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Changes in phenol-soluble nuclear proteins correlated with puff induction in Drosophila hydei
Cell Differentiation, 10 (1981) 229--235 Elsevier/North-Holland Scientific Publishers, Ltd.
229
CHANGES IN PHENOL-SOLUBLE NUCLEAR PROTEINS CORRELATED WITH PUFF INDUCTION IN DROSOPHILA HYDEI K A R L A BELEW and TOM BRADY *
Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, U.S.A. (Accepted 10 April 1981)
Two nonhistone chromosomal proteins accumulate in the nucleus o f Drosophila hydei salivary glands coincident with specific puff formation. A protein with a molecular weight of 40,000 appears in the nucleus with the induction of both the ecdysone puffs and puff II-48C. This protein appears to be associated with transcription in general or the phenomenon of puff formation. A protein with a molecular weight of 46,000 accumulates in salivary glands with the induction of II-48C b u t not with the induction of ecdysone puffs.
Drosophila hydei
puff induction
salivary glands
1. Introduction
Drosophila hydei and other members of the Diptera offer a unique opportunity to examine the regulation of gene activity in eucaryotes. Many of the tissues of these organisms have polytene chromosomes at various times during their development (Beermann, 1952). Both during normal development and as a result of experimental manipulation discrete puffs which are morphological evidence of intense gene activity occur on the chromosomes (Beermann, 1952; Ritossa, 1964; Berendes et al., 1965; Leenders and Beckers, 1972; Leenders and Berendes, 1972). A single large puff at region II-48C on the salivary gland chromosomes of D. hydei is induced in vitro by incubation of isolated salivary glands in 1.5 × 10 -2 M vitamin B6 (pyridoxine HC1) (Leenders et al., 1973). The ability to selectively induce this puff allows correlation of cytological and biochemical observations. One of the first events in puff formation is increased acidic protein~specific dye binding at the puff site (Berendes, 1968; Holt, 1970, 1971). This accumulation occurs * To whom reprint requests should be addressed.
phenol-soluble nuclear proteins
in the absence of a new protein synthesis (Holt, 1971 ; Goodman et al., 1976). The nonhistone chromosomal proteins (NHCP) are acidic and extremely heterogeneous (Elgin et al., 1973). Both quantitative and qualitative variability in these proteins occurs among different tissues within one organism and at different developmental stages within the same tissue (Gflmour and ,Paul, 1971; MacGfllivray et al., 1972; Elgin et al., 1973). However, Gilmour and Paul (1970), Kleinsmith et al. (1970), and Kostraba and Wang (1973) demonstrated that some nonhistone chromosomal proteins may function in gene activation. Goodman et al. (1976) indicated that the accumulation of acidic proteins at the puff site in response to ecdysterone is due to an influx of cytoplasmic proteins. Helmsing and Berendes (1971) and Helm_sing (1972)demonstrated the accumulation of a 41 kilodalton protein in response to ecdysterone treatment and a 23 kilodalton protein in response to heat shock or anaerobic shock in D. hydei salivary gland nuclei, although no alteration of the composition of nonhistones was reported by Elgin and Boyd (1975) in response to the same treatments. Both of these latter treatments induce the same series of puffs. Puff
0045-6039/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Scientific Publishers, Ltd.
230 II-48C, which is selectively induced b y vitamin B6 (pyridoxine HC1), is one of the six puffs resulting from heat shock or anaerobosis. Since Helmsing and Berendes (1971) found a change in the phenol-soluble nuclear protein pattern after induction of all six puffs, this work was undertaken to determine what changes (if any) in these proteins would be found when only p u f f II-48C was induced.
2. Materials and methods
2.1. Mass isolation o f salivary glands Gram quantities of salivary glands were isolated using a modification of the technique of B o y d et al. (1968). Mass cultures of D. hydei were raised under standard conditions at 24°C. Larvae were synchronous in growth, coming from eggs laid over a 2 h period. Sixty grams of third instar larvae (either 135 h or 184 h old) were used in each experiment. Each experiment reported was performed at least three times. The larvae were spread on a siliconized glass plate and oriented with a strong light. The internal organs were extruded b y rolling over the larvae from the rear with a steel rod. Tissues were washed from the rod and glass plate with cold (4°C) Ringers solution (Boyd et al., 1968). All subsequent operations were carried o u t at 4°C. The larvae were allowed to settle for 5 min. The fat bodies were poured off the t o p and the remaining suspension was then swirled and poured through a 1.1 mm mesh sieve to remove larval carcasses. The suspension was then swirled and allowed to settle for 30 s. The settling and sieving procedures were repeated four t i m e s . It was rapidly poured through a 0.85 mm mesh to remove large pieces of gut. Salivary glands preferentially adhere to t h e wails of the original beaker and were washed from the walls into a small beaker where contaminating tissues were removed with a Pasteur pipette. This procedure was repeated six times and the salivary glands pooled.
The salivary glands were pelleted at 60 × g for 5 min in a refrigerated centrifuge and the supernatant was poured off.
2.2. Incubation o f the glands A variety of incubation conditions were employed to induce the different series of puffs. In each case the isolated glands were divided into t w o equal portions. One of the portions was suspended in Poels medium (Poels, 1972) and incubated at 24°C for either 30 or 90 min. This portion served as the control for each experiment. The other portion was treated to induce the specific puffs desired. To induce the heat shock puffs the preparation was incubated in Poels medium at 37°C for 30 min. To induce p u f f II-48C the glands were incubated in 1.5 × 1 0 - 2 M vitamin B6 or 1.5 × 10 -2 M pyridoxal 5'-phosphate at 24°C for either 30 or 90 min. To induce ecdysone puffs the glands were incubated i n Poels medium with 1 mg/ml ecdysone for 90 min at 24°C. At the end of the incubation period the incubation medium was pipetted off and discarded. In each experiment, salivary glands were examined for the presence or absence of the appropriate p u f f or puffs.
2.3. Isolation o f nuclei Nuclei of the salivary glands were isolated and purified according to the procedure o f Elgin and B o y d (1975). The glands were transferred to a homogenizer and broken in 3 ml of nuclear isolation buffer ( 0 . 1 1 M N a C 1 , 0.002 M KC1, 0.0025 M MgC12 and 0 . 1 M Tris-HC1 at pH 7.2) which contained 0.001% spermidine and 0.2% Triton-X. Fragments were allowed to settle and the supernatant was removed. The homogenization was repeated with fresh buffer. The supernatants were combined and brought to 0.5% Triton-X. The nuclear suspension was filtered through a
231
53 ~m mesh and washed with 100 ml nuclear isolation buffer. The collected material was washed and centrifuged at 40 × g for 5 min to pellet the nuclei. Nuclei were resuspended in 10 ml of nuclear isolation buffer containing 1.67 M sucrose and layered on 2 ml buffer with 2.3 M sucrose in a polyaUomer tube. The tubes were centrifuged at 40,000 r.p.m, for 42 min in a Beckman L3-50 preparative ultracentrifuge (SW41 ROTOR) to form a nuclear pellet.
2.4. Protein extraction The nuclear proteins were extracted using the method of Konings et al. (1970). The nuclear pellet was suspended in 1 ml of Ringers solution (Leenders and Berendes, 1972) with 0 . 1 6 m M EDTA and 1% SDS. Three ml of phenol saturated with 0.25 M Tris-HC1 (pH 7.8) were added and the suspension vortexed for 10 min. The solution was centrifuged for 1 0 m in at 1 6 , 0 0 0 × g . The phenol layer was removed and reserved. The phenol extraction was repeated for the aqueous and interphase layers. Both phenol layers were then combined with 2 volumes of ice-cold acetone containing 0.1 M acetic acid. The solution was kept at 0°C for at least 2 h. The precipitate that formed was pelleted and washed with 1 : 1 ethanol:ether, then with ether and dried.
methanol and 9% acetic acid. The gels were stained overnight, rinsed in distilled water and electrophoretically destained for 30 min in 7.5% acetic acid and 5% methanol. A Beckman 24 spectrophotometer was used to scan the gels at 550 nm. The scans of the gels were examined for changes in the protein pattern. Gels were stored in 10% acetic acid.
2.6. Molecular weight determinations Molecular weights were determined by the method of Weber and Osborn (1969). The following molecular weight marker proteins were employed. Cytochrome c (12,400), myoglobin (17,500), chymotrypsinogen (24,000), ovalbumin (45,000). and bovine
B o w ~ Slwum AIDumm
~0
,
x
|
2.5. SDS polyacrylamide gel electrophoresis The dried precipitate was mixed with 20 ~1 of 0.01 M sodium phosphate buffer (pH 7.6) containing 0.1% SDS, 0.14 M ~-mercaptoethanol and 4 M urea and incubated for 2 h at 37°C. The protein was then heated to 100°C for 1.5 min. One drop of glycerol was added and the preparation was applied to the top of SDS acrylamide disc gels (Weber and Osborn, 1969) which had been modified to carry a 3% acrylamide, 0.1% SDS, phosphatebuffered stacking gel. The gels were run at 8 mA per gel for 4 h and stained in 0.25% Coomassie brilliant blue B dissolved in 45.5%
~
.1
,,I
,lJ
,4
,,J
,0
.Y
¢
J
J
i ~ o J ~ Idoee~t
Fig. 1. Standard molecular weight graph. The calculated relative mobility is plotted against the log of the molecular weight for each k n o w n molecular marker protein run. The arrows show the relative mobility of the 46,000 and 40,000 mol. wt. proteins found in the nuclear protein preparations.
232
serum albumin (68,000). The relative mobility of each protein in the gel was calculated and plotted against the log of the molecular weight to construct a standard molecular weight curve (Fig. 1).
3. Results
3.1. Isolation o f polytene nuclei The mass isolation techniques used yield salivary glands of normal morphology, free of other larval tissues except for a small fat body adhering to the isolated salivary glands (Fig. 2a): The isolated polytene nuclei appear normal (Fig. 2b). 3.2. Molecular weight determination The apparent molecular weight of the nuclear proteins was determined by referring to the standard curve of molecular weight (Fig. 1). The standard curve plots the relative mobility of a protein in the gel against the log of the molecular weight. The relative mobility of each protein was determined by the m e t h o d of Weber and Osborn (1969). The standard curve is a straight line, with little deviation of the molecular weight markers from the line. 3.3. Early third instar larvae Salivary gland mass isolated from early third instar larvae (136 h) do not yet have the complex series of ecdysone puffs associated with the normal pattern of development in Drosophila. These puffs, resulting from ecdysone secretions in the larva, occur during the late third instar (184 h). Puff II-48C is specifically induced by incubation of isolated salivary glands in Fig. 2. a) Mass isolated salivary glands. The glands are free from contamination by other larval tissues except for a small strip of fat b o d y adhering to the glands, b) Mass isolated polytene nucleus, free of cytoplasmic contamination.
233
1.5 X 10 -~ M vitamin B6 (pyridoxine HC1). This treatment resulted in the accumulation of two proteins in the nucleus (Fig. 3). These proteins have molecular weights of 40,000 and 46,000 and are never found in nuclear preparations from larvae at this stage in development which have no induced puffs (controls). To determine whether the accumulation of these proteins was specifically related to the induction of puff II-48C or associated with the general p h e n o m e n o n of puff induction, ecdysone puffs were induced. The early ecdysone puffs were induced by incubation of isolated salivary glands in 1 mg/ml ecdysone for 90 min. This group of puffs does not conrain II-48C. Examination of Fig. 4 reveals the accumulation of a 40,000mol.wt. nuclear protein in the glands incubated in the ecdysone, but not in the control glands. 3.4. Late third instar larvae The ecdysone puffs are present as a normal developmental p h e n o m e n o n in the salivary polytene nuclei from late third instar larvae (184 h after egg laying under our laboratory conditions).
~//t~
~
Ecdylone
Molecularweight 103 x
Fig. 4. A comparison o f d e n s i t o m e t r i c scans o f nuclear
protein run on SDS polyacrylamide gels from early third instar salivary gland controls and those incubated in I mg/ml ecdysone with nuclear protein from late third instar salivary gland controls.
Fig. 5 compares the salivary gland nuclear protein pattern from late third instar larvae with that obtained from early third instar salivary glands incubated in ecdysone. In both
Vitamin
Control
1(:10
504640 20 Molecular"weight 1O3
10
I III
x
Fig. 3. Densitometric scans of SDS polyacrylamide gels are compared. The nuclear protein was isolated from early third instar salivary gland controls and from those incubated for 30 rain in 1.5 x 10 -2 on vitamin Bs.
Molecular weight x 103
Fig. 5. A comparison of densitometric scans of nuclear protein run on SDS polyacrylamide gels from (a) late third instar salivary gland controls, and (b) early third instar salivary glands incubated in 1 mg/ml ecdysone.
234
D. hydei (Holt, 1970, 1971). This staining
Vilimirl 815
Icioxlll 5' II04
~fol b
Molecular
w e i g h t x 10 3
Fig. 6. Densitometric scans of nuclear protein run on SDS polyacrylamide gels. The proteins were extracted from late third instar control salivary glands and salivary glands treated with either vitamin B6, pyridoxal PO4 or heat shock. The arrow indicates the location of protein bands with molecular weight of 46,000.
the late third instar controls, containing naturally occurring ecdysone puffs, and ecdysone-incubated early third instar preparations the accumulation of a 40,000 mol.wt. peptide was observed. Puff II-48C was induced in salivary glands from 184 h old larvae either by vitamin Be, heat shock, or pyridoxal 5'-phosphate. This treatment resulted in the appearance of a major new protein (46,000 mol.wt.) band in the nuclear protein fraction (Fig. 6). This protein was never observed in control glands incubated for the same period of time under non-inducing conditions. 4. Discussion
Acidic proteins, as determined by fast green staining, accumulate in the puffs of
technique, however, gives no indication of the diversity, number or size of the accumulating proteins. The ability to induce specific puffs in polytene chromosomes of Drosophila salivary gland nuclei has allowed the correlation of acidic protein accumulation at the puff site with the qualitative changes in nonhistone chromosomal proteins. The two previous reports attempting to correlate changes in D. hydei nuclear proteins in response to puff induction are conflicting. Helmsing and Berendes (1971) observed the accumulation of a 23,000 mol.wt, protein in response to heat shock. Elgin and Boyd (1975) repeated these experiments but were unable to reproduce the results of Helmsing and Berendes (1971). While the gel scans presented by Elgin and Boyd (1975) fail to demonstrate the accumulation of a 23,000 mol.wt, protein, they do contain a minor peak in the region of 40,000 mol.wt, which is present in heat shock glands but not in controls. This difference was not discussed by the authors. The gels of Helmsing and Berendes (1971) do indeed show a band of protein in gels from heat-shocked preparations that is not found in the controls. The gels used by these authors are the same as reported in this paper. Although an exact comparison of the gels reported here and those of Helmsing and Berendes (1971) is not possible, the location of their 23,000 mol.wt, band in their gel is consistent with the location of the 46,000 mol.wt, band reported here. It should be noted that these authors neither list the marker proteins used for molecular weight determination nor present the method used for calculating the molecular weight of protein accumulating as a result of puff induction. Incubations of isolated salivary glands in vitamin B6 (pyridoxine-HC1) or pyridoxal 5'-phosphate induce only puff II48C, whereas heat shock results in a series of six puffs (II32A, II-36A, II48C, II-51B, III-58B and IV81B) one of which is II-48C. Our experiments
235
References
TABLE I Accumulation of nuclear proteins resulting from various experimental manipulations 40 K
46 K
Early third instar Control Vitamin B6 Ecdysone
-+ +
-+ --
Late third instar Control Vitamin B6 Pyridoxal 5t-phosphate Heat shock
+ + + +
-+ + +
demonstrated the accumulation of a 46,000 mol. wt. protein in the nucleus after treatment of salivary glands with vitamin B6, heat shock or pyridoxal 5'-phosphate, but not after incubation in ecdysone (Table I). This implicates its accumulation as being correlated with the induction of II-48C. The accumulation of the 40,000mol.wt. protein is noted in each case puffing is found in the nucleus (Table I) but not in the early third instar larvae which have no major puffs. This protein is likely to be of a general nature and function, perhaps a protein associated with the general formation of puffs or involved in a RNA-packaging function (a RNP particle protein).
Acknowledgements This work was supported by NSF grant (PMC78-16080) and a grant from the Institute for College Research (A& S) Texas Tech University to TB.
Beermann, W.: Chromosoma (Berlin) 25, 139--198 (1952). Berendes, H.D.: Chromosoma (Berlin) 2 4 , 4 1 8 - - 4 3 7 (1968). Berendes, H.D., F.M.A. van Brugel and T.K.H. Holt: Chromosoma (Berlin) 16, 35--47 (1965). Boyd, J.B., H.D. Berendes and H. Boyd: J. Cell Biol. 38, 369--376 (1968) Elgin, S.C.R., J.B. Boyd, L.E. Hood, W. Wray and F.C. Wu: CSHSQB 3 8 , 8 2 1 - - 8 2 4 (1973). Elgin, S.C.R. and J.B. Boyd: Chromosoma (Berlin) 5 1 , 1 3 4 - - 1 4 5 (1975). Gilmour, R.S. and J. Paul: FEBS Lett. 9 , 2 4 2 - - 2 4 4 (1970). Gilmour, R.S. and J. Paul: Proc. Natl. Acad. Sci. U.S.A. 70, 3440--3422 (1971). Goodman, R.M., E.C. Schmidt and W.B. Begamin: Cell Differ. 5 , 2 3 3 - - 2 4 6 (1976). Helmsing, P.J. and H.D. Berendes: J. Cell Biol. 50, 893--896 (1971). Helmsing, P.J.: Cell Differ. 1, 19--24 (1972). Holt, T.K.H.: Chromosoma (Berlin) 32, 64--78 (1970). Holt, T.K.H.: Chromosoma (Berlin) 32, 428--435 (1971). Kleinsmith, L.J., J. Hedima and A. Carroll: Nature (London) 226, 1025--1029 (1970). Konings, R.N.M., R. Ward, B. Francke and P.M. Hofschneider: Nature (London) 226, 604--607 (1970). Kostraba, N.C. and T.Y. Wang: Exp. Cell Res. 80, 291--296 (1973). Leenders, H.J. and P.J.A. Beckers: J. Cell Biol. 55, 257--265 (1972). Leenders, H.J. and H.D. Berendes: Chromosoma (Berlin) 3 7 , 4 3 3 - - 4 4 4 (1972). Leenders, H.J., J. Derksen, P.M.J.M. Mass and H.D. Berendes: Chromosoma (Berlin) 41, 447--460 (1973). MacGillivray, A.J., R.J. Krauze, H. Rickwood and J. Paul: Biochim. Biophys. Acta 277, 384--402 (1972). Poels, C.L2VI.: Cell Differ. 1, 63--69 (1972). Ritossa, F.: Exp. Cell Res. 3 5 , 6 0 1 - - 6 0 7 (1964). Weber, K. and M. Osborn: J. Biol. Chem. 244, 4406-4412 (1969).
229
CHANGES IN PHENOL-SOLUBLE NUCLEAR PROTEINS CORRELATED WITH PUFF INDUCTION IN DROSOPHILA HYDEI K A R L A BELEW and TOM BRADY *
Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, U.S.A. (Accepted 10 April 1981)
Two nonhistone chromosomal proteins accumulate in the nucleus o f Drosophila hydei salivary glands coincident with specific puff formation. A protein with a molecular weight of 40,000 appears in the nucleus with the induction of both the ecdysone puffs and puff II-48C. This protein appears to be associated with transcription in general or the phenomenon of puff formation. A protein with a molecular weight of 46,000 accumulates in salivary glands with the induction of II-48C b u t not with the induction of ecdysone puffs.
Drosophila hydei
puff induction
salivary glands
1. Introduction
Drosophila hydei and other members of the Diptera offer a unique opportunity to examine the regulation of gene activity in eucaryotes. Many of the tissues of these organisms have polytene chromosomes at various times during their development (Beermann, 1952). Both during normal development and as a result of experimental manipulation discrete puffs which are morphological evidence of intense gene activity occur on the chromosomes (Beermann, 1952; Ritossa, 1964; Berendes et al., 1965; Leenders and Beckers, 1972; Leenders and Berendes, 1972). A single large puff at region II-48C on the salivary gland chromosomes of D. hydei is induced in vitro by incubation of isolated salivary glands in 1.5 × 10 -2 M vitamin B6 (pyridoxine HC1) (Leenders et al., 1973). The ability to selectively induce this puff allows correlation of cytological and biochemical observations. One of the first events in puff formation is increased acidic protein~specific dye binding at the puff site (Berendes, 1968; Holt, 1970, 1971). This accumulation occurs * To whom reprint requests should be addressed.
phenol-soluble nuclear proteins
in the absence of a new protein synthesis (Holt, 1971 ; Goodman et al., 1976). The nonhistone chromosomal proteins (NHCP) are acidic and extremely heterogeneous (Elgin et al., 1973). Both quantitative and qualitative variability in these proteins occurs among different tissues within one organism and at different developmental stages within the same tissue (Gflmour and ,Paul, 1971; MacGfllivray et al., 1972; Elgin et al., 1973). However, Gilmour and Paul (1970), Kleinsmith et al. (1970), and Kostraba and Wang (1973) demonstrated that some nonhistone chromosomal proteins may function in gene activation. Goodman et al. (1976) indicated that the accumulation of acidic proteins at the puff site in response to ecdysterone is due to an influx of cytoplasmic proteins. Helmsing and Berendes (1971) and Helm_sing (1972)demonstrated the accumulation of a 41 kilodalton protein in response to ecdysterone treatment and a 23 kilodalton protein in response to heat shock or anaerobic shock in D. hydei salivary gland nuclei, although no alteration of the composition of nonhistones was reported by Elgin and Boyd (1975) in response to the same treatments. Both of these latter treatments induce the same series of puffs. Puff
0045-6039/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Scientific Publishers, Ltd.
230 II-48C, which is selectively induced b y vitamin B6 (pyridoxine HC1), is one of the six puffs resulting from heat shock or anaerobosis. Since Helmsing and Berendes (1971) found a change in the phenol-soluble nuclear protein pattern after induction of all six puffs, this work was undertaken to determine what changes (if any) in these proteins would be found when only p u f f II-48C was induced.
2. Materials and methods
2.1. Mass isolation o f salivary glands Gram quantities of salivary glands were isolated using a modification of the technique of B o y d et al. (1968). Mass cultures of D. hydei were raised under standard conditions at 24°C. Larvae were synchronous in growth, coming from eggs laid over a 2 h period. Sixty grams of third instar larvae (either 135 h or 184 h old) were used in each experiment. Each experiment reported was performed at least three times. The larvae were spread on a siliconized glass plate and oriented with a strong light. The internal organs were extruded b y rolling over the larvae from the rear with a steel rod. Tissues were washed from the rod and glass plate with cold (4°C) Ringers solution (Boyd et al., 1968). All subsequent operations were carried o u t at 4°C. The larvae were allowed to settle for 5 min. The fat bodies were poured off the t o p and the remaining suspension was then swirled and poured through a 1.1 mm mesh sieve to remove larval carcasses. The suspension was then swirled and allowed to settle for 30 s. The settling and sieving procedures were repeated four t i m e s . It was rapidly poured through a 0.85 mm mesh to remove large pieces of gut. Salivary glands preferentially adhere to t h e wails of the original beaker and were washed from the walls into a small beaker where contaminating tissues were removed with a Pasteur pipette. This procedure was repeated six times and the salivary glands pooled.
The salivary glands were pelleted at 60 × g for 5 min in a refrigerated centrifuge and the supernatant was poured off.
2.2. Incubation o f the glands A variety of incubation conditions were employed to induce the different series of puffs. In each case the isolated glands were divided into t w o equal portions. One of the portions was suspended in Poels medium (Poels, 1972) and incubated at 24°C for either 30 or 90 min. This portion served as the control for each experiment. The other portion was treated to induce the specific puffs desired. To induce the heat shock puffs the preparation was incubated in Poels medium at 37°C for 30 min. To induce p u f f II-48C the glands were incubated in 1.5 × 1 0 - 2 M vitamin B6 or 1.5 × 10 -2 M pyridoxal 5'-phosphate at 24°C for either 30 or 90 min. To induce ecdysone puffs the glands were incubated i n Poels medium with 1 mg/ml ecdysone for 90 min at 24°C. At the end of the incubation period the incubation medium was pipetted off and discarded. In each experiment, salivary glands were examined for the presence or absence of the appropriate p u f f or puffs.
2.3. Isolation o f nuclei Nuclei of the salivary glands were isolated and purified according to the procedure o f Elgin and B o y d (1975). The glands were transferred to a homogenizer and broken in 3 ml of nuclear isolation buffer ( 0 . 1 1 M N a C 1 , 0.002 M KC1, 0.0025 M MgC12 and 0 . 1 M Tris-HC1 at pH 7.2) which contained 0.001% spermidine and 0.2% Triton-X. Fragments were allowed to settle and the supernatant was removed. The homogenization was repeated with fresh buffer. The supernatants were combined and brought to 0.5% Triton-X. The nuclear suspension was filtered through a
231
53 ~m mesh and washed with 100 ml nuclear isolation buffer. The collected material was washed and centrifuged at 40 × g for 5 min to pellet the nuclei. Nuclei were resuspended in 10 ml of nuclear isolation buffer containing 1.67 M sucrose and layered on 2 ml buffer with 2.3 M sucrose in a polyaUomer tube. The tubes were centrifuged at 40,000 r.p.m, for 42 min in a Beckman L3-50 preparative ultracentrifuge (SW41 ROTOR) to form a nuclear pellet.
2.4. Protein extraction The nuclear proteins were extracted using the method of Konings et al. (1970). The nuclear pellet was suspended in 1 ml of Ringers solution (Leenders and Berendes, 1972) with 0 . 1 6 m M EDTA and 1% SDS. Three ml of phenol saturated with 0.25 M Tris-HC1 (pH 7.8) were added and the suspension vortexed for 10 min. The solution was centrifuged for 1 0 m in at 1 6 , 0 0 0 × g . The phenol layer was removed and reserved. The phenol extraction was repeated for the aqueous and interphase layers. Both phenol layers were then combined with 2 volumes of ice-cold acetone containing 0.1 M acetic acid. The solution was kept at 0°C for at least 2 h. The precipitate that formed was pelleted and washed with 1 : 1 ethanol:ether, then with ether and dried.
methanol and 9% acetic acid. The gels were stained overnight, rinsed in distilled water and electrophoretically destained for 30 min in 7.5% acetic acid and 5% methanol. A Beckman 24 spectrophotometer was used to scan the gels at 550 nm. The scans of the gels were examined for changes in the protein pattern. Gels were stored in 10% acetic acid.
2.6. Molecular weight determinations Molecular weights were determined by the method of Weber and Osborn (1969). The following molecular weight marker proteins were employed. Cytochrome c (12,400), myoglobin (17,500), chymotrypsinogen (24,000), ovalbumin (45,000). and bovine
B o w ~ Slwum AIDumm
~0
,
x
|
2.5. SDS polyacrylamide gel electrophoresis The dried precipitate was mixed with 20 ~1 of 0.01 M sodium phosphate buffer (pH 7.6) containing 0.1% SDS, 0.14 M ~-mercaptoethanol and 4 M urea and incubated for 2 h at 37°C. The protein was then heated to 100°C for 1.5 min. One drop of glycerol was added and the preparation was applied to the top of SDS acrylamide disc gels (Weber and Osborn, 1969) which had been modified to carry a 3% acrylamide, 0.1% SDS, phosphatebuffered stacking gel. The gels were run at 8 mA per gel for 4 h and stained in 0.25% Coomassie brilliant blue B dissolved in 45.5%
~
.1
,,I
,lJ
,4
,,J
,0
.Y
¢
J
J
i ~ o J ~ Idoee~t
Fig. 1. Standard molecular weight graph. The calculated relative mobility is plotted against the log of the molecular weight for each k n o w n molecular marker protein run. The arrows show the relative mobility of the 46,000 and 40,000 mol. wt. proteins found in the nuclear protein preparations.
232
serum albumin (68,000). The relative mobility of each protein in the gel was calculated and plotted against the log of the molecular weight to construct a standard molecular weight curve (Fig. 1).
3. Results
3.1. Isolation o f polytene nuclei The mass isolation techniques used yield salivary glands of normal morphology, free of other larval tissues except for a small fat body adhering to the isolated salivary glands (Fig. 2a): The isolated polytene nuclei appear normal (Fig. 2b). 3.2. Molecular weight determination The apparent molecular weight of the nuclear proteins was determined by referring to the standard curve of molecular weight (Fig. 1). The standard curve plots the relative mobility of a protein in the gel against the log of the molecular weight. The relative mobility of each protein was determined by the m e t h o d of Weber and Osborn (1969). The standard curve is a straight line, with little deviation of the molecular weight markers from the line. 3.3. Early third instar larvae Salivary gland mass isolated from early third instar larvae (136 h) do not yet have the complex series of ecdysone puffs associated with the normal pattern of development in Drosophila. These puffs, resulting from ecdysone secretions in the larva, occur during the late third instar (184 h). Puff II-48C is specifically induced by incubation of isolated salivary glands in Fig. 2. a) Mass isolated salivary glands. The glands are free from contamination by other larval tissues except for a small strip of fat b o d y adhering to the glands, b) Mass isolated polytene nucleus, free of cytoplasmic contamination.
233
1.5 X 10 -~ M vitamin B6 (pyridoxine HC1). This treatment resulted in the accumulation of two proteins in the nucleus (Fig. 3). These proteins have molecular weights of 40,000 and 46,000 and are never found in nuclear preparations from larvae at this stage in development which have no induced puffs (controls). To determine whether the accumulation of these proteins was specifically related to the induction of puff II-48C or associated with the general p h e n o m e n o n of puff induction, ecdysone puffs were induced. The early ecdysone puffs were induced by incubation of isolated salivary glands in 1 mg/ml ecdysone for 90 min. This group of puffs does not conrain II-48C. Examination of Fig. 4 reveals the accumulation of a 40,000mol.wt. nuclear protein in the glands incubated in the ecdysone, but not in the control glands. 3.4. Late third instar larvae The ecdysone puffs are present as a normal developmental p h e n o m e n o n in the salivary polytene nuclei from late third instar larvae (184 h after egg laying under our laboratory conditions).
~//t~
~
Ecdylone
Molecularweight 103 x
Fig. 4. A comparison o f d e n s i t o m e t r i c scans o f nuclear
protein run on SDS polyacrylamide gels from early third instar salivary gland controls and those incubated in I mg/ml ecdysone with nuclear protein from late third instar salivary gland controls.
Fig. 5 compares the salivary gland nuclear protein pattern from late third instar larvae with that obtained from early third instar salivary glands incubated in ecdysone. In both
Vitamin
Control
1(:10
504640 20 Molecular"weight 1O3
10
I III
x
Fig. 3. Densitometric scans of SDS polyacrylamide gels are compared. The nuclear protein was isolated from early third instar salivary gland controls and from those incubated for 30 rain in 1.5 x 10 -2 on vitamin Bs.
Molecular weight x 103
Fig. 5. A comparison of densitometric scans of nuclear protein run on SDS polyacrylamide gels from (a) late third instar salivary gland controls, and (b) early third instar salivary glands incubated in 1 mg/ml ecdysone.
234
D. hydei (Holt, 1970, 1971). This staining
Vilimirl 815
Icioxlll 5' II04
~fol b
Molecular
w e i g h t x 10 3
Fig. 6. Densitometric scans of nuclear protein run on SDS polyacrylamide gels. The proteins were extracted from late third instar control salivary glands and salivary glands treated with either vitamin B6, pyridoxal PO4 or heat shock. The arrow indicates the location of protein bands with molecular weight of 46,000.
the late third instar controls, containing naturally occurring ecdysone puffs, and ecdysone-incubated early third instar preparations the accumulation of a 40,000 mol.wt. peptide was observed. Puff II-48C was induced in salivary glands from 184 h old larvae either by vitamin Be, heat shock, or pyridoxal 5'-phosphate. This treatment resulted in the appearance of a major new protein (46,000 mol.wt.) band in the nuclear protein fraction (Fig. 6). This protein was never observed in control glands incubated for the same period of time under non-inducing conditions. 4. Discussion
Acidic proteins, as determined by fast green staining, accumulate in the puffs of
technique, however, gives no indication of the diversity, number or size of the accumulating proteins. The ability to induce specific puffs in polytene chromosomes of Drosophila salivary gland nuclei has allowed the correlation of acidic protein accumulation at the puff site with the qualitative changes in nonhistone chromosomal proteins. The two previous reports attempting to correlate changes in D. hydei nuclear proteins in response to puff induction are conflicting. Helmsing and Berendes (1971) observed the accumulation of a 23,000 mol.wt, protein in response to heat shock. Elgin and Boyd (1975) repeated these experiments but were unable to reproduce the results of Helmsing and Berendes (1971). While the gel scans presented by Elgin and Boyd (1975) fail to demonstrate the accumulation of a 23,000 mol.wt, protein, they do contain a minor peak in the region of 40,000 mol.wt, which is present in heat shock glands but not in controls. This difference was not discussed by the authors. The gels of Helmsing and Berendes (1971) do indeed show a band of protein in gels from heat-shocked preparations that is not found in the controls. The gels used by these authors are the same as reported in this paper. Although an exact comparison of the gels reported here and those of Helmsing and Berendes (1971) is not possible, the location of their 23,000 mol.wt, band in their gel is consistent with the location of the 46,000 mol.wt, band reported here. It should be noted that these authors neither list the marker proteins used for molecular weight determination nor present the method used for calculating the molecular weight of protein accumulating as a result of puff induction. Incubations of isolated salivary glands in vitamin B6 (pyridoxine-HC1) or pyridoxal 5'-phosphate induce only puff II48C, whereas heat shock results in a series of six puffs (II32A, II-36A, II48C, II-51B, III-58B and IV81B) one of which is II-48C. Our experiments
235
References
TABLE I Accumulation of nuclear proteins resulting from various experimental manipulations 40 K
46 K
Early third instar Control Vitamin B6 Ecdysone
-+ +
-+ --
Late third instar Control Vitamin B6 Pyridoxal 5t-phosphate Heat shock
+ + + +
-+ + +
demonstrated the accumulation of a 46,000 mol. wt. protein in the nucleus after treatment of salivary glands with vitamin B6, heat shock or pyridoxal 5'-phosphate, but not after incubation in ecdysone (Table I). This implicates its accumulation as being correlated with the induction of II-48C. The accumulation of the 40,000mol.wt. protein is noted in each case puffing is found in the nucleus (Table I) but not in the early third instar larvae which have no major puffs. This protein is likely to be of a general nature and function, perhaps a protein associated with the general formation of puffs or involved in a RNA-packaging function (a RNP particle protein).
Acknowledgements This work was supported by NSF grant (PMC78-16080) and a grant from the Institute for College Research (A& S) Texas Tech University to TB.
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