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Differentiation in the salivary glands of Drosophila melanogaster: Characterizaion of the glue proteins and their developmental appearance
Cell, Vol . 9, 3 65-373, November 1976, Copyright © 1976 by MIT
Differentiation in the Salivary Glands of Drosophila melanogaster : Characterization of the Glue Proteins and Their Developmental Appearance Steven K . Beckendorf* and Fotis C . Kafatos The Biological Laboratories Harvard University 16 Divinity Avenue Cambridge, Massachusetts 02138
Summary The larval salivary glands of Drosophila melanogaster, which are widely used for cytogenetic studies, are also useful for studying the regulation of specific protein synthesis during differentiation . These glands synthesize and secrete massive amounts of a glue which attaches the pupae to the substrate during metamorphosis. We find six major proteins in this glue . They show wide quantitative and qualitative variation among wild-type strains of D . melanogaster . Four of the proteins are glycosylated, and one of these is further modified so that its mobility on SDS polyacrylamide gels is greater than the mobility of its unmodified precursor . The glue proteins begin to be made at about 106 hr after egg deposition . The synthesis of four of the proteins begins coordinately, while one protein begins to be made slightly earlier and one slightly later . The proteins are synthesized for approximately 14 hr until puparium formation, when the glue is released from the salivary glands . Synthesis of five of the glue proteins stops abruptly within a few minutes after the glue is released . The sixth protein continues to be synthesized for at least 30 min after glue release . Introduction Near the end of their development, Drosophila melanogaster larvae crawl up the walls of their culture bottles and form puparia which later become pupae . As the puparium forms, the bloated salivary glands release their contents through the mouth . This secretion, which contains protein and carbohydrate, then hardens and becomes a glue which firmly attaches the pupa to the wall (Fraenkel and Brookes, 1953) . Several aspects of the production of the glue proteins are attractive for studies of developmental gene regulation . The proteins are produced in very large amounts in the salivary glands (Zhimulev and Kolesnikov, 1975), thereby facilitating biochemical analysis . The glands are noted for their giant polytene chromosomes which offer the possibility of cytogenetic analysis . Glue protein synthesis can be `Present address: Department of Molecular Biology, Stanley Hall, University of California, Berkeley, California 94720 . Reprint requests should be sent to this address .
analyzed genetically, and puffs in the polytene chromosomes correlated with individual proteins (Korge, 1975) . Korge (1975) has recently described four of the glue proteins and located two of them genetically . In this report, we identify six glue proteins, document their polymorphism in various Drosophila stocks, study the post-translational modifications of the proteins, and analyze their developmental expression . Results Identification of Proteins in the Glue Initial experiments were designed to determine how many proteins are present in the glue . As shown in Figure 1, there are at least eight major protein bands in glue from the Oregon R strain . All these proteins are present in bloated glands, and are largely or completely absent from prepupal glands which have released the glue . The absence of P2b, P5, and P6 from prepupal glands cannot be determined immediately from Figure 1, but can easily be demonstrated in strains which produce larger amounts of these proteins . Unlike the other proteins, P1 is very diffuse and often smears as though it has stuck to the edges of the sample slot . In addition, it stains quite weakly with Coomassie blue, usually with a magenta color rather than the dark blue or purple of most proteins . However, it does seem to be a protein and a component of the glue, since it is rapidly labeled by 3 H-proline (see below), is present in isolated glue, and is absent from prepupal glands . The bands P2a and P2b are specified by the X chromosome and appear to be allelic ; male larvae produce either P2a or P2b but never both (Figure 2) . Bands P4a and P4b also seem to be related to each other ; pulse-chase experiments show that they are made from the same precursor, or from two precursors of identical mobility on SDS gels (see below) . The apparent molecular weights of the proteins as determined by their mobility relative to a group of standard proteins are listed in Table 1 . These values are empirically useful, but for P1-P4 do not correspond to the molecular weight of the polypeptide chain since these proteins are extensively modified (see below) . In addition to the eight major bands that are labeled in Figure 1, there are several minor bands in the isolated glue . Of these, the band migrating just ahead of P3 is the best candidate for a glue protein . We have not studied it further because it is not reliably found in isolated glue (see Figure 3) and because it coincides with or is identical to a band which is still present in the glands after the glue is released (Figure 1) . Since the amounts of
Cdl 366
most of the other minor proteins vary from sample to sample, they are probably due to contaminating cellular proteins. Interstrain Variation To determine what types of natural variation exist among the glue proteins, we analyzed seven wildtype strains of D. melanogaster (Figure 3). Two
ABCD
E
PI P2a Ff”
kinds of variation are obvious: d’ifferences in amount and in electrophoretic mobility. The variations in mobility cannot be solely due to amino acid substitutions, since SDS gels separate normal proteins on the basis of molecular weight rather than charge differences (Weber and Osborn, 1968). Pl is present in similar amounts in all the strains, but its mobility varies. Proteins P2 and P3 are particularly variable in mobility; in some strains (for example, Urbana S and Lausanne S), it is difficult to determine which bands corresponds to P2 and which to P3. P2 and P3 also vary in amount from strain to strain. At the extreme, P3 appears to be missing from Hikone AW and Hikone AS. P4 is present in all the strains, and in most of them, it resolves into two bands corresponding to P4a and P4b. The different forms of this protein show large variations in amount in different strains. P5 and P6 seem to vary only in amount, P5 being prominent in only two of the seven strains. Protein Modification The presence of carbohydrate in the glue (Ashburner and Blumental, 1970) and the interstrain variations in electrophoretic mobility suggested that the proteins might be modified post-translationally. As a first test for modification, we looked for anomalous migration of the glue proteins on SDS gels of different acrylamide concentrations. To examine si-
Q
P2a P2b
P4a P4b
Ps P6 Figure 1. SDS Proteins
Polyacrylamide
Gel of Salivary
Glands
and
Glue
(A) bloated third instar glands; (B-D) three samples of isolated glue dissected from ethanol-fixed, bloated glands; (E) prepupal glands isolated just after the gluewas released. Each sample represents material from ten glands from Oregon R larvae.
Figure 2. Isolated of Oregon R
Glue
from
Individual
Male
and
Female
Larvae
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Table
Salivary
1. Apparent
Gland
Molecular
Proteins
Weights
of Oregon
R Glue
Proteins
Pl
130,000-160,000
P2a
120,000
P2b
105,000
P3
91,000
P4a
20,500
P4b
20,000
P5
15,000
P6
8,500
Values were calculated by comparing the mobilities of Oregon R glue proteins on a IO-20% acrylamide gradient gel with the mobilities of the following proteins: phosphorylase A, bovine serum albumin, pyruvate kinase, horseradish peroxidase, deoxyribonuclease I, a-chymotrypsinogen, trypsinogen, trypsin, myoglobin, hemoglobin, lysozyme, cytochrome c. Peptides produced by cyanogen bromide cleavage of myoglobin (Swank and Munkres, 1971) were used as standards for the low molecular weight ranqe.
multaneously a wide range of acrylamide concentrations, we electrophoresed salivary gland proteins vertically through a slab gel containing a horizontal gradient of acrylamide increasing exponentially from one end of the slab to the other (Figure 4). We reasoned that since most proteins migrate at rates proportional to their molecular weights even when the acrylamide concentration is varied (Weber and Osborn, 1968), such proteins would form a series of noncrossing lines when electrophoresed on the horizontal gradient gel. However, members of at least one class of modified proteins, the glycoproteins, migrate anomalously on SDS gels; the apparent molecular weight of some glycoproteins changes as the acrylamide concentration changes (Bretscher, 1971; Segrest and Jackson, 1972). On a horizontal gradient gel, the line formed by an anomalous protein should cross lines formed by unmodified proteins. We find that some but not all standard glycoproteins meet this expectation (data not shown). In Figure 4, salivary gland proteins from Oregon R are displayed on such a gel, revealing that P2a and b, P3, and P4a and b behave anomalously: they cross many of the lines formed by the majority of the proteins. Because it is of lower mobility than any other prominent protein, Pl does not cross any lines. In its course, however, it more nearly parallels P2a and b and P3 than it does the other proteins. The P5 of both Oregon R (Figure 4) and Hikone AW (not shown) behaves normally in this test; it does not cross any of the other proteins. Although there are few proteins with similar mobility for comparison, P6 from both strains also seems to behave normally. Pulse-chase experiments also suggested that the proteins are modified. Oregon R larvae were injected with 3H-proline, and their salivary glands were isolated at various times and analyzed by SDS gel
Figure Strains
3. Isolated
Glue
from
Larvae
of Seven
Different
Wild-Type
(0) Oregon R; (HW) Hikone AW; (F) Florida-g; (U) Urbana S; (S) Swedish C; (HS) Hikone AS; (L) Lausanne S. Each sample represents material from four glands. The proteins in one of the Oregon R slots are labeled for reference.
electrophoresis followed by autofluorography. As shown in Figure 5, at 2.5 min after injection, there is no material corresponding to PI, P2, or P3. Instead there is a broad labeled zone migrating faster than any of the three proteins. With increasing time after injection, the mobility of this zone decreases, and bands eventually appear at the positions of Pi,
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P2, and P3. By 40 min, these three proteins account for most of the radioactivity in the upper part of the gel. Their delayed appearance is probably not due to the time necessary for synthesis of the polypeptide chains, since other visible bands of high molecular weight show their normal mobility in samples as early as 5 min after injection. These data therefore suggest, although they do not prove, that following their synthesis, PI, P2, and P3 become modified in such a way that their mobility on SDS gels is decreased. One would expect a decrease in mobility as a protein became glycosylated (Bretscher, 1971; Segrest and Jackson, 1972). Figure 5 also shows that the apparent precursor of P4 (P4*) moves slightly slower than the finished product (mostly P4a in Oregon R). This difference, although minor, was consistently observed. The conversion of P4* to P4b as well as P4a in Lausanne S is also shown in Figure 5. At IO min after injection, P4” is labeled, while at 60 min, there are
two labeled bands corresponding to P4a and P4b. A similar experiment with Hikone AW showed that the positions of P5 and P6 are constant from 10 until 60 min after injection (Figure 5). To test directly for carbohydrate attached to the proteins, a gel was stained by the PAS reaction. As shown in Figure 6, there is a large smear of carbohydrate, which includes PI, at the top of the gel. P2a and b, P3, and P4a and b are also PAS-positive. P5 and P6 are unstained even in strains in which they are abundant. The hue of the PAS stain is characteristic for particular proteins. By using these characteristic hues, we can distinguish between P2 and P3 when their identification would otherwise be uncertain, as in Lausanne S. The Developmental Program of Glue Protein Synthesis To determine when the glue proteins are made, groups of Oregon R larvae were injected with 3H-
P4c1*
l Pl
P5
.
P6
.
l P2a .P2b
l P3
Figure
4. Behavior
When this gel was and an exponential of widely varying (see Experimental respectively, the the left end was sample containing
of Glue
Proteins
on an SDS-Polyacrylamide
Gel Containing
a Horizontal
Gradient
of Acrylamide
Concentration
being cast, the glass plates of the template were held in an orientation rotated clockwise 90” from the final orientation, gradient of 5-20% acrylamide was poured. The slope of the gradient was chosen so that the lines formed by proteins molecular weight would approach linearity. The gradient was formed by the same method as the vertical gradient gels Procedures), except that for this gel, the two gel solutions used to form the gradient contained 0% and 20% acrylamide, mixing chamber contained 3.6 ml, and the entire gel contained 5 ml. After polymerization, the gel was rotated, and sealed with a gel solution containing 5% acrylamide. A stacking gel was then poured with one long sample slot. A 150 bloated Oregon R salivary glands was layered across the top and electrophoresed at 10 mA for 3 hr.
Drosophila 369
Salivary
Gland
Proteins
l Pl,
P2
l P2b ,P3
1
p4*.
l P4a
I
I .P6
l P6
l P6 Figure
5. Modification
of Oregon
R Glue
Proteins
during
a Pulse-Chase
Experiment
Late third instar larvae were injected with ‘H-proline, and their salivary glands were isolated and prepared for electrophoresis at the times indicated (minutes after injection). After electrophoresis, the labeled bands were detected by autofluorography. (0) Oregon R; (L) Lausanne S; (HW) Hikone AW. The major band beneath PI, P2 in the 60 min HW sample may represent partially modified glue proteins which would move up to the position of Pi, P2 with a longer chase. Alternatively, this band may reveal that Hikone AW makes some P3 which can be detected by autofluorography, but which never accumulates in sufficient amounts to be detectable by Coomassie brilliant blue staining (see Figure 3 for the electrophoretic pattern of mature Hikone AW glue proteins).
Cell 370
proline at various times from early third instar until puparium formation at 120 hr after egg deposition. Their salivary glands were removed 30 min after injection, and the newly synthesized proteins analyzed by electrophoresis on SDS gels followed by autofluorography. As shown in Figure 7, the proteins are first clearly visible at about 106 hr after the egg is laid. They are then made continuously until the glue is released at puparium formation, 14 hr later.
0 C L HW
OCLHW
Since the larvae are only partially synchronized, one cannot determine precisely when a particular protein begins to be made. However, the order in which the synthesis of different proteins begins can be determined by analyzing many samples labeled at about 106 hr. Assume that once the synthesis of a protein begins, it continues until puparium formation. Then if a larva is found to be synthesizing just one of the proteins, that must be the protein whose synthesis begins first. A larva making two proteins must include the first protein and the protein whose synthesis begins second.This logic can be extended to order all the proteins. Figure 8 shows some typical results which indicate the order deduced for the glue proteins of Oregon R. P6 begins its synthesis first; P2, P3, and P4 begin coor-
Figure Figure 6. Comparison of Coomassie Salivary Gland Proteins Each of the four PAS-stained. Each and was stained (C) Canton S; (L)
Brilliant
Blue and PAS-Stained
slots at the left contained 25 glands and was of the four slots at the right contained 10 glands with Coomassie brilliant blue. (0) Oregon R; Lausanne S; (HW) Hikone AW.
7. Developmental
Program
of Glue
Protein
Synthesis
Oregon R larvae of the indicated ages (time after oviposition) were injected with XH-proline, and their salivary glands were isolated and prepared for electrophoresis 30 min later. After electrophoresis, the labeled bands ware detected by autofluorography. The last sample (WPP) came from a white prepupa injected within 15 min after puparium formation.
Drosophila 371
Salivary
Gland
Proteins
dinately slightly later; and Pl begins last. The beginning of P5 synthesis could not be determined in Oregon R, but in Hikone AW P5 begins to be synthesized at the same time as P2 and P4. To determine when the proteins cease being synthesized, we examined samples labeled just before or just after puparium formation. Fifteen mature larvae whose salivary glands were bloated with glue ready to be released were tested. All fifteen continued to synthesize all six glue proteins. In contrast, synthesis of either Pl, P2, or P3 was not detected in any of thirty labeled prepupae, even when they were injected with label within 1 min after the glue was released. Synthesis of P4 and P5 was occasionally detected at reduced levels in prepupae injected
PI
l
. P2b P3
l
l
-figure
8. Initiation
of Glue
Protein
P4a
l P6 Synthesis
Oregon R larvae 98-106 hr old were injected with 3H-proline, and their salivary glands were isolated and prepared for electrophoresis 30 min later. After autofluorography, the samples were arranged from left to right in the order in which the proteins begin to be made.
within 15 min after release of the glue (for example, Figure 7, slot wpp). P6 or another protein with the same mobility as P6 continues to be made for at least 30 min after release of the glue. Discussion Identification and Function of the Glue Proteins Eight proteins are prominent in glue isolated from Oregon R salivary glands. These all seem to be genuine components of the glue and not artifacts of dissection, since they disappear from the glands when the lumen empties at puparium formation. One pair of the proteins, P2a-P2b, is the result of a genetic polymorphism in the Oregon R stock. Another pair, P4a-P4b, are produced from a single precursor, P4*‘. Thus we believe there are six distinct proteins in Oregon R glue: Pl-P6. This basic pattern is confirmed by analysis of seven different wild strains. All produce at least five of the proteins or their variants, and none produces any additional proteins. In a recent study, Korge (1975) identified four glue-specific proteins and genetically localized two of them. His fraction 4, the gene for which is located by recombination at 3.5 on the X chromosome and is included in the Notch deletion N8, is probably identical with our P3. P3 is present in about the same relative amount as his fraction 4 and is included within the smaller Notch deletion N64il6 (S. K. Beckendorf, unpublished results). Both ends of the N64i16 deletion are within the region deleted by N* (Lefevre and Green, 1972). We have not yet attempted to correlate any of the other proteins with those identified by Korge. Though all six of the proteins described here are contained in the glue, it is not certain how many of them contribute to its stickiness. However, examination of the proteins made by the various wild-type strains (Figure 3) can reveal whether or not particular proteins are necessary for an effective glue. Since P3 is missing from the glue in Hikone AW and Hikone AS, and since P5 is missing or nearly missing in several strains, these proteins cannot be required for glue function. Similarly, no particular allele of PI, P2, or P4 is required. It seems probable that the stickiness of the glue is a result of several or all of its proteins. Modification of the Proteins Chemical analysis of glue has shown it to be composed of 30% carbohydrate and 70% protein (Ashburner and Blumenthal, 1970). We have shown here that some of the carbohydrate is covalently linked to four of the proteins (Pl-P4). Since glycosylation reduces the mobility of proteins on SDS gels (Bretscher, 1971; Segrest and Jackson, 1972), and
Cell 372
since P4a and P4b have a higher mobility than their precursor, P4*, this precursor must undergo some other modification in addition to glycosylation . Perhaps the polypeptide chain is cleaved during glycosylation . We have no evidence that two of the proteins, P5 and P6, are modified . However, the glue contains substantial amounts of glucosamine and galactosamine which are not detected by the PAS reaction . Either of these in small amounts might not have altered the mobility of the proteins enough to be detected on the horizontal acrylamide gradient gels or in the pulse-chase experiments . Developmental Control of Glue Protein Synthesis The overall pattern of glue protein synthesis can be summarized as follows . At about 106 hr, the proteins begin to be made : P6 first, then the group P2P5, and finally P1 . All the proteins continue to be synthesized until puparium formation, at which time all but P6 are turned off . Previous studies which followed glue synthesis by PAS staining of intact glands or by observing secretory granules in salivary gland cells concluded, in contrast to our results, that glue production begins at 90-95 hr (Lane, Carter, and Ashburner, 1972 ; Zhimulev and Kolesnikov, 1975) . To be certain that we were following the same developmental processes, we injected 96-106 hr larvae with 3H-proline and removed the salivary glands 30 min later . One gland from each larva was PAS-stained, and its twin was analyzed on a gel . In all cases, the onset of PAS staining coincided with the beginning of 3 H incorporation into glue proteins . Thus we can only explain the timing discrepancy as due to differences in the fly stocks or culture conditions . The termination of synethesis of all the glue proteins except P6 seems to be quite abrupt, probably occurring within a few minutes before and after the glue is released . In contrast, the two puffs known to make glue proteins, 3C11-12 and 68C (Korge, 1975), gradually decrease in size beginning at least 10 hr before puparium formation and release of the glue (Ashburner, 1967, 1969) . Thus the abrupt shutoff of glue protein synthesis does not simply reflect the end of transcription but must be under some type of post-transcriptional regulation . It is possible that, like many other events occurring shortly before or during puparium formation, cessation of glue protein synthesis is controlled by ecdysone . Preliminary experiments reported by Berendes (1974) suggest that glue protein synthesis is blocked when salivary glands are incubated in vitro with ecdysone . During normal development, however, any effect of ecdysone must be rather indirect . As indicated by the appearance of ecdysoneinduced puffs (Ashburner, 1967), the ecdysone
titer increases at least 10 hr before glue protein synthesis ceases . Initiation of glue protein synthesis is more difficult to study because of the lack of a marker like puparium formation which can be used to synchronize the larvae . Thus although we have established the sequence in which the synthesis of the various proteins begins, it is not possible to determine how much time elapses between the initiation of the first protein, P6, and the last protein, P1 . No survey of puffs present in the polytene chromosomes has been made prior to the beginning of glue protein synthesis . We do not yet know, therefore, how the beginning of transcription of the glue protein genes is related to the beginning of their translation .
Experimental Procedures Fly Stocks The Oregon R wild-type strain of D . melanogaster was used as the standard strain for these experiments . Other wild-type strains used were Canton S, Urbana S, Swedish C, Hikone AW, Hikone AS, Lausanne S, and Florida-9 . All flies were maintained at 25°C on a medium containing 90 g dry yeast, 380 g corn meal, 720 ml Karo syrup, 56 g agar, 160 g malt, 24 ml 95% ethanol, 5000 ml water, and 60 ml acid mix A . Acid mix A contained 42 ml concentrated H3 PO4 and 418 ml propionic acid in 1000 ml solution . Larval Synchronization For timed experiments, fertilized eggs were collected for 1-2 hr from a large population cage of well fed flies . 24 hr later, unhatched eggs were transferred to a small petri dish containing food . Larvae hatching during the next 2 hr (or in some cases, 1 hr) were placed in 16 x 50 mm petri dishes containing 10 ml of food prepared the same day and a drop of live yeast suspension . 20 larvae were placed in each dish . The mean time of puparium formation was 118-120 hr after the eggs were laid, with a range of approximately f 4 hr . Efforts to improve the synchrony, including collecting larvae as they molted from second to third instar (von Gaudecker, 1972), were ineffective . Polyacrylamide Gels Polyacrylamide gels containing SDS and a discontinuous buffer system were formulated according to Laemmli (1970), with the exception that the stacking gel contained 4 .5% rather than 3% acrylamide . Two sizes of slab gels were used : 9 x 6 .5 x 0 .1 cm, or 14 x 14 x 0 .1 cm . Except when noted otherwise in the figure legends, the gels contained an exponential gradient of acrylamide increasing from 10 .3-20% from top to bottom . The two solutions used to produce this gradient contained 10% and 20% acrylamide, respectively . To form one of the large gels, 5 ml of the 20% solution were placed in a 10 ml flask and stirred . Solution was removed from this mixing chamber and delivered to the gel template at the same rate as the 10% acrylamide solution was added to the mixing chamber . The final volume of the gel was 18 ml, so the concentration at the top of the gel was 10 .3% (for this calculation, see Noll, 1969) . Volumes for the small slabs were one third of those for the large slabs . The small gels were electrophoresed at 10 mA at room temperature . The large gels were run at either 15-20 mA at room temperature or 30 mA at 4-6°C (maintained by circulating refrigerated water through a plexiglass maze held in contact with the glass plates of the gel) . The resolution of the glycoproteins, especially P1, was better with the higher current and low temperature conditions .
Drosophila Salivary Gland Proteins 373
For protein detection, gels were fixed and stained in 0 .1% Coomassie brilliant blue R, 50% methanol, 10% acetic acid, and destained in 10% methanol, 7 .5% acetic acid . For carbohydrate detection, gels were stained by the periodic acid Schiff (PAS) reaction essentially as described by Fairbanks, Steck, and Wallach (1971), but with most of the times decreased .
Laemmli, U . (1970) . Nature 227, 680-685 . Lane, N ., Carter, Y . R ., and Ashburner, M . (1972) . Wilhelm Roux Arch . 169, 216-238 . Lackey, R. A ., and Mills, A . D . (1975) . Eur. J . Biochem . 56, 335-341 . Lefevre, G ., and Green, M . M . (1972) . Chromosome 36, 391-412 . Noll, H . (1969) . Tech . Protein Biosyn . 2, 101-179 .
Autofluorography Gels containing 3H-labeled proteins were subjected to autofluorography according to the method of Bonner and Laskey (1974) . The Kodak RP Royal film used was preexposed to 0 .1-0 .2 A560 according to Laskey and Mills (1975) .
Segrest, J . P., and Jackson, R . L . (1972) . Methods Enzymol . 28, 54-63 .
In Vivo Labeling In several experiments, the proteins were labeled with 3 H-proline . Proline was chosen because the glue has been shown to be rich in proline (Ashburner and Blumenthal, 1970), and because comparison between 3 H-leucine and 3H-proline showed that proline preferentially labels the glue proteins . For injection into larvae, 3 H-proline was evaporated to dryness under a stream of nitrogen and dissolved at a final concentration of 100 mCi/ml in Grace's medium lacking proline . Between 0 .2 and 0 .5 µl were injected into each larva . Larvae were maintained until dissection in small petri dishes containing Whatman 3MM filter paper discs moistened with a 10% sucrose solution . Larvae were dissected in Drosophila Ringer's solution (Ephrussi and Beadle, 1936), and the isolated glands were rinsed in Ringer's and dissolved by heating at 100°C for 1 min in electrophoresis sample buffer [0 .0625 M Tris-HCI (pH 6 .8), 1% SDS, 5% 2-mercaptoethanol] (Laemmli, 1970) .
Weber, K ., and Osborn, M . (1968) . J . Biol . Chem . 244, 4406-4412 .
Isolation of Secretion from Glands When we wanted to collect only the secreted glue, fully bloated glands were transferred to 95% ethanol . The secretion contracts and becomes a solid mass which can then be dissected free of the salivary gland cells (Kodani, 1948) . Isolated secretion masses were then dissolved in electrophoresis sample buffer . Acknowledgments We are grateful to Drs . Kalpana White and Michael Ashburner for helpful discussions, and to Drs . Argiris Efstratiadis and J . C . Regier for critical readings of the manuscript . Dr . William H . Petri graciously supplied the strains of D . melanogaster which we used . This research was supported by a grant from the NSF to F . C . K . During part of the time that this research was conducted, S . K . B . was a fellow of the Jane Coffin Childs Foundation for Medical Research . Received June 25, 1976 ; revised August 2, 1976 . References Ashburner, M . (1967) . Chromosoma 21, 398-428 . Ashburner, M . (1969) . Chromosoma 27, 47-63 . Ashburner, M ., and Blumenthal, A . (1970) . As cited in Ashburner, M . (1970) . Adv . Insect Physiol . 7, 1-95 . Berendes, H . D . (1974) . Proceedings IV International Congress of Endocrinology (Amsterdam : Excerpta Medica), pp . 311-314 . Bonner, W. M ., and Laskey, R . A . (1974) . Eur . J . Biochem . 46, 83-88 . Bretscher, M . S . (1971) . J . Mol . Biol . 58, 775-781 . Ephrussi, B ., and Beadle, G . W . (1936) . Am . Naturalist 70, 218-225 . Fairbanks, G ., Steck, T . L., and Wallach, D . F. H . (1971) . Biochemistry 10, 2606-2617 . Fraenkel, G ., and Brookes, V . J . (1953) . Biol . Bull . 105, 442-449 . Kodani, J . (1948) . Proc. Nat . Acad . Sci . USA 34, 131-135 . Korge, G . (1975) . Proc . Nat . Aced . Sci . USA 72, 4550-4554 .
Swank, R . T ., and Munkres, K . D . (1971) . Anal, Biochem . 39, 462477 . von Gaudecker, B . (1972) . Z . Zellforsch . 127, 50-86 .
Zhimulev, I . F ., and Kolesnikov, N . N . (1975) . Wilhelm Roux Arch . 178, 15-28 .
Differentiation in the Salivary Glands of Drosophila melanogaster : Characterization of the Glue Proteins and Their Developmental Appearance Steven K . Beckendorf* and Fotis C . Kafatos The Biological Laboratories Harvard University 16 Divinity Avenue Cambridge, Massachusetts 02138
Summary The larval salivary glands of Drosophila melanogaster, which are widely used for cytogenetic studies, are also useful for studying the regulation of specific protein synthesis during differentiation . These glands synthesize and secrete massive amounts of a glue which attaches the pupae to the substrate during metamorphosis. We find six major proteins in this glue . They show wide quantitative and qualitative variation among wild-type strains of D . melanogaster . Four of the proteins are glycosylated, and one of these is further modified so that its mobility on SDS polyacrylamide gels is greater than the mobility of its unmodified precursor . The glue proteins begin to be made at about 106 hr after egg deposition . The synthesis of four of the proteins begins coordinately, while one protein begins to be made slightly earlier and one slightly later . The proteins are synthesized for approximately 14 hr until puparium formation, when the glue is released from the salivary glands . Synthesis of five of the glue proteins stops abruptly within a few minutes after the glue is released . The sixth protein continues to be synthesized for at least 30 min after glue release . Introduction Near the end of their development, Drosophila melanogaster larvae crawl up the walls of their culture bottles and form puparia which later become pupae . As the puparium forms, the bloated salivary glands release their contents through the mouth . This secretion, which contains protein and carbohydrate, then hardens and becomes a glue which firmly attaches the pupa to the wall (Fraenkel and Brookes, 1953) . Several aspects of the production of the glue proteins are attractive for studies of developmental gene regulation . The proteins are produced in very large amounts in the salivary glands (Zhimulev and Kolesnikov, 1975), thereby facilitating biochemical analysis . The glands are noted for their giant polytene chromosomes which offer the possibility of cytogenetic analysis . Glue protein synthesis can be `Present address: Department of Molecular Biology, Stanley Hall, University of California, Berkeley, California 94720 . Reprint requests should be sent to this address .
analyzed genetically, and puffs in the polytene chromosomes correlated with individual proteins (Korge, 1975) . Korge (1975) has recently described four of the glue proteins and located two of them genetically . In this report, we identify six glue proteins, document their polymorphism in various Drosophila stocks, study the post-translational modifications of the proteins, and analyze their developmental expression . Results Identification of Proteins in the Glue Initial experiments were designed to determine how many proteins are present in the glue . As shown in Figure 1, there are at least eight major protein bands in glue from the Oregon R strain . All these proteins are present in bloated glands, and are largely or completely absent from prepupal glands which have released the glue . The absence of P2b, P5, and P6 from prepupal glands cannot be determined immediately from Figure 1, but can easily be demonstrated in strains which produce larger amounts of these proteins . Unlike the other proteins, P1 is very diffuse and often smears as though it has stuck to the edges of the sample slot . In addition, it stains quite weakly with Coomassie blue, usually with a magenta color rather than the dark blue or purple of most proteins . However, it does seem to be a protein and a component of the glue, since it is rapidly labeled by 3 H-proline (see below), is present in isolated glue, and is absent from prepupal glands . The bands P2a and P2b are specified by the X chromosome and appear to be allelic ; male larvae produce either P2a or P2b but never both (Figure 2) . Bands P4a and P4b also seem to be related to each other ; pulse-chase experiments show that they are made from the same precursor, or from two precursors of identical mobility on SDS gels (see below) . The apparent molecular weights of the proteins as determined by their mobility relative to a group of standard proteins are listed in Table 1 . These values are empirically useful, but for P1-P4 do not correspond to the molecular weight of the polypeptide chain since these proteins are extensively modified (see below) . In addition to the eight major bands that are labeled in Figure 1, there are several minor bands in the isolated glue . Of these, the band migrating just ahead of P3 is the best candidate for a glue protein . We have not studied it further because it is not reliably found in isolated glue (see Figure 3) and because it coincides with or is identical to a band which is still present in the glands after the glue is released (Figure 1) . Since the amounts of
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most of the other minor proteins vary from sample to sample, they are probably due to contaminating cellular proteins. Interstrain Variation To determine what types of natural variation exist among the glue proteins, we analyzed seven wildtype strains of D. melanogaster (Figure 3). Two
ABCD
E
PI P2a Ff”
kinds of variation are obvious: d’ifferences in amount and in electrophoretic mobility. The variations in mobility cannot be solely due to amino acid substitutions, since SDS gels separate normal proteins on the basis of molecular weight rather than charge differences (Weber and Osborn, 1968). Pl is present in similar amounts in all the strains, but its mobility varies. Proteins P2 and P3 are particularly variable in mobility; in some strains (for example, Urbana S and Lausanne S), it is difficult to determine which bands corresponds to P2 and which to P3. P2 and P3 also vary in amount from strain to strain. At the extreme, P3 appears to be missing from Hikone AW and Hikone AS. P4 is present in all the strains, and in most of them, it resolves into two bands corresponding to P4a and P4b. The different forms of this protein show large variations in amount in different strains. P5 and P6 seem to vary only in amount, P5 being prominent in only two of the seven strains. Protein Modification The presence of carbohydrate in the glue (Ashburner and Blumental, 1970) and the interstrain variations in electrophoretic mobility suggested that the proteins might be modified post-translationally. As a first test for modification, we looked for anomalous migration of the glue proteins on SDS gels of different acrylamide concentrations. To examine si-
Q
P2a P2b
P4a P4b
Ps P6 Figure 1. SDS Proteins
Polyacrylamide
Gel of Salivary
Glands
and
Glue
(A) bloated third instar glands; (B-D) three samples of isolated glue dissected from ethanol-fixed, bloated glands; (E) prepupal glands isolated just after the gluewas released. Each sample represents material from ten glands from Oregon R larvae.
Figure 2. Isolated of Oregon R
Glue
from
Individual
Male
and
Female
Larvae
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Table
Salivary
1. Apparent
Gland
Molecular
Proteins
Weights
of Oregon
R Glue
Proteins
Pl
130,000-160,000
P2a
120,000
P2b
105,000
P3
91,000
P4a
20,500
P4b
20,000
P5
15,000
P6
8,500
Values were calculated by comparing the mobilities of Oregon R glue proteins on a IO-20% acrylamide gradient gel with the mobilities of the following proteins: phosphorylase A, bovine serum albumin, pyruvate kinase, horseradish peroxidase, deoxyribonuclease I, a-chymotrypsinogen, trypsinogen, trypsin, myoglobin, hemoglobin, lysozyme, cytochrome c. Peptides produced by cyanogen bromide cleavage of myoglobin (Swank and Munkres, 1971) were used as standards for the low molecular weight ranqe.
multaneously a wide range of acrylamide concentrations, we electrophoresed salivary gland proteins vertically through a slab gel containing a horizontal gradient of acrylamide increasing exponentially from one end of the slab to the other (Figure 4). We reasoned that since most proteins migrate at rates proportional to their molecular weights even when the acrylamide concentration is varied (Weber and Osborn, 1968), such proteins would form a series of noncrossing lines when electrophoresed on the horizontal gradient gel. However, members of at least one class of modified proteins, the glycoproteins, migrate anomalously on SDS gels; the apparent molecular weight of some glycoproteins changes as the acrylamide concentration changes (Bretscher, 1971; Segrest and Jackson, 1972). On a horizontal gradient gel, the line formed by an anomalous protein should cross lines formed by unmodified proteins. We find that some but not all standard glycoproteins meet this expectation (data not shown). In Figure 4, salivary gland proteins from Oregon R are displayed on such a gel, revealing that P2a and b, P3, and P4a and b behave anomalously: they cross many of the lines formed by the majority of the proteins. Because it is of lower mobility than any other prominent protein, Pl does not cross any lines. In its course, however, it more nearly parallels P2a and b and P3 than it does the other proteins. The P5 of both Oregon R (Figure 4) and Hikone AW (not shown) behaves normally in this test; it does not cross any of the other proteins. Although there are few proteins with similar mobility for comparison, P6 from both strains also seems to behave normally. Pulse-chase experiments also suggested that the proteins are modified. Oregon R larvae were injected with 3H-proline, and their salivary glands were isolated at various times and analyzed by SDS gel
Figure Strains
3. Isolated
Glue
from
Larvae
of Seven
Different
Wild-Type
(0) Oregon R; (HW) Hikone AW; (F) Florida-g; (U) Urbana S; (S) Swedish C; (HS) Hikone AS; (L) Lausanne S. Each sample represents material from four glands. The proteins in one of the Oregon R slots are labeled for reference.
electrophoresis followed by autofluorography. As shown in Figure 5, at 2.5 min after injection, there is no material corresponding to PI, P2, or P3. Instead there is a broad labeled zone migrating faster than any of the three proteins. With increasing time after injection, the mobility of this zone decreases, and bands eventually appear at the positions of Pi,
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P2, and P3. By 40 min, these three proteins account for most of the radioactivity in the upper part of the gel. Their delayed appearance is probably not due to the time necessary for synthesis of the polypeptide chains, since other visible bands of high molecular weight show their normal mobility in samples as early as 5 min after injection. These data therefore suggest, although they do not prove, that following their synthesis, PI, P2, and P3 become modified in such a way that their mobility on SDS gels is decreased. One would expect a decrease in mobility as a protein became glycosylated (Bretscher, 1971; Segrest and Jackson, 1972). Figure 5 also shows that the apparent precursor of P4 (P4*) moves slightly slower than the finished product (mostly P4a in Oregon R). This difference, although minor, was consistently observed. The conversion of P4* to P4b as well as P4a in Lausanne S is also shown in Figure 5. At IO min after injection, P4” is labeled, while at 60 min, there are
two labeled bands corresponding to P4a and P4b. A similar experiment with Hikone AW showed that the positions of P5 and P6 are constant from 10 until 60 min after injection (Figure 5). To test directly for carbohydrate attached to the proteins, a gel was stained by the PAS reaction. As shown in Figure 6, there is a large smear of carbohydrate, which includes PI, at the top of the gel. P2a and b, P3, and P4a and b are also PAS-positive. P5 and P6 are unstained even in strains in which they are abundant. The hue of the PAS stain is characteristic for particular proteins. By using these characteristic hues, we can distinguish between P2 and P3 when their identification would otherwise be uncertain, as in Lausanne S. The Developmental Program of Glue Protein Synthesis To determine when the glue proteins are made, groups of Oregon R larvae were injected with 3H-
P4c1*
l Pl
P5
.
P6
.
l P2a .P2b
l P3
Figure
4. Behavior
When this gel was and an exponential of widely varying (see Experimental respectively, the the left end was sample containing
of Glue
Proteins
on an SDS-Polyacrylamide
Gel Containing
a Horizontal
Gradient
of Acrylamide
Concentration
being cast, the glass plates of the template were held in an orientation rotated clockwise 90” from the final orientation, gradient of 5-20% acrylamide was poured. The slope of the gradient was chosen so that the lines formed by proteins molecular weight would approach linearity. The gradient was formed by the same method as the vertical gradient gels Procedures), except that for this gel, the two gel solutions used to form the gradient contained 0% and 20% acrylamide, mixing chamber contained 3.6 ml, and the entire gel contained 5 ml. After polymerization, the gel was rotated, and sealed with a gel solution containing 5% acrylamide. A stacking gel was then poured with one long sample slot. A 150 bloated Oregon R salivary glands was layered across the top and electrophoresed at 10 mA for 3 hr.
Drosophila 369
Salivary
Gland
Proteins
l Pl,
P2
l P2b ,P3
1
p4*.
l P4a
I
I .P6
l P6
l P6 Figure
5. Modification
of Oregon
R Glue
Proteins
during
a Pulse-Chase
Experiment
Late third instar larvae were injected with ‘H-proline, and their salivary glands were isolated and prepared for electrophoresis at the times indicated (minutes after injection). After electrophoresis, the labeled bands were detected by autofluorography. (0) Oregon R; (L) Lausanne S; (HW) Hikone AW. The major band beneath PI, P2 in the 60 min HW sample may represent partially modified glue proteins which would move up to the position of Pi, P2 with a longer chase. Alternatively, this band may reveal that Hikone AW makes some P3 which can be detected by autofluorography, but which never accumulates in sufficient amounts to be detectable by Coomassie brilliant blue staining (see Figure 3 for the electrophoretic pattern of mature Hikone AW glue proteins).
Cell 370
proline at various times from early third instar until puparium formation at 120 hr after egg deposition. Their salivary glands were removed 30 min after injection, and the newly synthesized proteins analyzed by electrophoresis on SDS gels followed by autofluorography. As shown in Figure 7, the proteins are first clearly visible at about 106 hr after the egg is laid. They are then made continuously until the glue is released at puparium formation, 14 hr later.
0 C L HW
OCLHW
Since the larvae are only partially synchronized, one cannot determine precisely when a particular protein begins to be made. However, the order in which the synthesis of different proteins begins can be determined by analyzing many samples labeled at about 106 hr. Assume that once the synthesis of a protein begins, it continues until puparium formation. Then if a larva is found to be synthesizing just one of the proteins, that must be the protein whose synthesis begins first. A larva making two proteins must include the first protein and the protein whose synthesis begins second.This logic can be extended to order all the proteins. Figure 8 shows some typical results which indicate the order deduced for the glue proteins of Oregon R. P6 begins its synthesis first; P2, P3, and P4 begin coor-
Figure Figure 6. Comparison of Coomassie Salivary Gland Proteins Each of the four PAS-stained. Each and was stained (C) Canton S; (L)
Brilliant
Blue and PAS-Stained
slots at the left contained 25 glands and was of the four slots at the right contained 10 glands with Coomassie brilliant blue. (0) Oregon R; Lausanne S; (HW) Hikone AW.
7. Developmental
Program
of Glue
Protein
Synthesis
Oregon R larvae of the indicated ages (time after oviposition) were injected with XH-proline, and their salivary glands were isolated and prepared for electrophoresis 30 min later. After electrophoresis, the labeled bands ware detected by autofluorography. The last sample (WPP) came from a white prepupa injected within 15 min after puparium formation.
Drosophila 371
Salivary
Gland
Proteins
dinately slightly later; and Pl begins last. The beginning of P5 synthesis could not be determined in Oregon R, but in Hikone AW P5 begins to be synthesized at the same time as P2 and P4. To determine when the proteins cease being synthesized, we examined samples labeled just before or just after puparium formation. Fifteen mature larvae whose salivary glands were bloated with glue ready to be released were tested. All fifteen continued to synthesize all six glue proteins. In contrast, synthesis of either Pl, P2, or P3 was not detected in any of thirty labeled prepupae, even when they were injected with label within 1 min after the glue was released. Synthesis of P4 and P5 was occasionally detected at reduced levels in prepupae injected
PI
l
. P2b P3
l
l
-figure
8. Initiation
of Glue
Protein
P4a
l P6 Synthesis
Oregon R larvae 98-106 hr old were injected with 3H-proline, and their salivary glands were isolated and prepared for electrophoresis 30 min later. After autofluorography, the samples were arranged from left to right in the order in which the proteins begin to be made.
within 15 min after release of the glue (for example, Figure 7, slot wpp). P6 or another protein with the same mobility as P6 continues to be made for at least 30 min after release of the glue. Discussion Identification and Function of the Glue Proteins Eight proteins are prominent in glue isolated from Oregon R salivary glands. These all seem to be genuine components of the glue and not artifacts of dissection, since they disappear from the glands when the lumen empties at puparium formation. One pair of the proteins, P2a-P2b, is the result of a genetic polymorphism in the Oregon R stock. Another pair, P4a-P4b, are produced from a single precursor, P4*‘. Thus we believe there are six distinct proteins in Oregon R glue: Pl-P6. This basic pattern is confirmed by analysis of seven different wild strains. All produce at least five of the proteins or their variants, and none produces any additional proteins. In a recent study, Korge (1975) identified four glue-specific proteins and genetically localized two of them. His fraction 4, the gene for which is located by recombination at 3.5 on the X chromosome and is included in the Notch deletion N8, is probably identical with our P3. P3 is present in about the same relative amount as his fraction 4 and is included within the smaller Notch deletion N64il6 (S. K. Beckendorf, unpublished results). Both ends of the N64i16 deletion are within the region deleted by N* (Lefevre and Green, 1972). We have not yet attempted to correlate any of the other proteins with those identified by Korge. Though all six of the proteins described here are contained in the glue, it is not certain how many of them contribute to its stickiness. However, examination of the proteins made by the various wild-type strains (Figure 3) can reveal whether or not particular proteins are necessary for an effective glue. Since P3 is missing from the glue in Hikone AW and Hikone AS, and since P5 is missing or nearly missing in several strains, these proteins cannot be required for glue function. Similarly, no particular allele of PI, P2, or P4 is required. It seems probable that the stickiness of the glue is a result of several or all of its proteins. Modification of the Proteins Chemical analysis of glue has shown it to be composed of 30% carbohydrate and 70% protein (Ashburner and Blumenthal, 1970). We have shown here that some of the carbohydrate is covalently linked to four of the proteins (Pl-P4). Since glycosylation reduces the mobility of proteins on SDS gels (Bretscher, 1971; Segrest and Jackson, 1972), and
Cell 372
since P4a and P4b have a higher mobility than their precursor, P4*, this precursor must undergo some other modification in addition to glycosylation . Perhaps the polypeptide chain is cleaved during glycosylation . We have no evidence that two of the proteins, P5 and P6, are modified . However, the glue contains substantial amounts of glucosamine and galactosamine which are not detected by the PAS reaction . Either of these in small amounts might not have altered the mobility of the proteins enough to be detected on the horizontal acrylamide gradient gels or in the pulse-chase experiments . Developmental Control of Glue Protein Synthesis The overall pattern of glue protein synthesis can be summarized as follows . At about 106 hr, the proteins begin to be made : P6 first, then the group P2P5, and finally P1 . All the proteins continue to be synthesized until puparium formation, at which time all but P6 are turned off . Previous studies which followed glue synthesis by PAS staining of intact glands or by observing secretory granules in salivary gland cells concluded, in contrast to our results, that glue production begins at 90-95 hr (Lane, Carter, and Ashburner, 1972 ; Zhimulev and Kolesnikov, 1975) . To be certain that we were following the same developmental processes, we injected 96-106 hr larvae with 3H-proline and removed the salivary glands 30 min later . One gland from each larva was PAS-stained, and its twin was analyzed on a gel . In all cases, the onset of PAS staining coincided with the beginning of 3 H incorporation into glue proteins . Thus we can only explain the timing discrepancy as due to differences in the fly stocks or culture conditions . The termination of synethesis of all the glue proteins except P6 seems to be quite abrupt, probably occurring within a few minutes before and after the glue is released . In contrast, the two puffs known to make glue proteins, 3C11-12 and 68C (Korge, 1975), gradually decrease in size beginning at least 10 hr before puparium formation and release of the glue (Ashburner, 1967, 1969) . Thus the abrupt shutoff of glue protein synthesis does not simply reflect the end of transcription but must be under some type of post-transcriptional regulation . It is possible that, like many other events occurring shortly before or during puparium formation, cessation of glue protein synthesis is controlled by ecdysone . Preliminary experiments reported by Berendes (1974) suggest that glue protein synthesis is blocked when salivary glands are incubated in vitro with ecdysone . During normal development, however, any effect of ecdysone must be rather indirect . As indicated by the appearance of ecdysoneinduced puffs (Ashburner, 1967), the ecdysone
titer increases at least 10 hr before glue protein synthesis ceases . Initiation of glue protein synthesis is more difficult to study because of the lack of a marker like puparium formation which can be used to synchronize the larvae . Thus although we have established the sequence in which the synthesis of the various proteins begins, it is not possible to determine how much time elapses between the initiation of the first protein, P6, and the last protein, P1 . No survey of puffs present in the polytene chromosomes has been made prior to the beginning of glue protein synthesis . We do not yet know, therefore, how the beginning of transcription of the glue protein genes is related to the beginning of their translation .
Experimental Procedures Fly Stocks The Oregon R wild-type strain of D . melanogaster was used as the standard strain for these experiments . Other wild-type strains used were Canton S, Urbana S, Swedish C, Hikone AW, Hikone AS, Lausanne S, and Florida-9 . All flies were maintained at 25°C on a medium containing 90 g dry yeast, 380 g corn meal, 720 ml Karo syrup, 56 g agar, 160 g malt, 24 ml 95% ethanol, 5000 ml water, and 60 ml acid mix A . Acid mix A contained 42 ml concentrated H3 PO4 and 418 ml propionic acid in 1000 ml solution . Larval Synchronization For timed experiments, fertilized eggs were collected for 1-2 hr from a large population cage of well fed flies . 24 hr later, unhatched eggs were transferred to a small petri dish containing food . Larvae hatching during the next 2 hr (or in some cases, 1 hr) were placed in 16 x 50 mm petri dishes containing 10 ml of food prepared the same day and a drop of live yeast suspension . 20 larvae were placed in each dish . The mean time of puparium formation was 118-120 hr after the eggs were laid, with a range of approximately f 4 hr . Efforts to improve the synchrony, including collecting larvae as they molted from second to third instar (von Gaudecker, 1972), were ineffective . Polyacrylamide Gels Polyacrylamide gels containing SDS and a discontinuous buffer system were formulated according to Laemmli (1970), with the exception that the stacking gel contained 4 .5% rather than 3% acrylamide . Two sizes of slab gels were used : 9 x 6 .5 x 0 .1 cm, or 14 x 14 x 0 .1 cm . Except when noted otherwise in the figure legends, the gels contained an exponential gradient of acrylamide increasing from 10 .3-20% from top to bottom . The two solutions used to produce this gradient contained 10% and 20% acrylamide, respectively . To form one of the large gels, 5 ml of the 20% solution were placed in a 10 ml flask and stirred . Solution was removed from this mixing chamber and delivered to the gel template at the same rate as the 10% acrylamide solution was added to the mixing chamber . The final volume of the gel was 18 ml, so the concentration at the top of the gel was 10 .3% (for this calculation, see Noll, 1969) . Volumes for the small slabs were one third of those for the large slabs . The small gels were electrophoresed at 10 mA at room temperature . The large gels were run at either 15-20 mA at room temperature or 30 mA at 4-6°C (maintained by circulating refrigerated water through a plexiglass maze held in contact with the glass plates of the gel) . The resolution of the glycoproteins, especially P1, was better with the higher current and low temperature conditions .
Drosophila Salivary Gland Proteins 373
For protein detection, gels were fixed and stained in 0 .1% Coomassie brilliant blue R, 50% methanol, 10% acetic acid, and destained in 10% methanol, 7 .5% acetic acid . For carbohydrate detection, gels were stained by the periodic acid Schiff (PAS) reaction essentially as described by Fairbanks, Steck, and Wallach (1971), but with most of the times decreased .
Laemmli, U . (1970) . Nature 227, 680-685 . Lane, N ., Carter, Y . R ., and Ashburner, M . (1972) . Wilhelm Roux Arch . 169, 216-238 . Lackey, R. A ., and Mills, A . D . (1975) . Eur. J . Biochem . 56, 335-341 . Lefevre, G ., and Green, M . M . (1972) . Chromosome 36, 391-412 . Noll, H . (1969) . Tech . Protein Biosyn . 2, 101-179 .
Autofluorography Gels containing 3H-labeled proteins were subjected to autofluorography according to the method of Bonner and Laskey (1974) . The Kodak RP Royal film used was preexposed to 0 .1-0 .2 A560 according to Laskey and Mills (1975) .
Segrest, J . P., and Jackson, R . L . (1972) . Methods Enzymol . 28, 54-63 .
In Vivo Labeling In several experiments, the proteins were labeled with 3 H-proline . Proline was chosen because the glue has been shown to be rich in proline (Ashburner and Blumenthal, 1970), and because comparison between 3 H-leucine and 3H-proline showed that proline preferentially labels the glue proteins . For injection into larvae, 3 H-proline was evaporated to dryness under a stream of nitrogen and dissolved at a final concentration of 100 mCi/ml in Grace's medium lacking proline . Between 0 .2 and 0 .5 µl were injected into each larva . Larvae were maintained until dissection in small petri dishes containing Whatman 3MM filter paper discs moistened with a 10% sucrose solution . Larvae were dissected in Drosophila Ringer's solution (Ephrussi and Beadle, 1936), and the isolated glands were rinsed in Ringer's and dissolved by heating at 100°C for 1 min in electrophoresis sample buffer [0 .0625 M Tris-HCI (pH 6 .8), 1% SDS, 5% 2-mercaptoethanol] (Laemmli, 1970) .
Weber, K ., and Osborn, M . (1968) . J . Biol . Chem . 244, 4406-4412 .
Isolation of Secretion from Glands When we wanted to collect only the secreted glue, fully bloated glands were transferred to 95% ethanol . The secretion contracts and becomes a solid mass which can then be dissected free of the salivary gland cells (Kodani, 1948) . Isolated secretion masses were then dissolved in electrophoresis sample buffer . Acknowledgments We are grateful to Drs . Kalpana White and Michael Ashburner for helpful discussions, and to Drs . Argiris Efstratiadis and J . C . Regier for critical readings of the manuscript . Dr . William H . Petri graciously supplied the strains of D . melanogaster which we used . This research was supported by a grant from the NSF to F . C . K . During part of the time that this research was conducted, S . K . B . was a fellow of the Jane Coffin Childs Foundation for Medical Research . Received June 25, 1976 ; revised August 2, 1976 . References Ashburner, M . (1967) . Chromosoma 21, 398-428 . Ashburner, M . (1969) . Chromosoma 27, 47-63 . Ashburner, M ., and Blumenthal, A . (1970) . As cited in Ashburner, M . (1970) . Adv . Insect Physiol . 7, 1-95 . Berendes, H . D . (1974) . Proceedings IV International Congress of Endocrinology (Amsterdam : Excerpta Medica), pp . 311-314 . Bonner, W. M ., and Laskey, R . A . (1974) . Eur . J . Biochem . 46, 83-88 . Bretscher, M . S . (1971) . J . Mol . Biol . 58, 775-781 . Ephrussi, B ., and Beadle, G . W . (1936) . Am . Naturalist 70, 218-225 . Fairbanks, G ., Steck, T . L., and Wallach, D . F. H . (1971) . Biochemistry 10, 2606-2617 . Fraenkel, G ., and Brookes, V . J . (1953) . Biol . Bull . 105, 442-449 . Kodani, J . (1948) . Proc. Nat . Acad . Sci . USA 34, 131-135 . Korge, G . (1975) . Proc . Nat . Aced . Sci . USA 72, 4550-4554 .
Swank, R . T ., and Munkres, K . D . (1971) . Anal, Biochem . 39, 462477 . von Gaudecker, B . (1972) . Z . Zellforsch . 127, 50-86 .
Zhimulev, I . F ., and Kolesnikov, N . N . (1975) . Wilhelm Roux Arch . 178, 15-28 .