and electrophoretically indistinguishable from the unprocessed vitellogenin polypeptides (Vg, and Vg,) of locust fat bodies. Peptide patterns generated from these two translation products by Staphylococcus aureus V8 protease digestion were identical to those of Vg, and Vg,. RNA isolated from allatectomized female locusts treated with the juvenile hormone analog (ZR-515) was also able to direct the synthesis of vitellogenin in Xenopus oocytes, whereas RNA isolated from mature males or allatectomized females did not. The molecular weights of fat-body Vg, and Vg, were 235,000 and 225,000, respectively and the processed vitellogenin polypeptides were found to range from 126,000 to 52,000. Electrophoretic and chromatographic analysis of l%]methioninecontaining tryptic peptides of Vg, and Vg, showed two different tryptic peptide fingerprints Distinctly different peptide patterns were also observed when Vg, and Vg, were partially digested with V8 protease or papain. However, tryptic peptide mapping and V8 protease limited digestion mapping of fat-body X, and X, revealed that these two polypeptides were derived from Vg, and Vg,. This suggests that Vg, and Vg, are products of two different vitellogenin structural genes.

The production of eggs in insects involves the synthesis in the fat body and secretion into the hemolymph of massive amounts of vitellogenins, the female-specific yolk precursor proteins (l-3). These proteins are then selectively taken up by the developing oocytes and deposited, sometimes in slightly modified forms, as vitellins, the major proteins of the yolk (2). Analogous to the estrogen-controlled vitellogenin synthesis in the liver of amphibians and birds (4, 5), the synthesis of vitellogenins in many insect species is controlled by juvenile hormone (JH)’ (3, 6, 7); and, therefore, ’ Abbreviations used: SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; EGTA, ethylene glycol bis @aminoethyl ether) N’, N’-tetraacetic acid; IgG, immunoglobulin G; JH, juvenile hormone; Vg,, Vg,, polypeptides of vitellogenin; tic, thin-layer chromatography. 0003-9861/80/050266-11$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

these systems are favorable for the analysis of mechanisms of hormone-controlled gene expression. Vitellin and vitellogenin from the African migratory locust (Locusta migratoria) have been purified and characterized as lipoglycoprotein with a native molecular weight of about 550,000 (8-10). Upon denaturation of vitellin or vitellogenin with SDS, eight polypeptides of molecular weights reported as ranging from 140,000 to 52,000 are released (8, 10). Chen et al. (10) further showed, by pulse labeling of locust fat body in vitro with 3H-labeled amino acids and by analysis of vitellin-immunospecific products, that there was rapid incorporation of label into two polypeptides with molecular weights about 265,000 and 250,000, followed by transfer of the label to smaller polypeptides, and then final accumulation in polypeptides identical to those of vitellin. 266

TRANSLATION

OF VITELLOGENIN

It was suggested that these two large rapidly labeled polypeptides (now called Vg, and Vg,) may represent products of two vitellogenin structural genes. However, there was no direct chemical evidence for the presence of two primary vitellogenin gene products. The juvenile hormone-controlled synthesis of insect vitellogenins has been studied at the subcellular level in a cockroach, Leucophcrea maderae (6), and a locust, Locusta migratotia (7, 8). In the locust, vitellogenin is found only in reproductively mature females, and its production can be prevented by removal of the corpora allata (the source of JH) and then restored by treatment with JH or a synthetic analog, ZR-515 (7, 8). Biochemical and ultrastructural studies of vitellogenin synthesis induced in the fat body by JH showed that the action of the hormone may be at the gene level (8, 11). To elucidate the mechanism of ,JH control of vitellogenin synthesis, isolation of vitellogenin mRNA and its translation into complete products are essential. I now report the translation of locust vitellogenin mRNA into polypeptides in oocytes of Xenopus laevis, and the determination of the identity of the translation products with the authentic polypeptides (Vg,, Vgs) of vitellogenin. MATERIALS

AND

METHODS

Illsects. Stocks of African migratory locusts (Locusta w!igrutoria) were obtained from Dr. G. R. Wyatt (Queen’s University, Kingston, Ontario) and were reared in the laboratory under crowded conditions as previously described (IO). Allatectomy was performed, by a procedure described by Strong (12), on adult females l-2 days after eclosion. The absence of vitellogenin in the hemolymph of each individual was confirmed 2 weeks after allatectomy by immunodiffusion with antiritellin serum (10). Antibody. Antiserum against vitellin, which is immunologically identical with vitellogenin, was prepared and rendered specific by adsorption with adult male locust hemolgmph as described previously (10). The antiserum was stored in 0.5.ml portions at -70°C. Synthesis of vitellogenin in cultured fufizt body. Fat-body tissue was carefully removed from adult female locusts in sterile locust Ringer solution at 4°C as described previously (13). After rinsing three times in the Ringer solution, fat bodies from six to eight fe-

mRNA

AND PEPTIDE

ANALYSIS

267

males were incubated in 3 ml of the Ringer solution containing 100 &i of [“Hlleucine (100 Ciimmol, New England Nuclear Corp.) or 100 &i of [““Slmethionine (500 Ci/mmol, New England Nuclear Corp.) at 30°C with gentle shaking for 30 min or 3 h. At the end of the labeling period, fat bodies were homogenized in 5 ml of 0.05 M Tris-HCl buffer, pH 8.1, containing 0.4 M NaCI, 1 mM PMSF, 1% Triton X-100, 1% deoxycholate, and 0.2%’ SDS, combined with the incubated medium, and then centrifuged at 30,OOOg for 1 h at 4°C. The labeled vitellogenin was isolated by precipitation with ritellin-specific antiserum as described before (10) or with vitellin antiserum anti goat anti-rabbit IgG. The immune precipitate, after washing in sodium phosphate buffer (0.1 M, pH 7.5) containing 0.9% NaC’l, 1%’ Triton X-100, ant1 1% deoxycholate, was dissolved in Tris- HCl buffer (0.1 M, pH 8.0), containing 3% SDS, 1% /&mercaptoethanol, and 1O’Z glycerol and heated at 100°C for 3 min. Polyacrylmnide gel electroph,oresis. Electrophoresis in polyacrylamitle gel containing SDS was performed in a slab gel apparatus using conditions described previously (10). Vg, and Vg, \vere routinely separated in 5% gels. For the analysis of peptitles generated by proteolysis. 15% gels were used, RNA extmctiou a?ld isolution qf‘ poly(A)+-R,VA. Total cytoplasmic RNA was extracted from fat bodies of reproductively active adult females b> the method of Palmiter (14) with some modifications. To 5 g of fat bodies was added 10 ml of 0.1 M sodium acetate buffer (pH 5.0) containing 25 mM NaCl, 35 mM MgCl,, 25 mM EGTA, 25 pg/ml polyvinyl sulfate, 35 pg spermine, 0.5% SDS, and 10 ml of buffer-saturated phenol. This was homogenized for 5 min at 4°C with the Dounce homogenizer and centrifuged at SOOOgfor 10 min. The mixture was then shaken with 20 ml chloroform and centrifuged. The aqueous phase was further extracted with 10 ml of chloroform five times. The RNA was precipitated by the addition of 2-2.5 vol of ethanol at -20°C. The crude RNA pellet was washed five times with 3 M sodium acetate (pH 6.0), twice with 70% ethanol containing 0.1 M sodium acetate, dissolved in H,O, and lyophilized. The poly(A)+-RNA was isolated by affinity chromatography on oligo(dT)-cellulose by the method of Aviv and Leder (15) or by poly(U)-Sepharose 4B according to Lizardi (16). Oocyte microi?ljectkm and product analysis. Batches of 30 oocytes each were injected with 40-60 nl of RNA solution in injection medium (17), and then incubated in 0.3 ml of culture medium (17) containing 80 &i of [“Hlleucine (110 &i/mmol) or l%]methionine (500 Ciimmol) for 18 h at 20°C. Control oocytes were injected with the same volume of the injection medium. After incubation, oocytes were homogenized in 1.0 ml of 0.05 M Tris-HCl buffer (pH X.1) containing 0.4 M NaCl, 1 mM PMSF, 1% Triton X-100, 1% deoxycholate, and 0.2% SDS. The homogenate was

268

THOMAS T. CHEN

centrifuged at 30,OOOgfor 1 h, and the vitellogenin was immunoprecipitated by the method described in the previous section. The immunoprecipitate, after washing, was analyzed on 5% SDS-polyacrylamide slab gels and the labeled polypeptides were visualized by fluorography (10). Typtic peptide analysis. Tryptic digestion of [YSlmethionine-labeled Vg, and Vg, was carried out by the method of Morrison and Lodish (18). Vg, and Vg, from fat body cultured in medium were isolated by immunoprecipitation and resolved on 5% SDSpolyacrylamide gels. The location of each protein within the gel was determined by autoradiography on fixed, dried gel. Trypsin digestion of the individual proteins was accomplished without prior elution from the gel slices. The region of the gel containing each individual protein band was excised, cut into small pieces, and placed in 5 ml of 0.2 M ammonium bicarbonate solution (pH 8.5) containing 50 pg of trypsin. The suspension was incubated at 37°C for 20 h with gentle shaking. The digestion was continued for two further 6-h incubations with 5 ml of fresh trypsin-ammonium bicarbonate solution each. The digested solutions were pooled and lyophilized. The [a”S]methionine tryptic peptides were analyzed by high voltage electrophoresis at pH 3.5 and chromatography on thin-layer sheets of silica gel (20 x 20 cm, 0.2 mm thick; polygram SIL N-HR, Mackerey-Nagel Co.) as described by Dobos and Rowe (19). Peptide mapping by limited proteolysis. Peptide mapping of Vg, and Vg, by limited proteolysis was carried out by the method of Cleveland et al. (20). [aYS]Methionine-labeled Vg, and Vg, isolated from fat body cultured in vitro or from Xenopus oocytes microinjected with vitellogenin mRNA were separated on 5% SDS-polyacrylamide slab gels as described above. Digestion of Vg, or VgZ was accomplished by placing gel slices containing each individual protein in the sample wells of a second SDS-gel (15% acrylamide), overlaying each slot with various amounts of Staphylococcus aureus V8 protease or papain in 0.125 M Tris-HCl buffer, pH 7.6, containing 10% glycerol, 0.1% SDS, and 0.1% bromophenol blue as indicated, and allowing hydrolysis to proceed for 30 min at room temperature. Electrophoresis was then commenced at 60 V and the peptide patterns were visualized by fluorography (10). RESULTS

Translation of Vitellogenin Xenopus Oocytes

mRNA

in

Total RNA or poly(A)+-RNA was prepared from fat bodies of mature male or reproductively active female locusts and injected into Xenopus oocytes. Figure 1

a

b

cde -rrl

f9

Q FIG. 1. Synthesis of vitellogenin inxenopus oocytes injected with locust fat-body RNA. Batches of 30 oocytes were injected with 40-60 ml of RNA dissolved in injection medium or with injection medium alone, and labeled with 100 &i rH]leucine at 20°C for 18 h. Soluble oocyte proteins from each batch were precipitated with antivitellin serum (see Materials and Methods). Immunoprecipitates were analyzed on a slab gel containing 5% acrylamide and 0.1% SDS and then fluorographed. Slot a: oocytes injected with injection medium; slot b: oocytes injected with RNA (260 A,,Jml) from fat body of mature males; slot c: oocytes injected with oligo(dT)-cellulose flow-through RNA (260 A&ml) from fat body of mature females; slot d: poly(A)-containing RNA (38 A&ml) of mature females isolated by oligo(dT)-cellulose; slot e: oocytes injected with poly(a)-containing RNA (38 A&ml) of mature females isolated by poly(U)-Sepharose 4B; slots f-g: vitellogenin isolated from mature female fat body pulse labeled with rYS]methionine.

shows the translation products of total and poly(A)-enriched fat body RNAs as revealed by immunoprecipitation of rH]leutine-labeled soluble oocyte proteins with highly specific vitellin antibody. Injection of poly(A)+-RNA of reproductively active female locusts resulted in the synthesis of two polypeptides which were immunologically and electrophoretically identical to authentic unprocessed vitellogenin polypeptides, Vg, and Vgz (Fig. 1, slots d-g). Occasionally two smaller polypeptides (X, and X,) were also detected in lesser quantities. Total fat-body RNA of mature male locusts or poly(A)--RNA of

TRANSLATION

OF VITELLOGENIN

females failed to direct the synthesis of vitellogenin polypeptides in Xenopus oocytes (Fig. 1, slots b and c). To demonstrate that the induction of vitellogenin synthesis by JH involves the appearance of vitellogenin mRNA in the fat body, total cytoplasmic RNA was isolated from allatectomized female locusts treated with 250 pg of ZR-515 (in olive oil) and injected into Xenopus oocytes for translation. As shown in Fig. 2, RNA from hormone-treated females directed the syna b c .9-a-‘--

d

mRNA AND PEPTlDE ANALYSIS

thesis of vitellogenin polypeptides in Xen,omRNA activity was detected in RNA prepared from allatectomized females that had not been treated with ZR-515.

pus oocytes. No vitellogenin

Molecular

Weights

The molecular weights of Vg,, Vg,, and their processed products were determined by electrophoresis on SDS-polyacrylamide gels (5 and 7.5% acrylamide) and the results are presented in Figs. 3A-C. From three independent determinations on SDSpolyacrylamide slab gels (5% acrylamide) using myosin, Escherichia coli P-galactosidase, rabbit muscle phosphorylase b, bovine serum albumin, and ovalbumin as marker proteins, the molecular weights of primary vitellogenin polypeptides (Vg, and Vg,) are found to be 235,000 and 225,000 (Figs. 3A and C). The molecular weights of the processed radioactive vitellogenin polypeptides, determined in three independent experiments on SDS-polyacrylamide slab gels (7.5% acrylamide), are found to be (a) 126,000, (b) 117,000, (c) 112,000, (d) 104,000, (f) 64,000, and (h) 54,000 (Figs. 38 and C). These polypeptides are indistinguishable from those observed in stained SDS-gels of vitellin, but vitellin shows two additional polypeptides of 96,000 (e) and 57,000 (g), respectively (Fig. 3B). Tryptic

FIG. 2. Accumulation of vitellogenin mRNA in allatectomized female locusts treated with ajuvenile hormone active analog, ZR-515. RNA was extracted from fat bodies of allactectomized females treated with ZR515 for 72 h. Batches of 30 Xenopus oocytes were injected with 40-60 ml of RNA dissolved in injection medium or with injection medium alone, and were incubated with 100PCi p%]methionine at 20°C for 18 h. Soluble oocyte proteins from each batch were precipitated and analyzed as described under Materials and Methods. Slot a: oocytes injected with RNA (260 A,,Jml) isolated from reproductive active females; slot b: oocytes injected with RNA (260 A&ml) isolated from allatectomized but ZR-515 treated females; slot c: oocytes injected with RNA (260A,,dml) isolated from allatectomized females; slot d: oocytes injected with injection medium. The R, values for Vg, and Vg, are 0.23 and 0.25, respectively.

269

Peptide Analysis

Since vitellogenins are glycoproteins, it is conceivable that Vgl and Vg2 are the same polypeptide but containing different amounts of carbohydrate residues, resulting in different mobilities on SDS-polyacrylamide gels. To test for differences between these two polypeptides, Vgl and Vg, prepared by pulse labeling of the fat body in vitro with [35S]methionine were analyzed by tryptic peptide mapping. r5S]Methionine-labeled Vg, and Vg, were excised from preparative SDS-polyacryIamide slab gels, digested with trypsin (150 pg), and then the tryptic peptides were analyzed by high voltage electrophoresis and chromatography on a thinlayer plate coated with silica gel. As shown in Fig. 4A, though there were some com-

270

THOMAS

My

a

T. CHEN

bc

MW -A.

I+

13 9.4 6.8

4.3

B

0

0.2

0.4

C

0.6

0.8

1.0

Rf

FIG. 3. Molecular weight determination of Vg,, Vg, and their processed products by SDS-polyacrylamide gel electrophoresis. Vitellogenin was prepared by incubation of locust fat body in locust Ringer solution containing [W]methionine for 20 min or 3 h at 3o”C, and precipitated under vitellin antibody and goat anti-rabbit IgG as described under Materials and Methods. Protein markers used are myosin (200,000), E. coli P-galactosidase (130,000), phosphorylase b (94,000), bovine serum albumin (SS,OOO),and ovalbumin (43,000). (A) Polypeptide patterns of vitellogenin on SDS-polyacrylamide gels (5% acrylamide). A constant amount of radioactive proteins (lo5 cpm) was loaded on slots b and c. slot a, protein markers; b, vitellogenin (3-h labeling); c, vitellogenin (20-min labeling). (B) Polypeptide patterns of vitellogenin on SDS-polyacrylamide gels (7.5% acrylamide). Slot a, protein markers; b, vitellin; c, vitellogenin (3-h labeling). (C) Semilog plot of molecular weights of marker proteins, vitellin, and vitellogenin. R, is the relative mobility so that of bromophenol blue, and which was determined from three independent runs. Slot a, 5% acrylamide gel and slot b, 7.5% acrylamide gel. Arrows (a-h) indicate the R, values of processed vitellogenin polypeptides.

mon peptides present in Vg, and Vg,, the overall peptide fingerprint of Vg, was substantially different from that of the Vg,.

Since X, and X2 were also occasionally detected in the vitellogenin immunoprecipitate prepared from fat body maintained

TRANSLATION

OF VITELLOGENIN

ill zjitro or from Xenopus oocytes injected with female locust RNA, these two polypeptides might also represent two additional vitellogenin structural gene products. To test this possibility, [YS]methioninelabeled X, and X, isolated from fat body cultured in vitro were subjected to tryptic peptide mapping, and the results are presented in Figs. 4B and C. All of the tryptic peptides in X, or X, could be found in Vg, and Vg,, respectively. This suggests that X, was the proteolytic cleavage products of Vg, and X, was that of Vg,. Limited Proteolytic Vg?, X,, and X,

Digestion

of Vg,,

In order to provide an additional line of evidence regarding differences in chemical structure of Vg, and Vg,, radiolabeled polypeptides were subjected to peptide mapping by the method of Iimited proteolysis developed by Cleveland et al. (20). When [:‘“S]methionine-labeled authentic Vg, and Vg, were excised from a preparative gel and digested with different amounts of S. aureus V8 protease or papain, notable differences in digestion pattern from each polypeptide were obtained (Figs. 5A and B). The digestion patterns of each authentic polypeptide contained many peptides that were not present in the other. Vg, and Vg, isolated from Xenopus oocytes injected with fat-body RNA containing vitellogenin messenger activity were also digested with S. aureus V8 protease, and the digestion patterns are shown in Fig. 6. The patterns derived from these two polypeptides were indistinguishable from those derived from authentic Vg, and Vg,, respectively. To rule out the possibility that X, and X, were primary products of additional vitellogenin structural genes, these two polypeptides isolated from fat body cultured in vitro were also digested with S. aureus V8 protease. As presented in Figs. 7A and B, with the exception of two to three peptides missing from X, and X,, the peptide patterns derived from these polypeptides were identical to those of Vg, and Vg,, respectively. Therefore, it is concluded that Vg, and Vgz represent products from two

mRNA

AND PEPTIDE

ANALYSIS

271

different vitellogenin structural genes and that X, and X, are secondarily derived from these. DISCUSSION

The injection of mRNA into a living cell can yield two kinds of information: (a) the nature and amount of the injected mRNA, and (b) the specificity of the translational machinery and the presence of post-translational modification systems of the cell (Ref. (21), for review). In this paper I have demonstrated the translation of locust vitellogenin mRNA into two large polypeptides in Xenopus oocytes. These two polypeptides are immunologically and electrophoretically identical to Vg, and Vg, isolated from the fat body of reproductively active females. Further confirmation of the chemical identities of these two translation products as vitellogenins was obtained from peptide analysis using 5’. aureus V8 protease (Fig. 6). In their studies of the translation of Xenopus vitellogenin mRNA in Xenopus oocytes, Berriclge and Lane (22) reported that newly synthesized vitellogenin molecules were phosphorylated, proteolytically processed, and assembled into yolk platelets. However, as shown in Fig. 1, though each vitellogenin mRNA is translated faithfully into its product in Xenopus oocytes, no evidence of processing of these two large precursors into vitellin polypeptides was observed. This suggests that the enzyme responsible for the conversion of the primary vitellogenin polypeptides into the derived polypeptides in fat-body cells is not present in Xenopus oocytes. The molecular weights of the primary translational products of vitellogenin, Vg, and Vg,, were found to be 235,000 and 225,000, respectively. These values are more reliable than the previous estimates of 265,OOO:and250,000 which were based on an extrapolated standard curve from 7.5% acrylamide gels (10). The molecular weights of the processed polypeptides, ranging from 128,000to 54,000, also differ somewhat from the earlier report, which used a less satisfactory set of protein standards. The radioactive, newly formed vitellogenin polypep-

272

THOMAS

T. CHEN

TLC

3 +-----TLCFIG. 4. Tryptic peptide maps of pYS]methionine-labeled Vg,, Vg,, X,, and X2 isolated from locust fat body cultured in vitro. r5S]methionine-labeled Vg,, Vg,, X,, and X,, prepared by pulse labeling of fat bodies in vitro, were digested with 150 pg of trypsin. Tryptic peptides from each sample were subjected to electrophoresis (at pH 3.5) and chromatography on thin layer plates coated with silica gel. 0, origin of sample application. a, Vg, and Vg,, b, Vg, and X,, and c, Vg, and X,.

I

Al

2

3

4

5

6

912

“g1 -c

3

n

4

"g2

5

6

FIG. 5. Limited proteolytic digestion of Vg, and Vg, isolated from locust fat body cultured in vitro. Gel slices containing pYS]methionine-labeled proteins were applied to a second SDS-gel (15% acrylamide) in the presence of S. aureus V8 protease or papain. After electrophoresis, the peptide patterns were visualized by fluorography. (A) Peptide patterns generated by limited V8-protease digestion. Slots 1 and 4: 1.25 pg of protease; slots 2 and 5: 6.5 pg of protease; slots 3 and 6: 20 pg of protease. (B) Peptide patterns generated by limited papain digestion. Slots 1 and 4: 5 pg of papain; slots 2 and 5: 15 pg of papain; slots 3 and 6: 50 pg of papain. 273

274

THOMAS

T. CHEN

Vg, indeed are two different polypeptides. The same conclusion was arrived at when Vg, and Vg, isolated from Xenopus oocytes microinjected with poly(A)+-RNA of reproductively active female locusts were analyzed by limited proteolysis with V8 protease (Fig. 6). Two additional high molecular weight polypeptides, X, and X2, occasionally found in fat body pulse labeled with radioactive amino acids and in samples prepared from Xenopus oocytes injected with vigellogenin mRNA preparations, might conceivably be primary translational products of yet another pair of vitellogenin genes. However, peptide mapping of X, and X, isolated from fat body labeled with [35S]methionine showed that they were proteolytic cleavage products of Vg, and Vg,, respectively, as a result of the natural course of processing or an artifact during sample preparation. Therefore, this study confirms that locust vitellogenin is a dimer of two primary subunits with molecular 12 3 4 5 6 weights of about 235,000 (Vg,) and 225,000 FIG. 6. Limited S. aureus V8 protease digestion of WgZ) which are probably the products of Vg, and Vg, made in Xenopus oocytes microinjected two types of vitellogenin structural genes. with vitellogenin mRNAs. [3SS]Methionine-labeled Vg, These two primary polypeptides are conand Vg,, isolated from oocytes injected with locust verted to the final peptides of 126,000 to fat-body poly(A)-containing RNA, were separated on a 52,000 M, as the result of proteolytic cleavSDS-gel (5% acrylamide). Gel slices containing Vg, and Vg, were excised and loaded on to a second SDSage, during post-translational processing gel (15% acrylamide) in the presence of various amounts within the fat-body cells. If all of the susof Wprotease. After electrophoresis, the peptide patceptible sites in the vitellogenin molecules terns were visualized by fluorography. Slots 1 and 4: are not cleaved in every molecule, then 1.25 pg of protease; slots 2 and 5: 6.5 pg of protease; nonintegral proportions of the processed slots 3 and 6: 25 pg of protease. products will be expected (10). Proteolytic cleavage from large pretides isolated from locust fat body main- cursors may similarly account for the prestained in vitro are lacking two components ence of the multiple polypeptides (or sub(M, 96,000 and 57,000) which are seen in units) observed in other insect vitellins stained SDS-polyacrylamide gels of vitello- and vitellogenins (2). In the cockroach, genin and vitellin. This may reflect further, Leucophaea maderae, a complex pattern of delayed processing, or possibly an absence polypeptides (with apparently integral of methionine in these regions of the ratios) released from vitellogenin or vitellin upon denaturation has been described protein. In the previous studies by pulse labeling by Koeppe and Ofengand (23). Data from of fat body in Ringer solution with 3H- experiments involving incorporation of in fat labeled amino acids, it was suggested that amino acids for 5 h into vitellogenin the rapidly labeled polypeptides (now called bodies maintained in culture have led these Vg, and Vg,) may represent products of authors to suggest that the observed polytwo vitellogenin structural genes (10). I peptides originated from two separately precursors, believed to reprehave now shown by tryptic peptide mapping translated and mapping with limited papain or S. sent separate structural genes. Recently, that vitellins aureus V8 protease digestion that Vg, and Harnish et al. (24) reported

TRANSLATION

Al2

3

OF VITELLOGENIN

4

5

6

mRNA

AND PEPTIDE

Bl

27.5

ANALYSIS

2345

6

FIG. 7. Comparison of peptide patterns generated from fat-body Vg,, Vg,, X,, and X, by limited digestion with S. aureus V8 protease. (A) Peptide patterns generated from Vg, and X,; (B) peptide patterns generated from Vg, and X,. Slots 1 and 4: 1.25 kg of protease: slots 2 and 5: 6.5 pg of protease; slots 3 and 6: 20 pg of protease.

of representatives of six insect orders appear to be derived from primary polypeptides of about 225,000 M,, and that, in some species at least, more than one gene is involved. On the other hand, Warren et al. (25) and Bownes (26) reported recently that the vitellogenin of Drosophila melanogaster consisted of three polypeptides of molecular weights 44,000, 45,000, and 46,000, respectively which are only slightly smaller than the primary products of translation. Multiple genes coding for vitellogenin may also exist in Xexopus Laevis. By employing recombinant DNA methods to study the vitellogenin gene, Wahli et al. (27) have concluded that at least four genes for this protein may exist in the frog genome. Intracellular proteolytic processing of newly synthesized proteins is well known in many systems such as in viruses (28), polypeptides hormones, and other secretory proteins (29). The initially translated forms of these proteins are larger proproteins which probably exercise a role in

vectoral transport into the cisternae of endoplasmic reticulum (30). Most of the vitellogenins are synthesized as large polypeptides and then processed into smaller polypeptides either in the cell where they are synthesized as in locusts (10) or in oocytes as in Xenopus (4, 31). However, there is no evidence as to the functional significance of intracellular processing, but the possibility of some role in secretory transport or selective uptake by oocytes seems worth exploring. ACKNOWLEDGMENTS I thank Dr. Y. Masui for teaching me the technique of Xenopus oocyte microinjection. I am also grateful to Drs. G. R. Wyatt, D. M. Davies, and G. J. Sorger for critically reading the manuscript. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the McMaster University Research Board. REFERENCES 1. TELFER, W. H. (1965) Ann7~. 10, 161-184

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THOMAS

2. WYATT, G. R., AND PAN, M. L. (1978) Annu. Rev. Biochem. 47, 779-817. 3. HAGEDORN, H. H., AND KUNKEL, J. G. (1979) Annu. Rev. Entomol. 24, 475-505. 4. CLEMENS, J. M. (1974) Progr. Biophys. Mol. Biol. 28, 71-107. 5. TATA, J. R. (1976) Cell 9, 1-14. 6. ENGELMANN, F. (1974) Amer. Zool. 14, 11951206. 7. CHEN, T. T., COUBLE, P., ABU-HAKIMA, AND WYATT, G. R. (1979) Develop. Biol. 69, 59“”

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