Current Genetics

Current Genetics 1, 63-74 (1979)

© by Springer-Verlag 1979

Altered Patterns of Protein Synthesis Induced by Heat Shock of Yeast L. McAlister, S. Strausberg, A. Kulaga, and D. B. Finkelstein

Department of Biochemistry,Universityof Texas Health Science Center at Dallas, Dallas, Texas 75235, USA

Summary. The products of protein synthesis from exponential phase cultures of Saccharomyces cerevisiae grown at 23 °C or at 36 °C appear to be essentially identical. However, yeast cells respond to a shift in culture temperature from 23 °C to 36 °C with the rapid de novo synthesis of a polypeptide species of molecular weight 100,000. Within 6 0 - 9 0 rain after the shift this polypeptide represents approximately 2.5% of the total cellular protein, a 5 - 1 0 fold increase over the preshift level. The level of this polypeptide then decreases with continued growth of the cells at 36 °C. Analyses by SDS-polyacrylamide gel electrophoresis of polypeptides obtained from cells pulse labeled with [3s S]methionine demonstrate that following a temperature shift from 23 °C to 36 °C the synthetic rate of the 100,000 molecular weight polypeptide (as well as a number of other polypeptide species) increases to a level at least 10 fold higher than that observed prior to the shift. A concomittant decrease is observed in the synthesis of a large number of polypeptide species which were actively synthesized before the shift. Maximum changes in synthetic rates are observed 2 0 - 3 0 rain after the shift and preshift synthetic patterns are regained within 6 0 - 9 0 min. Synthetic changes of the same magnitude and time course can be produced by short ( 2 0 - 3 0 min) exposures to 36 °C implicating a heat shock response. Several of the transiently induced polypeptides, including the 100,000 molecular weight species, show an affinity for DNA as determined by DNA-cellulose chromatography. Key words: Saccharomyces cerevisiae - Translation Coordinate regulation - Electrophoresis.

Offprint requests to: D. B. Finkelstein

Introduction

Events essential to cellular growth and reproduction are very often examined in organisms harboring conditionally lethal temperature sensitive mutations (Horowitz and Leupold, 1951). Such strains grow normally at one temperature (the permissive temperature) and exhibit the defective phenotype only at another temperature (the restrictive temperature). In choosing the temperature range for such mutant studies, care is taken to remain within the physiological level for the system being examined. Conditions are chosen such that non mutant cells grow "normally" when cultivated under both sets of conditions. Thus, while 2"3 °C (permissive) and 36 °C (restrictive) have been used in the examination of growing Saccharomyces cerevisiae (Hartwell, 1967), the restrictive temperature must be lowered to 33.5 °C for sporulation studies as this yeast fails to spomlate readily at 36 °C (Simchen, 1974). An unstated assumption in working with temperature sensitive mutants appears to be that if cells grow normally under both permissive and restrictive conditions then a change from one condition to the other, i.e., a change within the physiological growth range will have no untoward effects on the cells. That the above assumption is not tree may be seen from the work of Warner and Oorenstein (1977) who demonstrated a transient inhibition of transcription of mRNA coding for ribosomal proteins upon a shift of wild type S. cerevisiae from 23 °C to 36 °C. During the course of experiments using certain temperature sensitive cell division cycle (cdc) mutants (Hartwell et al., 1974), we have observed that yeast cells undergo a remarkable number of changes in the products of protein synthesis during very early time points following a 23 °C to 36 °C temperature shift. Moreover, this response is in no way limited to mutant strains. It is reproducible in a large number of wild type strains and O172-8083/79/0001/00063/$02.40

64

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast

appears t o be a g e n e r a l i z e d r e s p o n s e o f S . cerevisiae cells s u b j e c t e d t o this t e m p e r a t u r e shift. As d e s c r i b e d in this p a p e r , t h e r e s p o n s e o f y e a s t t o a t e m p e r a t u r e s h i f t is similar in m a n y respects t o t h e h e a t s h o c k r e s p o n s e d e s c r i b e d for Drosophila (Tissi6res et al., 1 9 7 4 ; M c K e n z i e et al., 1 9 7 5 ; Lewis et al., 1975). T h e y e a s t r e s p o n s e is c h a r a c t e r i z e d b y t h e t r a n s i e n t de novo s y n t h e s i s o f a specific s m a l l ' s u b s e t o f p o l y p e p t i d e s a n d b y a c o n c o m i t t a n t r e d u c t i o n in s y n t h e s i s o f a large n u m b e r o f o t h e r cellular p r o t e i n s . H o w e v e r , u n l i k e

Drosophila larvae or cells w h i c h are n o t viable at h i g h e r t e m p e r a t u r e s (Lewis et al., 1975), wild t y p e y e a s t cells are c o m p l e t e l y viable a n d e v e n t u a l l y r e s u m e n o r m a l synt h e t i c p a t t e r n s at the e l e v a t e d t e m p e r a t u r e .

Material a n d M e t h o d s

Culture Conditions and Temperature Shifts Most experiments were conducted with the haploid S. cerevisiae strain A364A (a gall adel ade2 ura2 ural his71ys2 tyrl), which was obtained from the Yeast Genetic Stock Center, Berkeley, California. Other strains tested for the heat shock response included the diploid X131 (kindly provided by R. Pigon), the a mating type haploid X2180-1A, the ~ mating type haploid X2180-1B, and a rho" petite derived by treatment of A364A with ethidium bromide as previously described (Finkelstein and Butow, 1976). All cultures were routinely grown in a low-sufate medium (Douglas et al., 1979) buffered with 10 mg/ml succinate and 6 mg/ml NaOH and supplemented to aceomodate requirements of strain A364A with 80 ~zg/ml adenine, 80 #g/ml uracil, 160 #g/ml histidine, 160 #g/ml lysine, and 40 #g/ml tyrosine. Other additions included SO D- to 20 #l/ml and 2% glucose. For experiments utilizing alternate carbon sources, the glucose was replaced with 1.5% ethanol or 3% glycerol. Experimental cultures were inoculated from a diluted stationary phase culture and grown at least 5 - 1 0 generations to mid log phase (approximately 2 x 107 cells/ml). Cell growth was monitored turbidimetrically with a Klett-Summerson colorimeter using a red (660 nm) filter. One Klett unit equals approximately 5 x 105 cells/ml. Cultures were grown in flasks filled to less than 20% of their stated capacity in a Lab Line gyrorotary shaker with the water bath set at 23 °C. Temperature shifts were conducted by pipetting cells from the 23 °C culture into flasks prewarmed in a second shaker with the water bath set at 36 °C. A Hewlett Packard 3802-A thermometer was used to verify that culture medium shifted in this manner equibrated at the higher temperature within one minute. For intermediate temperatures, the water baths were appropriately adjusted and equilibrated.

Labeling Procedures To uniformly label cellular proteins, cultures were inoculated into medium containing SO D- reduced to 8 ~g/ml and 40 #Ci/ml H235SO4 (New England Nuclear, carrier free). Such a protocol provides an isotope incorporation directly proportional to culture growth. For chase experiments, the cells were pelleted following labeling, washed once with sterile water, and resuspended in medium lacking isotope and supplemented with 100 ~g/ml each SOD- and L-methionine.

For pulse labeling experiments, 1.0 ml of a log phase culture was transfered to a preequifibrated 12 x 75 mm glass test tube containing 100 ~1 of diluted isotope. As noted in the text and figure legends, pulse labeling was conducted with either "10 #Ci of [35Slmethionine (Amersham, 570 Ci/mM) or with 100/A of 1 : 2 - 1 : 4 0 dilutions of a 35S-hydrolysate of yeast cells. The hydrolysate was obtained by growing 5 ml yeast cultures (strain X2180-1B) to late stationary phase in the presence of 1 mCi/ml H~Sso4 plus 8 tag/ml SO~-. The cells were pelleted, resuspended in 0.5 ml H20, and the pellet heated under a nitrogen atmosphere for 10 min at 100 °C. Digestion with pancreatin was conducted according to procedures described by Graham and Stanley (1972). The supernatant (0.5 ml) obtained by centrifuging the digest for 10 min on a Clay-Adams tabletop centrifuge was made 0.1% 2-mercaptoethanol. Aliquots of the supernatant were stored at -80 °C and used for labeling without further purification. Use of either [35S]methionine or the 35S-hydrolysate produced essentially identical pulse labeling patterns; the hydrolysate dilutions were empirically chosen to allow autoradiographic exposure times of 1 - 3 days. For use in double label experiments maximal 3H-labeling of the temperature inducible proteins was obtained by pulse labeling a culture of A364A with 10 #Ci/ml L-[4,5-3Hlleucine (ICN, 39 Ci/mM) for 40 min beginning 5 min after a shift from 23 °C to 36 °C. Aliquots of this [3H]leucine labeled culture were stored as frozen cell pellets for use as reference samples. In all cases, labeling was terminated by simultaneous addition of sodium azide to 20 mM and cycloheximide to 200 #g/ml and immediately placing the sample on ice. After 5 rain on ice, the cells were pelleted, washed once with cold 2 mM sodium azide, and frozen as pellets at - 2 0 °C.

Sample Preparation and Electrophoretie Procedures To prepare samples for electrophoresis, frozen cell pellets were thawed on ice in 200 ~1 of SDS-sample buffer (50 mM Tris C1 pH 6.8, 2% sodium dodecyl sulfate, 2mM EDTA, 1% 2-mercaptoethanol, 10% glycerol (v/v), and 0.001% bromophenol blue). An equivalent volume of 0.45-0.55 mm glass beads and 10 ~1 0.1 M phenylmethylsulfonyl fluoride were added to each sample and the cells immediately broken by 3 cycles of vortexing for 20 sec intervals and returning the samples to ice between vortexing. An additional 100 #1 volume of SDS-sample buffer was added to aid in transfering the lysate to Beckman microfuge tubes. The lysate was cleared of debris by centrifuging for 10 min in a Beckman microfuge set at maximum speed. The supernatant was transfered to a new microfuge tube and heated for 90 sec in a boiling water bath. 25 ~1 samples were placed in each gel slot for electrophoresis. Samples for double label analysis were similarily prepared except that each experimental 35Sqabeled pellet was combined with a reference [3H]leucine labeled pellet (to be used as an internal standard) before breaking to minimize any differences in protein composition due to breakage. Proteins were fractionated on 8% or 10% polyacrylamide-SDS slab gels as described previously (Finkelstein and Butow, 1976) using the discontinuous buffer system of Laemmli (1970). The dimensions of the separating gels were 1.25 mm (thickness) x 140 mm (width) x 135 mm (length). Gels were electrophoresed at room temperature at 30 mA until the tracking dye reached 1 cm from the end of the gel. Following electrophoresis, gels were fixed and stained for 30 min in 0.1% Coomassie brilliant blue, 50% methanol and 10% acetic acid and subsequently destained overnight in 10% acetic acid plus 5% methanol. After drying on a BioRad gel dryer, the gels were autoradiographed using Kodak RP.R2 X-ray film. Molecular weight standards used for gel elec-

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast trophoresis were: rat liver ATP citrate synthase (molecular weigth 110,000), rabbit muscle phosphorylase A (molecular weight 92,500), bovine serum albumin (molecular weight 68,000), rat liver malic enzyme (molecular weight 60,000), pig heart fumarase (molecular weight 49,000), and yeast glyceraldehyde phosphate dehydrogenase (molecular weight 36,000). Citrate synthase and malic enzyme were kindly provided by R. Frenkel. Two-dimensional electrophoretic analysis followed the procedure of O'Farrell (1975) with the modification that the ampholytes used for the first-dimensional isoelectric focusing consisted of 2% broad range ampholytes (pH 3-10). Samples were prepared by diluting 10 ~1 of the SDS-solubilized protein samples with 15 ~1 lysis buffer (9.5 M urea, 2% (w/v) NP-40, 2% ampholytes, pH 3-10, 5% 2-mercaptoethanol). Following focusing at 400V for 18 h at room temperature, the isoelectric focusing gels were equilibrated for 1 h at room temperature in 5 ml SDS-sample buffer then fused with 1% agarose in SDSsample buffer to the top of polyacrylamide slab gels. Electrophoresis in the second dimension and autoradiography were as described above. Following autoradiography of two-dimensional gels, protein spots of interest were cut from the dried gels, hydrated and counted as previously described (Finkelstein and Butow, 1976). Each sample analyzed by a two-dimensional gel included pulse 35S-labeled proteins and reference [3H]leucine labeled proteins prepared as described above. The 3H-label was used to determine an appropriate internal standard: isotope ratios were determined for several spots and normalized back to the 35S/3H ratio of the SDS sample loaded on the gel (set as 1.0). Changes in synthetic rates of the proteins between the 23 °C and 36 °C were then expressed as ratios of the normalized values. Of the major spots which showed no synthetic change (i.e., showed no variations in normalized isotope ratios), the one which comigrates with commercially available yeast glyceraldehyde phosphate dehydrogenase consistently represented 5-6% of the total synthetic activity and was chosen as an internal standard because of the low levels of synthesis of other proteins of the same molecular weight. For densitometric measurements of 35Sqabeling patterns from one-dimensional gels, a Joyce Loebl Microdensitometer was used to provide scans of each lane. Peaks corresponding to the bands of interest were cut from the scans to a baseline determined by the density of the stacking gel and the weight of each peak was compared to the peak weight for glyceraldehyde phosphate dehydrogenase in the same scan, or, analogously, to the weight of the total scan. In lanes where no peak was observable for a particular band (i.e., inducible bands at 23 °C and repressible bands at 36 °C), the maximum possible value was assumed by superimposing the peak shape of that band over the background. The exposure times for autoradiographs were chosen to be within the linear tange of the film, thus providing scan peak weights which are directly proportional to siotope.

DNA-Cellulose Chromatography For analysis of DNA binding proteins, 10 ml of logarithmically growing cells were pelleted, washed with sterile water, and resuspended in the same volume of medium lacking SO~-. The culture was shifted to 36 °C and after 5 rain, 2.5 mCi of H~5SO4 added to the culture. Labeling was terminated after 45 min and a 1 ml aliquot was processed to prepare SDS-soluble proteins as described above. The remaining cells were pelleted. The cell pellet was resuspended in 200/A of a low salt breaking buffer (20 mM Tris-HC1 pH 7.4, 10 mM MgC12, 1 mM EDTA,

65 50 mM NaCI, 1 mM 2-mercaptoethanol, 200 /~g/ml lysozyme, and 1 mM phenylmethylsulfonyl fluoride) and broken by vortexing with glass beads. 50 ~g pancreatic DNase (Sigma) was added to the lysate and the sample incubated on ice for 20 rain. This was followed by an addition of 50 #1 5 M NaC1 and a further 70 rain incubation on ice. The lysate was transfered to a microfuge tube and centrifuged for 5 min at maximum speed in a Beckman microfuge to remove debris and beads. The supernatant was then centrifuged for 20 min at maximum speed in a Beckman airfuge. The resulting supernatant was desalted by loading directly onto a 1 c m x 50 cm Sephadex G-25 column preequilibrated with the DNA-cellulose buffer (50 mM NaC1, 1 mM EDTA, 1 mM 2mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 mM Tris-HC1 pH 7.4, 10% (w/w) glycerol, and 25 /~g/ml lysozyme). Appropriate fractions were pooled and a portion was retained at this point for comparison with the SDS-extracted protein samples. The remainder was loaded onto a 1 ml (packed volume) DNAcellulose column preequilibrated with the same buffer and containing a 1 ml cellulose plug to reduce non-specific protein binding as previously described (Finkelstein and Butow, 1976). After loading the sample and removing the cellulose plug, the column was washed with 6 ml of the 0.05 M NaC1 buffer to remove unbound material. Proteins bound to the column were then eluted in a stepwise fashion using the above buffer contalning increasing concentrations of NaC1. All samples were concentrated by trichloroacetic acid precipitation and redissolved in SDS-sample buffer for gel electrophoresis.

Results

Appearance of a 100,000 Molecular Weight Polypeptide Following a Temperature Shift Cultures o f Saccharomyces cerevisiae grow n o r m a l l y at 36 °C w i t h a generation time o f 2.7 h as c o m p a r e d to 3.3 h at 23 °C. SDS-solubilfzed proteins e x t r a c t e d f r o m cultures grown c o n t i n u o u s l y at 23 °C or at 36 °C show essentially identical patterns w h e n fractionated on one dimensional SDS-polyacrylamide gels. However, w h e n cultures growing at 23 °C are shifted to 36 °C, we observe that a major n e w p o l y p e p t i d e band o f apparent molecular weight 100,000 n o t present at 23 °C appears after 2 h at the elevated temperature. Figure 1A shows the isotope pattern o f SDS-solubilized proteins o b t a i n e d from cells u n i f o r m l y labeled at 23 °C.1 The pattern f r o m a p o r t i o n o f the same culture following a 2 h shift to 36 °C in the c o n t i n u e d presence o f isotope is shown in Fig. lB. The large increase in isotope i n c o r p o r a t e d i n t o the 100,000 molecular weight band (indicated by the arrow in Fig. 1) is the major difference b e t w e e n the control (23 °C) and shifted (36 °C) cultures at this level o f resolution.

Although assessing protein patterns on the basis of uniform 35SO2- labeling assumes an equivalence of sulfur containing amino acids among different proteins, we observe no major differences between patterns of proteins stained with Coomassie blue and autoradiographic (or fluorographic) patterns of proteins uniformly labeled with 35SO~- (or[3H] leucine).

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast

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Fig. 1. Appearance of a 100,000 molecular weight polypeptide following a shift of yeast cultures from 23 °C to 36 ° C. A culture of A364A was uniformly labeled by growth at 23 °C in the presence of H35SO4 (as described in Materials and Methods). Samples of the culture were taken at 23 °C (A), at 2 h following a shift of a portion of the culture to 36 ° C (B), and at 2 h following removal of the isotope and a subsequent shift to 36 °C of another portion of the culture (C). The isotopic labeling and chase procedures as well as sample preparations were as described in Materials and Methods. SDS-solubilized proteins were fractionated on 8% polyacrylamide slab gels and examined by autoradiography. Molecular weight standards were coelectrophoresed on the same slab and visualized by staining with Coomassiebrilliantblue. The arrow marks the position of the 100,000 molecular weight band

If isotope is removed from the uniformly labeled 23 °C culture the cells resuspended in a chase medium just prior to the shift to 36 °C, no increase in isotope incorporation in the region of the 100,000 molecular weight band is detectable after 2 h (Fig. 1C). In this case the new band is visible only with Coomassie blue staining (data not shown). The appearance of the 100,000 molecular weight polypeptide band is therefore due to de novo synthesis of the polypeptide in response to the temperature shift and not to processing of some preexisting precursor molecule. This response, although characterized here and in the following experiments for strain A364A, has been reproduced in a number of other wild type strains of S. cerevisiae (listed in Materials and

Fig. 2. Levels of the 100,000 molecular weight band in A364A cells following a shift from 23 °C to 36 °C. A culture of A364A was uniformly labeled by cultivation to mid log phase in the presence of H2"~SO4. The culture was shifted to 36 °C and maintained in logarithmic growth by frequent dilutions with prewarmed medium containing 2% glucose and H35So4 to the same constant specific radioactivity. At the indicated times, 1 ml samples were taken and SDS-soluble proteins analyzed by onedimensional electrophoresis. The autoradiogxaphic pattern of each sample was scanned with a Joyce Loebl Microdensitometer and the peak weight of the 100,000 molecular weight ~protein expressed as a percentage of the peak weight for glyceraldehyde phosphate dehydrogenase (molecular weight 36,000). The latter remained a constant percentage (approximately 6 %) of the total scan weight; therefore the percent total cellular isotope represented by the 100,000 molecular weight band at each time = (peak wt of the 100,000 molecular weight band / peak wt of glyceraldehyde phosphate dehydrogenase) x 6

Methods) and thus appears to be a generalized response of yeast cells subjected to a 23 °C to 36 °C shift. At the 2 h time point following the temperature shift, the 100,000 molecular weight band represents a major component of the total cellular isotope pattern. To allow closer examination of the amounts of this band present at various times following a shift, we have quantitated autoradiographs of one-dimensional SDS gels by densitometry. From such analyses of a culture uniformly labeled by growth in the presence of H 3 s SO4 at 23 °C and shifted to 36 °C (Fig. 2) it is apparent that isotope rapidly appears in the 100,000 molecular weight band following the shift. Isotope in the 100,000 molecular weight band reaches a maximum within 6 0 - 9 0 min at a level at least 6-fold higher than the level at 23 °C. At the maximum, the 100,000 molecular weight band represents approximately 2.5% of the total cellular protein. The band thus reaches a level comparable to that of glycolytic enzymes and 10-fold figher than estimated levels of individual ribosomal proteins (Warner,

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast

67

Fig. 3. Transient changes in protein synthetic patterns of yeast cells shifted from 23 ° C. Exponentially growing cultures of A 364A were pulse labeled with [35S]methionine at 23 °C (lanes C) and at the times indicated below each lane following a shift to 36 °C. Labeling procedures and sample preparations are described in Materials and Methods. The autoradiographic patterns represent equal aliquots of SDS-soluble proteins labeled during 10 min pulses (A) or during 2 min pulses (B) starting at the times shown and subsequently resolved on 10% polyacrylamide slab gels. The bands identified by letters, numbers, or asterisks are explained in the text

1971). After 2 h at the elevated temperature, isotope in the 100,000 molecular weight band declines with continued cultivation at 36 °C and approaches the preshift level by 24 h. This observed decline is not due to descreased growth rates of the culture or to isotope depletion (see legend for Fig. 2). Also, the level of the internal standard, glyceraldehyde phosphate dehydrogenase, remains constant during this time since, as described in the legend for Fig. 2, glucose deprivation is prevented (Hommes, 1966). In a related experiment (data not presented), the stability of isotope associated with the 100,000 molecular weight band was examined in protein samples of ceils pulse labeled from 1 0 - 2 0 min following a shift to 36 °C then chased as described in Materials and Methods. The isotope had been incorporated into the 100,000 molecular weight band during the pulse remained stable after a 1 h chase whether the cells were retained at 36 °C or shifted back to 23 °C. Therefore, the previous observation that isotope disappears from the 100,000 molecular weight protein with extended tittle at 36 °C in a uni-

formly labeled culture (Fig. 2) is not due to a rapid turnover rate. In addition, no new bands appeared following a pulse and a 1 h chase. Thus, the 100,000 molecular weight band does not appear to be a precursor from of other cellular proteins. These observations collectively suggest that the 100,000 molecular weight band is a stable protein, the synthesis of which is rapidly induced for a period of approximately 6 0 - 9 0 min following a 23 °C to 36 °C shift.

Changes in Protein Synthesis Following a 23 °C to 36 °C Temperature Shift To analyze the synthesis of the 100,000 molecular weight polypeptide, shifted cell samples were pulse labeled with [aS S]methionine at various times following a shift from 23 °C to 36 °C. Figure 3A shows the autoradiographic pattern of whole cell proteins synthesized during 10 min pulses at the times indicated following a shift to 36 °C. It is apparent that the changes in protein synthetic pat-

68

L. McAhster et al.: Protein Synthesis Induced by Heat Shock of Yeast

Fig. 4. Two-dimensional analysis of protein synthetic patterns of yeast cultures before and after a temperature shift. An exponentially growing culture of A364A was pulse labeled with a 35S-hydrolysate for 10 min at 23 °C (A) and from 10-20 min following a shift to 36 °C (B). Prior to breaking the cells, the samples were mixed with reference [3H]leucine labeled cells (prepared as described in Materials and Methods). Sample preparation and two-dimensional electrophoretic procedures are described in Materials and Methods. While the 3H-labeled polypeptides do not contribute to the autoradiographic patterns of the 35S-pulse labeled polypeptides presented here, they do permit identification of the location of inducible proteins in both samples by Coomassie brilliant blue staining of the gels. The locations of inducible proteins are indicated by triangles (zx);those of repressible proteins are indicated by inverted triangles (V). Notations follow the nomenclature assigned in Fig. 3. The pH gradient of the isoelectric focusing gel was measured as described by O'Farrell (1975)

terns of cells shifted to 36 °C are much more profound than the induction of a single new band. The pattern of protein synthesis during the 1 0 - 2 0 mi pulse at 36 °C is distinctly different from that observed at 23 °C and many o f these changes are apparent even in the 0 - 1 0 min pulse. Within 20 min at 36 °C there is a preferential synthesis of a small group of polypeptide species which are absent or synthesized only at very low levels at 23 °C. Among these are species of apparent molecular weights of 100,000 (a, corresponding to the band previously detected in uniformly labeled samples), 90,000 (b), 79,000 (e), and 38,000 (d). Other inducible bands are less apparent on one-dimensional gels. The induction of synthesis of this group of polypeptides appears to correspond temporally with a decrease in synthesis of a large number of cellular proteins normally made at 23 °C. The repressible (synthetically depressed) bands span a wide range of molecular weights. The lower molecular weight bands in this group include several which comigrate on one-dimensional gels with proteins obtained from purified yeast ribosomes (indicated by asterisks in Fig. 3A), in

agreement with the results of Gorenstein and Warner (1976). We also detect a number of high molecular weight repressibl e band; several of these are identified by the numbers I, 2, and 3 in Fig. 3A. Between 60 and 90 min following a shift to 36 °C the pattern of protein synthesis approaches that observed in preshift samples. Most of the inducible proteins are no longer synthesized at high levels by 90 rain and the synthesis of proteins repressed at earlier time points is resumed. After 180 rain at 36 °C the synthetic pattern is almost indistinguishable from that of 23 °C samples. Thus, it appears that the translational capacity of cells shifted to 36 °C is immediately but only transiently directed toward synthesis of the 36 °C inducible proteins. The onset of changes in synthetic patterns can be more closely examined using shorter pulse labeling periods. Figure 3B presents the autoradiographic patterns of cellular proteins pulse labeled with [3 s S]methionine for 2 min periods before and following a 23 °C to 36 °C temperature shift. Isotope incorporation for inducible bands a, b, and d is significant within only 4 min after

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast

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Time at 56"C (min) Fig. 5. Densitometric analysis of altered synthetic patterns of yeast proteins following a 23 °C to 36 °C shift. The autoradiographic patterns of 10 min 35S-pulse labeled proteins similar to those presented in Fig. 3 were analyzed by densitometry (see Materials and Methods). The peak wt/total scan wt was used to estimate percent of total synthesis for each band. These values are expressed relative to the maximum observed value for each band. Points are drawn at the midpoint of the pulse period. The 0 time point corresponds to the 23 °C sample. (A) Synthetic rates of inducible bands a (o), b (~), and d (z~)relative to respective maximum values in the 20-30 min pulse of 3.34, 3.68, and 1.41% total synthesis. (B) Synthetic rates of repressible bands 1 (o), 2 (n), and 3 (z~) relative to respective maximum values in the 23 °C pulse of 1.68, 3.03, and 2.87 % total synthesis. (C) Synthetic rate of glyceraldehyde phosphate dehydrogenase (o) relative to a maximum in the 10 20 rain pulse of 7.3% of total synthesis

the shift. (Inducible band c was not well resolved on this gel.) Changes in synthetic rates of the repressible bands appear to be somewhat slower; significant reduction in isotope incorporation is obvious after 6 min. Within the classes of inducible and repressible bands, the initial changes in synthetic rates appear to be very closely related. The changes are so rapid, however, that it is impossible to determine whether the bands within either class exhibit exact coordinate control. To examine the range of changes in translation products, pulse labeled polypeptides were analyzed by twodimensional gel electrophoresis. The synthetic pattern from the resulting autoradiograph of proteins from cells pulse labeled for 10 min at 23 °C is presented in Fig. 4A and the corresponding pattern from cells pulse labeled from 1 0 - 2 0 min following a shift to 36 °C in Fig. 4B. While we have chosen proteins for further analysis which represent the major isotopic species at their particular molecular weight, m a n y other differences are also obvious. Although it is theoretically possible to quantitate the synthetic rates for individual proteins as 35 S-cpm incorporated into the two-dimensional spot relative to the total trichloroacetic acid precipitable 35S-cpm of the sample, unsystematic variation in isotope losses during preparation of two-dimensional gels may give large errors. We therefore used a double label procedure (see Materials and Methods) to validate that synthetic rate of glyceraldehyde phosphate dehydrogenase remains essentially constant following a temperature shift. This allowed comparisons of 3SS-cpm incorporation into other proteins to be expressed relative to an internal standard on the same two-dimensional gel. Synthetic rates estimated by this method for two-dimensional, gels are compared

Table 1. Synthetic rates for individual polypeptides. A logarithmically growing culture of A364A was divided and pulse labeled with a 35S-hydrolysate, one portionfor 10 min at 23 °C, the other from 10-20 min following a shift to 36 °C. SDS-solubilizedproteins of each culture were resolved by both one- and two-dimensional gel electrophoresis. Nomenclature follows that of Fig. 3

Polypeptide

a b d 1 2 Glyceraldehyde phosphate dehydrogenase

Percent total synthesis Two-dimensional analysisa 23 °C 36 °C

One-dimensional analysisb 23 °C 36 °C

0.06 0.14 ND 1.07 1.07

4.46 4.48 ND 0.25 0.26

0.39 0.39 0.19 3.61 1.78

5.62 5.29 1.76 0.87 0.34

6.20

6.00

6.00

7.58

a Spots were cut from dried two-dimensional gels and counted as described in Materials and Methods. (The inclusion of 3Hlabeled reference proteins .provided an isotopic method for assertaining that the same protein on two different gels was properly located and cut.) The percent total synthetic activity of glyceraldehyde phosphate dehydrogenase by two-dimensional analysis was determined as 35S-epm in glyceraldehyde ~5hosphate dehydrogenase/trichloroacetic acid-precipitable S-cpm in the sample loaded on the isoelectric focusing gel. The percent total synthetic activities of the other polypeptides were determined as (35S-cpm in the spot / 35S-cpm in glyceraldehyde phosphate dehydrogenase) x 6 h Peaks corresponding to each protein band were cut from densitometric tracings of autoradiographs of one-dimensional gels (see Materials and Methods). Synthetic levels for each band were expressed as (peak weight of the band / total weight of the scan) x 100

70 with rates quantitated from scans of autoradiographs from one-dimensional gels in Table 1. It may be seen that the limitation of one-dimensional analysis is an overestimation of basal synthetic values for the inducible proteins at 23 °C and for the repressible proteins at 36 °C. To facilitate the analysis of multiple time points, we have chosen to use one-dimensional analyses, accepting the resulting underestimation of changes in synthetic rates of a given band. In order to quantitate changes in protein synthesis following a temperature shift, densitometric analysis was applied to the isotopic patterns from a pulse labeling experiment similar to that presented in Fig. 3A. The bands which were examined may be grouped into three distinct categories (Fig. 5). The inducible bands a, b, and d show very similar synthetic patterns (Fig. 5A). Incorporation of isotope into these bands is maximal during the 2 0 - 3 0 min pulse and at this point represents' synthetic rates 9 to 11 times higher than preshift rates. Synthetic rates for these bands then decline and approach preshift rates by 90 rain. Synthesis of the inducible bands decreases exponentially from the maximum levels with t 1/2 values between 20 and 40 min. Quantitation of the second synthetic category, the repressible proteins (bands 1, 2, and 3 in Fig. 3A), is presented in Fig. 5B. The rates of synthesis of these bands decline to minimal levels between 5 and 15 fold lower than 23 °C rates. The temporal changes are very similar among the repressible bands. Minimal rates are observed in the 2 0 - 3 0 m i n pulse at 36 °C, corresponding to the time of maximal synthesis of the inducible bands. However, the repression of synthesis of bands 1, 2, and 3 is not obvious in the 0 - 1 0 min pulse following the shift; this is in contrast to the induction of bands a, b, and d which is already apparent in the 0 - 1 0 min pulse (Fig. 5A). Within the limits of this analysis, synthetic rates of the repressible bands 90 min after the 36 °C shift are indistinguishable from preshift rates. The synthetic rates of several bands are not significantly altered by a 23 °C to 36 °C shift. Glyceraldehyde phosphate dehydrogenase is representative of this category (Fig. 5C). Synthetic rates for the band corresponding to this enzyme measured during the 10 min pulse periods represent 6.4% -+ 0.4% (SEM) of the total protein synthesis.

Parameters o f the Temperature Shift Our observations of the response of yeast cultures to a temperature shift have for the most part been restricted to cultures grown at 23 °C and shifted to 36 °C. These temperatures reflect those most frequently used in studies with temperature sensitive strains (Hartwell, 1967) and were originally chosen for the isolation of

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast Table 2. Responses of yeast cultures to various temperature shifts. Cultures of A364A were grown to midqog phase at the indicated temperatures. The cells were pulse labeled with a 35S-hydrolysate for 10 min at the cultivation temperature and from 10-20 min following a shift of a portion of the culture to the elevated shift temperature. SDS-solubilized proteins were resolved on one-dimensional gels and the" resulting autoradiographs subjectively scored for induction and repression responses as equivalent (++) or of lower magnitude (+) than changes observed in a 23 °C to 36 °C shift, or as (-) if no change was apparent Cultivation temperature

Shift temperature

Induction response

Repression response

23 °C 23 °C 23 °C 30 °C 30 °C

30 °C 33 °C 36 °C 36 °C 39 °Ca

+ ++ ++ ++

+ ++ ++ ++

A temperature of 39 °C is above the physiological range of cultivation, but, despite a decrease in total protein synthesis, the heat shock responses were still observable

temperature sensitive mutants because growth is apparently normal at both temperatures (Pringle, 1975). We have determined that the temperature shift response may be elecited by a less severe temperature shift of cultures grown at 30 °C and subsequently shifted to 36 °C (Table 2), an increase of only 6 degrees. The absolute magnitude of the temperature increase is not the determinant for the elicitation of the heat shock response, however, because a 7 degree shift from 23 °C to 30 °C does not result in any major alteration of protein synthetic patterns. Instead, the response we describe appears to require rapid(?) passage through a specific transitional temperature. We estimate that this temperature falls between 30 °C and 33 °C since a shift from 23 °C to 33 °C does in fact result in altered synthetic patterns similar to but of lesser magnitude than those elicited by a 23 °C to 36 °C shift. A second important parameter of the temperature shift is the duration of exposure to the elevated temperature. It is obvious from the preceding experiments that maintaining a shifted culture at 36 °C does not indefinitely alter synthetic patterns. Instead, it appears that the observed response may be a function of the initial shock of the shift rather than of the elevated temperature level per se. This possibility was investigated by quantitating the effect of Short exposures to 36 °C on (a) the level of 100,000 molecular weight inducible protein and on (b) the synthetic rates of several inducible and repressible bands. To measure the appearance of the 100,000 molecular weight inducible polypeptide in response to brief exposures to 36 °C, a culture uniformly labeled by growth in the presence of H 3sSO4 at 23 °C was shifted to 36 °C.

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast

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Fig. 6. Appearance of the 100,000 molecular weight protein following heat shock. A log phase culture of A364A uniformly labeled with H~SO 4 was shifted to 36 °C in the continued presence of isotope. At various times after the shift, 1 ml samples of the 36 °C culture were returned to 23 °C and allowed to continue incubation at 23 °C. These samples were processed at the 60 min time point to determine the relative capacity for synthesis of the 100,000 molecular weight protein attained during short exposures to 36 °C (o). Samples of the culture maintained at 36 °C were taken at the times indicated for analysis of the appearance of the 100,000 molecular weight protein (o). The levels of the protein were determined as described in the legend for Fig. 2 and expressed relative to the maximum observed in each curve (1.48% of the total isotope in the synthetic capacity curve, 1.78% of the total isotope in the appearance curve). Variation in absolute maximum values is attributed to differences in nonspecific background isotope levels in the gels, and also to some day-to-day variation we have observed in culture responses to a 23 °C to 36°C shift

Fig. 7. Changes in protein synthesis following heat shock. A log phase culture of A364A growing at 23 °C was shifted to 36 °C and divided into three flasks. One flask was maintained at 36 °C (.), the second was returned to 23 °C after 2 min (o), and the third was returned to 23 °C after 10 min (o). Aliquots were taken from each culture and pulse labeled for 5 rain intervals with a 35S-hydrolysate at the indicated times. The pulse labeled samples were analyzed as described in Materials and Methods and autoradiographs quantitated as described in the legend for Fig. 2. Points are drawn at the midpoint of the pulse labeling period. The 0 time point corresponds to a 23 °C sample pulse labeled just before the start of the experiment. Synthetic levels of inducible bands a (A) and b (B) are expressed relative to the maximum levels observed (2.6% and 2.7% of the total synthesis, respectively). For respressible bands 2 (C) and 3 (D), rates are expressed relative to those observed at 23 °C (1.4% and 1.7% of the total synthesis, respectively)

At various times from 1 to 60 rain following the shift, an aliquot of the culture was returned to 23 °C and allowed to continue incubation at that temperature until the end of the 60 rain period. The levels of isotope in the 100,000 molecular weight protein in these samples thus represent the capacity for production of this band attained during short exposures to 36 °C. The results of this experiment, presented in Fig. 6, suggest that an exposure of cells to 36 °C of as short as 1 5 - 2 0 min is sufficient to result in production of the 100,000 molecular weight band to the same level as observed in cells held at 36 °C for 60 rain. To test the effect of brief exposures to 36 °C on translational patterns, the time courses of synthetic changes were compared in cultures shifted to 36 °C for 2 min or 10 rain and returned to 23 °C before pulse labeling. The synthetic patterns of inducible bands a and b following such a reciprocal shift are presented in Fig. 7 (A and B, respectively). For both bands, substantial synthetic capacity is attained within a 2 rain exposure of a culture to 36 °C, a time only slightly longer than the equilibration of the culture to the higher temperature. For band a, a 2 min exposure is sufficient to induce" a rate, measured 10

rain later, comparable to that of a culture retained for 10 min at 36 °C. The induction of band b after a 2 min exposure to 36 °C continues increasing for 20 min to a rate as high as 50% of the maximum rate observed. Comparison of the time courses of synthesis following a 10 min exposure with those following a 2 min exposure shows that both the extent and duration of induction are increased with time of exposure to 36 °C but the 10 min exposure still does not result in the synthetic levels attained in ceils held at 36 °C. Combining these results with the previous measurements of the level of the 100,000 molecular weight protein in cultures held at 36 °C for brief periods (Fig. 6), a time of 2 0 - 3 0 min at 36 °C is estimated to be the requisite exposure time to generate an induction of full magnitude. The decline in synthetic rates of repressible bands 2 and 3 (Fig. 7, C and D, respectively) is much less dramatic following brief exposures to 36 °C. In fact 2 min at 36 °C appears to result in some subsequent stimulation of synthesis of both bands 2 and 3. A 5 min exposure (not shown) results in very little alteration in the synthetic rates of these bands. Only following a 10 min exposure to

72

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast response may be more appropriately termed a heat "shock" response,even though we are perturbing the culture by alteration of temperature within the-normal growth range.

Affinity of Heat Shock Inducible Proteins for DNA

Fig. 8. Binding to DNA-cellulose by yeast heat shock inducible proteins. A log phase culture of A364A was shifted from 23 °C to 36 °C and pulse labeled with 38SO2-as described in Materials and Methods. The autoradiographic pattern of SDS-solubilized proteins from a portion of the shifted cells was analyzed (lane 1) and the remaining cellsbroken for DNA-cellulosechromatography (see Materials and Methods). Lane 2 shows the input pattern of isotopic species loaded onto the DNA-ceUulose column. Those species not bound were present in the flow-through fraction (lane 3). Proteins bound to the column were eluted with 4.0 ml of a 0.1 M NaC1 buffer wash (lane 4) followed by 4.0 ml of a 0.25 M NaC1 buffer wash (lane 5).

36 °C is any significant repression observable, and for both bands the level of repression is much less than that observed in a culture held at 36 °C. One conclusion suggested by these results is that the induction and repression events may be independently controlled responses to a temperature shift. Repression may require continued exposure to 36 °C. Alternatively, the repression of certain bands may follow and depend on attaining some prior minimum level of induction. This interpretation could explain the previously observed temporal lag of repression. Commitment to the temperature shift response, at least in terms of the induction events, is very rapid. Returning the culture to 23 °C after 2 0 - 3 0 min does not reduce the magnitude of the response. Thus, the temperature shift

Full understanding of the nature of the heat shock response ultimately depends upon knowledge of the function of the proteins which are produced during such a shock. It is obvious that normal patterns of protein synthesis are drastically altered during a temperature shift and that an enormous amount of cellular energy is directed toward immediate synthesis of the inducible class of proteins. It is not unreasonable to assume that these proteins may have key metabolic or regulatory functions. We have examined the possibility that one or more of the inducible proteins may interact with DNA by asking if any of the proteins has an inherent affinity for DNA. DNAcellulose chromatography was conducted as described in Materials and Methods using an extract of cells pulse-labeled with 3SSO24- for 40 min beginning 5 min after a shift to 36 °C. The resulting autoradiographic patterns are shown in Fig. 8. The 100,000 molecular weight temperature inducible protein (labeled a) is retained on DNA cellulose almost quantitatively as may be seen by comparing the isotopic species present in the input sample (lane 2) with the isotopic species present in the flow-through volume (lane 3). This inducible protein binds relatively tightly to DNA since it is eluted only by a buffer containing 0.25 M NaC1. The identity of this protein has been confirmed by analysis using two-dimensional gel electrophoresis (not shown). While it should be noted that not all inducible proteins bind to DNA-cellulose as may be seen for protein b, two other inducible proteins of apparent molecular weights 65,000 and 56,000 (as indicated in Fig. 8) also bind to DNA-cellulose (lane 4). Since in SDS-solubilized whole cell proteins analyzed by one-dimensional gel electrophoresis, there are no major differences in this molecular weight range between 23 °C and 36 °C samples, it is concluded that polypeptides migrating with these apparent molecular weights in whole cell samples represent multiple species. The three inducible proteins which bind to DNAcellulose constitute the major differences between the complement of DNA-binding proteins from cells shifted to 36 °C and that from cells at 23 °C (not shown). Twodimensional analysis has established that the 65,000 and 56,000 molecular weight DNA-binding proteins have isoelectric points falling within a neutral to slightly acidic range (not shown). These inducible DNA-binding proteins show a lesser affinity for DNA than the 100,000 molecular weight protein as manifest in the salt concentration required to elute them. The basis for the affinity of the

73

L. McAlisteret al.: Protein Synthesis Induced by Heat Shock of Yeast inducible proteins for DNA is unknown, but the possibility of tight binding due only to nonspecific ionic interactions seems unlikely considering the relatively neutral isoelectric points of these proteins.

Discussion

While it is intuit.ively obvious that exposure of an organism to potentially lethal temperatures would result in drastic shock, it was not expected that manipulation of temperature within the normal physiological range used for cultivation would constitute an environmental insult requiring an adaptive response at the level of translation. Even though yeast grow "normally" at either 23 °C or at 36 ° C, the pattern of protein synthesis in Saccharornyces cerevisiae is drastically, but only transiently, altered following a shift from 23 °C to 36 °C. Synthesis of a group of inducible proteins begins within 4 - 6 min, peaks between 2 0 - 3 0 min, and returns to preshift levels within 6 0 - 9 0 min. Although this synthetic period is relatively short (less than half the generation time of the cells), several of the inducible proteins are produced to high levels. Once the synthetic period is over, the proteins are eventually diluted out by normal protein synthesis as the cells continue to grow (Fig. 2). Therefore growth at 36 °C does not require that these proteins be present at high levels. It seems plausible to suggest that proteins required for homeostatic adaptation to a rapid but physiological environmental change would show just this pattern of synthesis. The major precedent for response of eucaryotic cells to a temperature increase is the heat shock response of Drosophila. Since the now classical observation by Ritossa (1962) that shifting larvae from 25 °C to 30 °C results in a distinctly altered pattern of puffs in the polytene chromosomes of Drosophila, the heat shock response has been intensively studied as a model for coordinate transcriptional control in eucaryotes. The mRNA species which code for several induced proteins have been isolated from polyribosomes of heat shocked Drosophila cell lines or salivary glands and shown to (a) hybridize to DNA in the heat induced puffs (McKenzie et al., 1975; Spradling et al.; 1977) and to (b) code for the inducible proteins in in vitro protein synthesizing systems (Sondermeijer and Lubsen, 1978). During heat shock in Drosophila, RNA polymerase B molecules accumulate at heat shock puffs and decrease in puffs which were transcriptionally active before the shock (Jamrich et al., 1977), suggesting transcriptional control of both induction and repression. While cytogenetic analysis is not possible in yeast since the chromosomes are too small to be visualized by standard techniques (Robinow, 1975), we do have preliminary evidence for controls operative at a transcriptional level. Populations of translationally a~tive mRNA species

in heat shock yeast cells are distinctly different from those of preshock cells, z In addition, the mutant strain ts 136 (isolated from A364A) which is presumably defective in transport of RNA from the nucleus to the cytoplasm at 36 °C (Hutchison et al., 1969) does not exhibit the normal induction associated with the heat shock response implicating an absence of cytoplasmic mRNA species coding for the heat shock inducible proteins in 23 °C cells. 2 Finally, the repression we observe correlates well temporally with the previously coordinate decrease in synthesis of ribosomal proteins following a temperature shift which has been attributed to decreased levels of corresponding mRNA species (Warner and Gorenstein, 1977). Studies of the Drosophila heat shock response have implicated a correlation between the response and certain metabolic parameters. Ashburner (1970) observed that a puffing response similar to that induced by heat shock occurs in salivary glands during recovery from anoxia. Compton and McCarthy (1978) also report induction of heat shock puffs when hydrogen peroxide is added to an in vitro system consisting of salivary glands disrupted in cytoplasm of normal cells. They suggest that the primary event in initiation of the heat shock response may be a disruption of the normal cellular oxidation-reduction potential. Since several uncouplers of oxidative phosphorylation also induce the heat shock puffs (Leenders and Berendes, 1972), mitochondrial functions have been repeatedly implicated in the Drosophila response. We have looked for similar correlates in the S. cerevisiae heat shock response. Since yeast are facultative anaerobes, mitochondrial functions may be altered by culture conditions. We have observed no major differences in the heat shock response of cultures grown on either fermentable or non-fermentable carbon sources. In addition, the usual response was observed in a 23 °C to 36 °C shift of a respiratory deficient strain (arho- mutant derived from strain A364A). 3 These observations suggest that neither mitochondrial respiratory function nor mitochondrial protein synthesis is essential for the heat shock response in yeast. We have not yet determined, however, whether dynamic changes in mitochondrial functions can independently result in the altered synthetic patterns characteristic of the heat shock response. Although the response of yeast cultures shifted from 23 °C to 36 °C is similar in many respects to the heat shock response described for Drosophila, the elevated temperature is not physiologically detrimental as has been described for Drosophila tissues (Lewis et al., 1975). Because of the continued viability of shifted yeast cultures, it should be possible to determine the final subcellular localization of the inducible proteins as a first step in 2 Manuscript in preparation. 3 Unpublished observations.

74 determining the functional roles o f these proteins. Of particular interest in this respect is the apparent affinity for DNA shared by several o f the inducible proteins (Fig.

8). The alterations in translation pattern following heat shock provides an excellent system for the study o f an adaptive mechanism involving the coordinate regulation o f gene expression. It is als6 important that investigators using temperature sensitive mutants be aware o f the fact that manipulations within the physiological growth range may represent an environmental insult and it is possible that effects attributed to a particular temperature sensitive lesion may overlap effects attributable to a generalized heat shock response.

Acknowledgements. We are grateful to Dr. H. Bremer and Ms. N. Shephard for assistance in the use of their Joyce Loebl Microdensitometer. This research has been supported by grants PCM 76-17208 from the NSF and 1 RO1 GM2529-01 from the NIH (to D.B.F.). A. K. was the recipient of a Chilton Foundation summer research fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References Ashburner, M.: Chromosoma 31,356 376 (1970) Compton, J. L., McCarthy, B. J.: Cell 14, 191-201 (1978) Douglas, M., Finkelstein, D., Butow, R.: Meth. Enzymol. 56, 58-66 (1979) Finkelstein, D. B., Butow, R. A.: Arch. Biochem. Biophys. 174, 52-65 (1976)

L. McAlister et al.: Protein Synthesis Induced by Heat Shock of Yeast Gorenstein, C., Warner, J. R.: Proc. Nat. Acad. Sci. USA 73, 1547-1551 (1976) Graham, R., Stanley, W. M.: Anal. Biochem. 47, 505-513 (1972) Hartwell, L. H.: J. Bacteriol. 93, 1662-1670 (1967) Hartwell, L. H., Culotti, J., Pringle, J. R., Reid, B. J.: Science 183, 46-51 (1974) Hommes, F. A.: Arch. Biochem. Biophys. 114,231-233 (1966) Horowitz, N. H., Leupold, U.: Cold Spring Harbor Symp. Quant. Biol. 16, 65-74 (1951) Hutchison, H. T., Hartwell, L. H., McLaughlin, C.S.: J. Bacteriol. 99, 807-814 (1969) Jamrich, M., Greenleaf, A. L., Bautz, E.K.F.: Proc. Nat. Acad. Sci. USA 74, 2079-2083 (1977) Laemmli, U.K.: Nature 227,680-685 (1970) Leenders, H.J., Berendes, H.D.: Chromosoma 37, 433-444 (1972) Lewis, M., Helmsing, P.J., Ashburner, M.: Proc. Nat. Acad. Sci. USA 72, 3604-3608 (1975) McKenzie, S. L., Henikoff, S., Meselson, M.: Proc. Nat. Acad. Sci. USA 72, 1117-1121 (1975) O'Farrell, P.H.: J. Biol. Chem. 250, 4007-4021 (1975) Pringle, J.R.: Methods Cell Biol. 12, 149 184 (1975) Ritossa, F.: Experientia 18,571-573 (1962) Robinow, C.F.: Methods Cell Biol. 11, 1-22 (1975) Simchen, G.: Genetics 76,745-753 (1974) Sondermeijer, PJ.A., Lubsen, N.H.: Eur. J. Biochem. 88, 331339 (1978) Spradling, A., Pardue, M.L., Penman, S.: J. Mol. Biol. 109,559 587 (1977) Tissifires, A., Mitchell, H.K., Tracy, U.M.: J. Mol. Biol. 84,389 398 (1974) Warner, J.R.: J. Biol. Chem. 246,447-454 (1971) Warner, J.R., Gorenstein, C.: Cell 11,201-212 (1977)

Communicated by F. Kaudewitz Received June 28, 1979