Cell, Vol.

17. 241-254,

June

1979,

Copyright 0

1979

by MIT

The Induction of Gene Activity Drosophila by Heat Shock

Michael Ashburner* and J. Jose Bonnert Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143

During the normal development of the larval salivary gland of Drosophila melanogaster, considerable changes occur in the patterns of puffing activity. These can be seen as changes in the puffs of the gland’s polytene chromosomes, and occur as a consequence of changes in the titer of the insect’s growth and moulting hormone, ecdysone (for review see Ashburner and Richards, 1976). In addition to the changes in gene activity normal to development, there are changes in the activities of a set of genes that occur as a direct consequence of subjecting animals to a wide variety of experimental insults, for example, a brief heat shock. The discovery of the induction of a unique set of puffs by heat shock (Ritossa, 1962) has led the way to an analysis of gene function and structure in Drosophila that is, so far, unique. The cytological facts can be summarized briefly (Ritossa, 1962, 1963, 1964a; Berendes and Holt, 1964; Berendes, Van Breugel and Holt, 1965; van Breugel, 1966; Ashburner, 1970; Ellgaard, 1972; Lewis, Helmsing and Ashburner, 1975). If Drosophila larvae or their excised tissues are subjected to a brief heat shock (for example, 40 min at 37”C, the normal culture temperature being 25”C), puffs are induced at a few specific sites (Figure 1). In D. melanogaster there are nine heat-inducible puffs, (33B, 63C, 64F, 678, 70A. 87A, 87C, 93D and 95D); in D. hydei there are six (32A, 36A, 48BC and 81 B; 31 C and 858 are small and variable in their response). The in vivo induction of the puffs by the heat shock is very rapid; it occurs within 1 min of the temperature increase although the puffs continue to increase in size for some 30-40 min (at 37°C) before regressing. The maximum sizes of the induced puffs are a function of the severity of the temperature shock, at least until lethal temperatures are met (Figure 2). The induction requires RNA, but not protein synthesis. In the absence of protein synthesis, however, the induced puffs fail to regress unless the temperature is returned to normal (puff 48BC of D. hydei is an exception; Leenders and Beckers, 1972). Prolonged (for example, more than 1 hr) temperature shock results in additional changes in puffing activity; most remarkable is the fact that all other puffs, puffs active at the time the temperature shock began, regress. It was the discovery that heat shock also results in the induction of the synthesis of a set of polypeptides ’ Permanent address: Department of Genetics, University bridge, Cambridge, England. t Present address: Department of Biology. Indiana University. ington. Indiana 47401.

of CamBloom-

in

Review

(TissiBres, Mitchell and Tracy, 1974) which resulted in the heat shock response graduating from a cytological curiosity to an object of more general interest. It is now known that at the level of specific RNA and protein synthesis, the heat shock response is shown by all tissues of the fly, including the permanent tissue culture cell lines. Heat shock results in the production of a specific set of RNAs transcribed from the genes which, in polytene chromosomes, form the heat shock puffs. Some of these RNAs are preferentially translated into a set of polypeptides (the heat shock polypeptides or “hsp’s”). RNA Synthesis after Heat Shock At the level of RNA synthesis, there are at least three distinct responses of Drosophila tissues to heat shock: the induction of the synthesis of a class of heat shock RNAs some of which, although not all, are translated into the hsp’s; the suppression of the synthesis of most other messenger RNAs, although not those coding for the histones or those coded by the mitochondrial genome; and the disruption of the normal processing of the primary transcription products of the 5s and 18s + 28s ribosomal RNA cistrons (Ellgaard and Clever, 1971; Lengyel and Pardue, 1975; Rubin and Hogness, 1975; Jacq, Jourdan and Jourdan, 1977). The basis for the specificity in the control of both transcription and RNA processing is unknown. The changes in RNA synthesis resulting from heat shock can be shown in several ways. Autoradiography of the polytene chromosomes, after incorporation of 3H-uridine into RNA, shows that the heat shock puffs are newly induced sites of RNA synthesis, and that as a consequence of the heat shock, preexisting puffs cease to incorporate the precursor (Ritossa, 1964b; Berendes, 1968; Tissi&es et al., 1974; Belyaeva and Zhimulev, 1976; Bonner and Pardue, 1976). The changing pattern of transcription resulting from heat shock is reflected in a change in the distribution of RNA polymerase II and other chromosomal proteins, detected immunochemically or interferometrically. There is a marked migration of these components into the heat shock puff sites (Holt, 1970; Plagens, Greenleaf and Bautz, 1976; Silver and Elgin, 1977; Jamrich, Greenleaf and Bautz, 1977a; Elgin, Serunian and Silver, 1978; Greenleaf et al., 1978; Mayfield et al., 1978; Spruill et al., 1978; Silver and Elgin, 1979). Heat shock also results in the depletion of RNA polymerase II from non-heat shock puff chromosomal regions (Greenleaf et al., 1978) and an increase in the level of fluorescent staining for this enzyme in the nucleoplasm (Jamrich et al., 1977b). The accumulation of specific heat shock RNA sequences at the heat shock puff sites can be demonstrated by the cytological hybridization of complementary DNA sequences to the puff RNA (Livak et al., 1978). In view of these results, it is not surprising to find that inhibitors of

Cell 242

Figure 1. Induction by Heat Shock and 87Cl in D. melanogaster (a) Control, data.)

unshocked;

(40 min at 37°C)

(b) heat-shocked.

of Puffs at 87A7

(M. Ashburner,

original

RNA synthesis (for example, actinomycin D or CXamanitin) block the induction of the heat shock puffs and the migration of RNA polymerase II to puff sites (Berendes, 1968; Ellgaard, 1968; Ellgaard and Celver, 1971; Holt and Kuijpers, 1972; Compton and McCarthy, 1978; J. J. Bonner, A. L. Greenleaf and K. R. Yamamoto, manuscript in preparation). In tissue culture cells, the heat shock RNAs have been analyzed by electrophoresis (Figure 3; Spradling, Pardue and Penman, 1977; Mirault et al., 1978; Moran et al., 1978) and by cytological hybridization to polytene chromosomes (McKenzie, Henikoff and Meselson, 1975; Spradling, Penman and Pardue, 1975; Henikoff and Meselson, 1977; Lubsen et al., 1978). The kinetics of synthesis of the RNAs have been studied by Spradling et al. (1977) and by McKenzie (1976). The rates of accumulation of most of the heat shock RNAs increase with an increase in the severity of the shock, with a maximum which is rather dependent upon tissue culture media and cell growth conditions (Spradling et al., 1977; S. L. McKenzie, personal communication). The polysomal RNA from heat-shocked cells has been rather extensively studied. There is now no doubt that this polysomal RNA includes messengers for the heat shock polypeptides. Polyadenylated polysomal RNA from heat-shocked tissue culture cells fractionates into two major size classes on sucrose density gradients, one at 20s and the other at 12s (McKenzie and Meselson, 1977; Spradling et al., 1977; Mirault et al., 1978; Moran et al., 1978). These RNA sequences are either absent from, or present at much lower concentration in, the polysomal RNA of control cells. Further fractionation of the heat shock polysomal RNA has been achieved by electrophoresis under denaturing conditions. Of the 20s RNAs of D. melanogaster, the largest species (Al) hybridizes in situ preferentially to puff 63BC, the second largest (A2) to both puffs 87A and 87C, and the smallest (A3) to puff 95D (Table 1;

0

,

,

,

,

10

20

30

40

J

I 60

Figure 2. Size of the Heat Shock Puff 87Cl of Shock and Time in D. melanogaster

I 120m

as a Function

of Severity

(A) 29%; 0 33%; (0) 37’C; the growth temperature was 25°C. Puff size is the ratio of the diameter of 87Cl to that of an unpuffed reference band, 87D1.2. (From Lewis et al., 1975.1

Spradling et al., 1977). The minor species A4 hybridizes to 87A and 87C, although with a >4-fold preference for 87C. Electrophoretic resolution of the 12s RNAs has not been as good; this peak certainly includes sequences complementary to the 676 heat shock puff. The five puffs mentioned are the largest puff8 of D. melanogaster. As yet seheat-induced quences hybridizing to the smaller heat shock puffs (that is, 338, 64F and 70A) have not been clearly identified (beyond the fact that they are polyadenylated), perhaps because the concentration of these RNAs is relatively low. The ninth heat shock puff, 93D, presents a rather different picture since its cytoplasmic transcripts would appear to be either poly(A)- or to have poly(A) tails too short to allow effective retention by an oligo(dT) column (Spradling et al., 1975, 1977; Henikoff and Meselson, 1977). The 93D puff is a unique heat shock puff in other ways. For example, no RNA polymerase II migration into it can be detected by immunoperoxidase staining after induction (J. J. Bonner, unpublished results), and it can be induced (in vitro) independently of the other heat shock puffs by benzamide (Lakhotia, 1971) and “aged” culture medium (Bonner and Pardue, 1976). A fascinating story has emerged with respect to the RNA sequences transcribed from the large heat-inducible puffs 87A and 87C. Spradling et al. (1977) found that the RNA species A2 hybridized to both sites. Henikoff and Meselson (1977) observed that the ratio of silver grains hybridizing to these sites, while 1:1.7 (A:C) for polysomal RNA, was 1:3.7 for nuclear RNA. The hybridization competition experi-

Heat Shock 243

in Drosophila

ments of Henikoff and Meselson (1977), coupled with the biochemical and genetic data discussed below, indicate that the 87A and 87C sites share sequences which are represented in polysomal RNA. There are,

25

35

36

however, additional sequences transcribed at 87C. Thus 87A and 87C may represent a gene duplication event, coupled with the acquisition of additional sequences at 87C. In D. hydei, the equivalents of 87A and 87C-a gene duplication-are probably not to be found (but see van Breugel et al., 1978). 48BC, however, showed similar hybridization-competition behavior to that of the D. melanogaster 87C (Lubsen et al., 1978). While hybridization of nuclear RNA to 32A was effectively competed by polysomal RNA, 80% of hybridization at 48BC remained. Thus not all the sequences transcribed from 48BC are equally represented in polysomes.

OC

-

A4

-

A5,A6

Figure 3. Cytoplasmic Poly(A)+ RNA Synthesized peratures in D. melanogaster Cultured Cells

at Different

Protein Synthesis after Heat Shock Two dramatic changes in the pattern of protein synthesis occur as a consequence of heat-shocking Drosophila tissues (Figure 4). The synthesis of a small number of heat shock polypeptides (hsp’s) is induced, and the synthesis of most other proteins ceases (Tissieres et al., 1974; Lewis et al., 1975; McKenzie et al., 1975; Koninkx, 1976). A very rapid response to heat shock of tissue culture cells is the breakdown of almost the entire population of polysomes [McKenzie et al., 1975; Biessmann, Levy and McCarthy, 1978; see also Sondermeijer and Lubsen (19781, who find that this response is not so dramatic in heat-shocked tissue culture cells of D. hydei]. This breakdown of polysomes is insensitive to the actinomycin D inhibition of RNA synthesis (S. L. McKenzie, manuscript in preparation), suggesting that it cannot be the result of a simple competition for ribosome binding by heat shock-induced RNAs. Only some 10 min after the start of the heat shock do new polysomes appear, and these contain newly synthesized RNA transcribed from the induced heat shock genes. Thus a response to heat shock is the cessation of translation of preexisting mRNAs. The basis for this response is unknown. The preexisting mRNAs decay during severe heat shock (to a temperature above the optimum for induction of the heat shock RNAs; Spradling et al., 1977; S. L. McKenzie, personal communication). During

Tem-

RNA synthesized at 25°C (left lane) contains RNA species of a wide variety of sizes; at 35°C (center lane) or 36°C (right lane), the heat shock-specific RNAs are induced and most other RNA synthesis declines. The RNA species A4 is detected onlv at 35°C. Electrophoretie analyses of A. Spradling.)

Table

1. Correlation

Puff

33B

RNA”

-

of D. melanogaster

Heat Shock

Puff Sites with Gene

Products

63BC

64F

676

70A

67A/87C

Alb

-

ASb

-

AZ”

87C

930

95D

apa=

-

A3”

-

68”

(A4Y hsp

-

0z4

-

27”

-

-

70. 726

23a. e

’ By in situ hybridization. ’ Nomenclature of Spradling et al. (1977). ’ Nomenclature of Lis et al. (1978). d By hybrid-arrested translation (see text). e By genetic mapping of size polymorphism

(N. S. Peterson,

G. Moller and H. K. Mitchell,

manuscript

in preparation).

Cell 244

23O 26O 29O 31’ 33’

35” 370 38’

- 36,000

.-

-

alnm

--

Figure

4.

Protein

Autoradlograms the temperatures (S. L. McKenzie,

Synthesis

during

Heat Shock

27,000 26,000 23,000 22,000

to Various

Tempera-

of SDS-polyacrylamide gels of proteins labeled at indicated in D. melanogaster tissue culture cells. original data.)

heat shock at the optimum temperature, however, the preexisting RNAs are relatively stable, and some of them, at least, can be isolated from shocked cells and translated in a cell free protein synthesis system (Mirault et al., 1978). They will renew translation after return of the cells to 25°C (S. L. McKenzie, personal communication). About 10 min after the start of the heat shock, the synthesis of the hsp’s can be detected by 35S-methionine incorporation and separation of the polypeptides on SDS-acrylamide gels. There is some confu-

sion in the literature concerning the precise number of hsp’s and their molecular weights. The most economical interpretation of the data suggests that there are eight major different polypeptides in D. melanogaster and six in D. hydei. The apparent molecular weights of the D. melanogaster proteins have been estimated from SDS-acrylamide gels to be approximately 82,000, 70,000, 68,000, 36.000, 27,000, 26,000, 23,000 and 22,000 daltons (although estimates may vary by as much as 6%). We will arbitrarily call them hsp82, hsp70 and so on. The chief source of confusion concerns hsp’s with molecular weights in the 70,000 dalton range. The two-dimensional gels of Mirault et al. (1978) provide evidence that there are several polypeptides in this region, ranging in molecular weight from 70,000 to 72,000 daltons. The evidence, both genetic and biochemical, indicates that these are very similar proteins. One interpretation is that of post-translational modification of a parent polypeptide. Alternatively, since there is good evidence that hsp70 is encoded by the 87A/87C heat shock puff complex and that there are multiple copies of the coding sequences at these sites, it may be that each of these proteins is the product of a particular gene copy. For now we call the “two major” polypeptides in this region hsp70 and hsp72. In addition to the eight major hsp’s, there are minor hsp’s seen only after long exposure of autoradiograms (S. L. McKenzie, personal communication). The broad features of the synthesis of hsp’s after heat shock are not tissue-specific (Tissieres et al., 1974; Lewis et al., 1975). Some tissue differences, however. have been detected (for example, see Sondermeijer and Lubsen, 1978). Charge heterogeneity and size polymorphism in some of the proteins probably result from post-translational protein modifications as well as transcriptional differences (Sondermeijer and Lubsen, 1978; Mirault et al., 1978). In D. melanogaster, synthesis of aft eight hsp’s starts within the first 10 min following an increase in temperature. Maximum rates of synthesis are reached about 1 hr later, by which time the hsp’s account for at least 50% of total precursor incorporation. The rates of hsp synthesis then decline so that by 3 hr (at 37°C in tissue culture cells) they are half-maximal. If the cells are transferred back to 25°C (for example, after 1 hr at 37”C), the rate of hsp synthesis declines much faster than would have been the case at the high temperature [see also Koninkx (1976) for similar data from salivary glands of D. hydei]. With continuous high temperature treatment, the hsp’s accumulate, since they have long half-lives, so that by 6-8 hr they form some 10% of the cell’s total proteins (Moran et al., 1978). Control of the synthesis of the different hsp’s, while similar, is not necessarily “coordinate.” Whereas induction of synthesis of the hsp’s appears simultaneous in D. melanogaster (Lewis et al., 1975; Mirault et

Heat Shock 245

in Drosophila

al., 1978) and may (Koninkx, 1976) or may not (Lewis et al., 1975) be in D. hydei, Mirault et al. (1978) point out that asynchrony of the order of a few minutes would not have been detected in these experiments. Furthermore, S. L. McKenzie (manuscript in preparation) finds significant differential temperature effects on the production of hsp’s in D. melanogaster tissue culture cells. For example, the maximum rate of synthesis of hsp82 occurs at a temperature some 4°C lower than that required for the maximum rate of hsp70 synthesis. At the T,,, for hsp82, hsp70 is made at only some 10% of its maximum rate. S. L. McKenzie argues that these differences occur through the control of mRNA concentration. We should emphasize the extraordinary rapidity of the heat shock response. At the level of the puffs, induction can be seen (at 87C) within 1 min of the temperature shift. By the addition of actinomycin D, which blocks production of the hsp’s if added before the shock, Lewis et al. (1975) found little effect on the subsequent hsp synthesis when the drug was added 5 min after the shock. S. L. McKenzie (personal communication) finds qualitatively similar results in tissue culture cells. In addition to the time required for synthesis of its mRNA, it would presumably take approximately 3-4 min (at 37°C) to synthesize a polypeptide of molecular weight 70,000. Thus since hsp70 can be detected 8-l 0 min after shock (Mirault et al., 1978), the processing of the primary transcript must also be very rapid. We have mentioned the fact that polysomal RNA from heat-shocked cells will hybridize to the sites of the heat shock puffs. In addition, these RNAs can be translated in cell-free protein synthesis systems into polypeptides of identical electrophoretic mobility and, in some cases, identical tryptic digest patterns to hsp’s synthesized in vivo (McKenzie and Meselson, 1977; Sondermeijer and Lubsen, 1978; Mirault et al., 1978; Moran et al., 1978). In several cases, there is direct evidence that particular heat shock puffs code for particular hsp’s. Cloned DNA sequences complementary to the 63BC and 95D (R. Holmgren, K. F. Livak, R. Morimoto, R. Freund and M. Meselson, manuscript in preparation), 678 (E. A. Craig, S. C. Wadsworth and B. McCarthy, manuscript in preparation) and 87A/87C (Livak et al., 1978; McCarthy et al., 1978; Schedl et al., 1978) puff sites have been hybridized to heat shock polysomal RNA before the translation of this RNA in a cell-free system. These clones remove from the translatable RNA those sequences which would have been translated into hsp82 (for 63BC), hsp68 (for 95D), hsp23 and hsp26 (for 6781, and hsp70 and hsp72 (for 87A/87C). The relationships between individual puffs and hsp’s have also been studied by genetic techniques. Ish-Horowitz. Holden and Gehring (1977; also personal communication) have analyzed the effects on the pattern of hsp synthesis of deletions for the 87A/

87C puff complex. Several deletions are available including those which delete both of these puffs, or only one or the other. Homozygotes for these deficiencies are recognizable and, while lethal, survive as embryos or first instar larvae. The surprising result was that homozygotes deficient for either 87A [that is, Df(3R)229] or 87C [that is, Df(3R)kar3J] alone synthesized all the hsp’s after heat shock. It was necessary to delete simultaneously both puffs [that is, in Df(3R)karlW homozygotes or in Df(3R)229 Df(3R)kar3J/Df(3R)karD3 heterozygotes] before any effect could be seen; hsp70 was then completely absent. In view of the data concerning the hybridization of heat shock polysomal RNA to these puff sites and the data from the studies of the DNA sequence organization of recombinant clones hybridizing to these sites, the most reasonable interpretation of this result is that the genes coding for hsp70 are repeated at 87A and 87C. Whether or not they are identical copies of each other or whether they code for different but similar polypeptides (perhaps hsp70 and hsp72) is an open question. A recent report claiming that the 93D heat shock puff codes for hsp70 (Scalenghe and Ritossa, 1977) was in error (F. Scalenghe and F. Ritossa, personal communication). Organization of the Heat Shock Genes The abundance of the mRNA for the heat shock polypeptides in the polysomal RNA of heat-shocked cells has facilitated the isolation of recombinant plasmids from their complementary DNA sequences. Genomic clones, containing heat shock gene sequences, have been isolated in four laboratories by colony hybridization screens using either labeled heat shock RNA directly (Lis, Prestidge and Hogness, 1978; Livak et al., 1978; Schedl et al., 1978) or cloned cDNA prepared against heat shock polysomal RNA (Craig, McCarthy and Wadsworth, 1979). So far, clones have been obtained complementary to the 63BC, 678, 95D and 87A/87C sites, although we have results of detailed analyses only for the latter. The results of the analyses of the clones can be summarized briefly. The first conclusion is that the region coding for hsp70 is found at both 87A and 87C, an observation substantiated by genetic data (discussed above). The second conclusion is that there are multiple copies of the coding region at both 87A and 87C. The third conclusion is that there are repeated sequences at 87C that are transcribed as a response to heat shock but which appear not to be translated. The fourth conclusion is that there are DNA sequences near the 5’ ends of the RNA coding regions which are common to both the 87A and 87C sites. These points will now be considered in somewhat more detail. We will first discuss the sequences complementary to RNA which does not code for hsp70. Lis et al. (1978) described a repeated sequence family whose

Cell 246

transcription is induced by heat shock. A schematic representation of this sequence is shown in Figure 5D. By heteroduplex analysis of several clones containing homologous regions, Lis et al. (1978) identified sequence elements which they named (Y (a 0.4-0.5 kb element), p (1.1 kb) and y (0.9 kb). These elements may be found as short tandem arrays of alternating (Y and p elements, although there are also copies of (Y and ,LI which are not found in such tandem arrays. In situ hybridization indicates that (Y and p elements are found at 87C, but not at 87A, and also in several other chromosomal locations (for example, the chromocenter and lOBC on the X chromosome). Only the a/3 sequences at 87C, however, are in tandem arrays; flies with a homozygous deficiency for 87Cl [DF(3R)kar3J] lack the 1.5 kb Hind Ill fragment characteristic of a/3 tandem arrays. The @ sequence is transcribed in response to heat shock. The most abundant poly(A)+ RNA homologous to C@ is 1.9 kb and corresponds to an a& sequence. This RNA is probably equivalent to A4 of Spradling et al. (1977; see Figure 3). It is transcribed only from the

B

I

i

87C a/3 arrays (Livak et al., 1978). The function of the a/la RNA is unknown at present. The cloned 1.5 kb repeat fails to affect the translation of the hsp mRNAs in a cell-free system (by a hybrid-arrested translation assay; Livak et al., 1978). Thus it would appear that aj?a RNA does not code for any of the major hsp’s. A regulatory role, as suggested by Henikoff and Meselson (1977) and Lis et al. (1978) seems improbable, since flies deleted for the 87C C@ sequences synthesize all of the hsp’s (Ish-Horowitz et al., 1977) and show normal induction and regression of the heat shock puffs (M. Meselson, personal communication; S. Henikoff, personal communication). The hsp70 coding regions have been cloned by Schedl et al. (1978; see also Moran et al., 1979; Artavanis-Tsakonas et al., 1979) McCarthy et al. (1978; see also Craig et al., 1979) and Livak et al. (1978). Clones containing all, or part, of the structural genes for hsp70, whether genomic DNA fragments or cDNA copies of mRNA, hybridize in situ to both 87A and 87C. The coding sequence is approximately 2.2 kb long and lacks detectable intervening sequences

Xho Xbo \ Sal Bgl “‘A 8amPs+ / Sal (111 A”’ ’ j/ 5’ 3’

i

56H8

II

C7

Figure

i

TI I T I, I ’ __--_-_----------

5. Organization

of the 67A/87C

4

Heat Shock

TI

Xba

I: Xho II ,I 11 11

Pst I 5’

ps+\

Barn lPs+ 11,

Sal

c

G5

3’

Genes

Simplified restriction enzyme maps of several cloned genes, aligned at the common sequence containing Xho and Xba restriction sites. It is speculated that this common sequence represents a regulatory locus. In addition to the labeled restriction sites, Hint II sites ( 1 ) and Hind III sites ( 7 ) are shown. Open bars represent the cloning vector. Regions encoding hsp70 are indicated by solid bars beneath the maps; regions encoding &‘a RNA are indicated by stippled bars. Some additional homology with a/3 sequences may be found on clones 132E3 and G5. as indicated by dashed lines. The limits of homologous sequences and the extent of homology of a/3 sequences of these and other clones may be quite variable; the limits shown here may not be exact. The ap gene shown is purely schematic and combines features of several cloned & regions: that the y sequence. partly homologous to the common sequence of the hsp70 genes, may be found at the 5’ end of an RNA coding region; and that ap units may be found in tandem arrays. [Redrawn from Lis et al. (1978); Livak et al. (1978); Craig et al. (1979): Artavanis-Tsakonas et al. (1979); Moran et al. (1979); and L. Moran (personal communication).]

Heat Shock 247

in Drosophila

in heteroduplex electron micrographs. Two of the copies on plasmid 132E3 of Schedl et al. (1978) and three on clone G3 of Craig et al. (1979) are in a direct tandem array, separated by a short (just under 1 kb) noncoding (but not necessarily nontranscribed) region. On each clone, the multiple copies of the coding region have identical restriction enzyme maps, but variation has been observed among the various clones (see below). Homology of the hsp70 gene sequences appears to extend beyond the 5’ end of the coding region. A short DNA stretch with two characteristic restriction sites Xba I and Xho I is found adjacent to the hsp70 mRNA coding sequences in all clones examined so far (Livak et al., 1978; Artavanis-Tsakonas et al., 1979; Craig et al., 1979; Moran et al., 1979). This common sequence is at least partly homologous to the y sequence of Lis et al. (1978). Moreover, a second region of homology more distal to the 5’ end of each of three hsp70 genes was revealed in the detailed comparison of clones 56H8 and 132E3 (Artavanis-Tsakonas et al., 1979; Moran et al., 1979). It has been speculated that the combination of such elements at the 5’ end of the different genes might play a role in their coordinate expression (Lis et al., 1978; Artavanis-Tsakonas et al., 1979; Moran et al., 1979). It is interesting that this putative control sequence is found on clone G5 (from tissue culture cells; Craig et al., 1979) not immediately adjacent to the 5’ end of the coding region, but displaced some 1.3 kb distally (see Figure 5C). Whether G5 encodes a variant of hsp70 (hsp72?) or is not transcribed in vivo is not known. Since the hsp70 genes occur at both 87A and 87C, it is of interest to ask which of the cloned DNA fragments are derived from which site. Clones containing (Yor p sequences (132E3, pPW232, pPW248 and G5) are undoubtedly derived from 87C, since no a or /3 sequences can be detected at 87A. The restriction maps of the hsp70 coding sequences on these clones all have a Pst restriction site 0.1 kb from the 5’ end (Figure 5A). The three coding sequences on G3 also contain this Pst site; G3, like 132E3, lacks the 1.5 kb repeat, but does hybridize with a/3 sequences. Of clones lacking a/3 repeats, 56H8 of Schedl et al. (1978) lacks the 5’ Pst site and has, instead, a Sal site almost at the 5’ end and a Bgl I site 0.3 kb proximal (Figure 5B). Clone pPW231 of Livak et al. (1978) also has the Bgl I site, but the cloned fragment may stop short of the possible Sal site. It is tempting to suggest that the clones lacking the ap repeats and lacking the 5’ terminal Pst site, such as 56H8, may be derived from 87A. However, the finding that there are a number of variant forms of the gene sequence may suggest that there is genetic polymorphism at these sites within the different fly stocks used as sources of DNA for cloning. None of the stocks were made homozygous prior to the isolation of DNA. For example, a 6.6 kb Rl fragment

present in 56H8 (and also present in the Oregon R stock from which the DNA was obtained) is not present in other stocks of D. melanogaster (for example, the cu, kar, Sb 3rd chromosome marker stock; D. IshHorowitz, personal communication). Thus it is not a simple task to assign an origin of 87A or 87C for the plasmids. We have stated that the coding regions for hsp70 are repeated at 87C. There is quite independent evidence for more than one hsp70 coding region at 87A. Homozygotes for Df(3R)kaP2completely lack the 87C region but do retain the puff at 87A. The pattern of hsp’s made by these homozygotes is abnormal, however, for they show, in addition to hsp70, a new hsp of molecular weight 40,000 daltons (F. Ritossa et al., manuscript in preparation). Since the proximal breakpoint of Df(3R)karD’ is at the 87A7 band that forms the puff, the possibility arises that the deletion breakpoint is actually within the more distal of two hsp70 coding regions at this site. That this may be so is seen from a comparison of the tryptic fingerprints of hsp70 and the mutant hsp40, which indicate that hsp40 is a fragment of the larger protein. This result indicates that there are at least two, and possibly more, hsp70 regions at 87A. How many copies of the hsp70 coding region are there at 87A and 87C? Estimates of gene number vary from four copies at 87A and seven at 87C (M.-E. Mirault et al., manuscript in preparation) to two copies at 87A and three at 87C (Henikoff and Meselson, 1977; R. Holmgren et al., manuscript in preparation; D. Ish-Horowitz, personal communication). Differences in the apparent gene number may reflect experimental variation among different laboratories, although there are complicating factors. Genetic polymorphism (discussed above) requires that only homozygous stocks be used for copy number estimation, especially if the method used involves restriction analysis (by transfer of restricted DNA to nitrocellulose filters for hybridization). Furthermore, tandemly repeated genes may undergo unequal crossing over (Sturtevant, 1925; see also Russel et al., 1970); this would give rise to recombinant products with fewer or more copies than on the parental chromosomes. As with the sequences of the genes themselves, the number of copies may well vary among different stocks of Drosophila. Clones have also been isolated, again by hybridization to poly(A)+ heat shock RNA, to the 63BC and 95D sites (R. Holmgren et al., manuscript in preparation). At neither site is there evidence for more than one copy of the coding region, but this does not mean that the surprises the heat shock system has in store are at an end. The fact that the 95D region codes for hsp68 is clear from a hybridization-arrested translation assay. Neither this assay nor those with hsp70 clones gave any indication of homology between the hsp68 and hsp70 genes. Under less stringent condi-

Cell 248

tions of a DNA/DNA hybridization between the 95D clones and two hsp70 clones (pPW232.1, which is roughly the 5’ half of the coding region, and pPW229.1, which is roughly the 3’ half of the coding region), it is clear that there is homology between the hsp70 and hsp68 coding regions. The DNA hybrid is by no means perfect since it has a ATm of about 15°C. Indications of this homology can also be seen in in situ hybridization experiments since both pPW232.1 and pPW229.1 will hybridize to 95D (and more inexplicably to a site in 87D near, or perhaps even at, rosy) (Livak et al. 1978). It is probably too early to attempt to draw any far reaching and general conclusions from these studies of cloned heat shock genes. Until we have information on the organization of other genes in Drosophila for comparison, we will not know which, if any, features of the heat shock gene’s organization are general and which features are special. Induction Mechanism So far, we have discussed the response to heat shock without indicating the wide variety of agents that can induce a similar response. The first study of the heat shock response of the polytene chromosomes also showed that an identical set of puffs could be induced by other agents (Ritossa, 1962). Those now known are listed in Table 2. It can be seen from this table that these agents include uncouplers of oxidative phosphorylation, inhibitors of electron transport, hydrogen acceptors, and inhibitors of various enzymes and other cellular functions. An important, but unanswered, question is whether all these agents act via a common pathway with respect to their action as puff inducers, or whether they act via independent or converging pathways. Leenders et al. (1974a) have been able to rule out at least one possible common denominator of these agents: that the puffing response is one to a decrease in the ATP pool size or an altered ATP/ADP ratio (Ellgaard and Maxwell, 1975; also as implied by Ashburner, 1970). For example. arsenite is a good inducer of the puffs and has no discernable effect on the ATP concentration or ATP/ADP ratio (Vossen et al., 1977). On the other hand, oligomycin or KCN, which do reduce the ATP concentration, fail to induce the puffs, at least by themselves. Oligomycin plus KCN will induce the puffs (Leenders and Berendes, 1972; see also Behnel and Rensing, 1975). The primary targets of many of the inducing agents would appear to be mitochondrial electron transport and oxidative phosphorylation. Leenders et al. (1974b) have proposed that the inducers possess the common property of forcing the electron transport chain into an oxidized state, and that some consequence of this effect results in puff induction. It is difficult, however, to interpret the results of experiments with inhibitors, since they may have a well

known primary site of action within the cell, but may also have effects on other targets or may produce a complex pleiotropic response to the primary effect. For example, it has been found that exogenous ATP or ITP will inhibit the induction of the heat shock puffs (Leenders and Berendes, 1972; Behnel and Rensing, 1975). Similarly, inhibition of the puffing response of D. hydei has also been found by the application of 10 mM malate + 10 mM succinate (Leenders and Berendes, 1972) at pH 7.1 (H. J. Leenders, personal communication). These latter compounds, however, are ineffective in blocking the heat shock response of D. melanogaster unless the culture medium is poorly buffered (Table 3). Thus unless the two Drosophila species respond to heat shock by very different biochemical mechanisms, which appears improbable, it is difficult to extrapolate from these data to a functional model of the response. Despite difficulties in interpretation of inhibitor experiments, the evidence to date favors the idea that the mitochondria are the primary target to the inducing stimuli (Leenders et al., 1974b). If so, it is pertinent to inquire how the effects of the inducers on the mitochondria are transmitted to the genome. The failure of inhibitors of protein synthesis to affect heat shock puff induction indicates that if the agents that signal from the mitochondria to the genome are proteins, these agents must exist in the cell prior to the application of the inducing stimulus. Perhaps inducers bring about a change in the conformation or subcellular compartmentalization of these signal molecules. Evidence favoring this general idea comes from an interesting experiment by Sin (1975). Mitochondria were isolated from normal salivary glands of D. hydei and then either maintained at 25°C in vitro or subjected to a heat shock. From the supernatant of the shocked but not control mitochondria, Sin obtained a nondialyzable heat-labile activity which, when injected into the cytoplasm of salivary gland cells, would induce two heat shock puffs. This result should encourage attempts to reconstitute the responding system using isolated nuclei and fractions from heat-treated cells (isolated nuclei free of cytoplasm do not respond to heat shock). Compton and Bonner (1978) (see also Compton and McCarthy, 1978; McCarthy et al., 1978) and T. Kornberg (personal communication) have obtained the induction of heat shock puffs in isolated salivary gland nuclei after incubation in cytosol from heat-shocked but not control tissue culture cells. This bodes well for the fractionation and reconstitution of those factors which signal between the inducers themselves and the genome. This exciting prospect offers the opportunity to analyze in detail the interaction between specific genes and substances that control their transcription. Function of the hsp’s Little is known about

the nature

of the heat shock

Heat Shock 249

Table

in Drosophila

2. Inducers

of the Heat Shock

Puffs

Inducer

Concentration

Ammonium

chloride

0.01

-

M

Drosophila

Species

Puffs Induced

Reference

melanogaster

At least 930.

hydei

All

11

busckii

None

3

melanogaster

None

8

Saturated

hydei

All but 81 B

11

Arsenite

7.5 x 10-‘M

hydei

All

11

Atractyloside

lo-4M

hydei

None

14

melanogaster

None

16

busckii

All

3

hydei

All

11

melanogaster

93D only

9

virilis

20CD

18

hydei

None

14

melanogaster

None

16

Amytal

3.4 x 10m4 M

Anoxia

Antimycin

A

Azide

3 x 10m3M

Benzamide

1.3%

w/v

Colchicine

0.1 mg/ml

Cyanide

10-6-10~2

M

87A,87C

17

Dicoumarol

10-3M

busckii

All

3

Dinactin

lo-EM

melanogaster

All

13

Dinitrophenol

IO-3M

busckii

All

1

hydei

All

11

melanogaster

All

2, 10.13

busckii

None

3

busckii

All

1

virilis

All

18

All

4. 5. 6

melanogaster

All

2. 8, 10

melanogaster”

All

19

Fluoride

lo-’

M

Heat shock

hydei (and species)

Hydrogen

10m3-2.5

peroxide

Hydroxylamine 2-heptyl-4-hydroxyquinoline (HONO) lodoacetate Menadione Methylene

blue

Oligomycin

N-oxide

X lo-*

M

related

0.01

M

melanogaster

At least 93D. 87A. 87C

17

0.25

mg/ml

hydei

All but 81 B

11

10m3M

busckii

None

3

10-3M

hydei

All

11

lO-‘M

hydei

Allb

11

10m3 M

melanogaster

None

21

Saturated

hydei

None

11

>10-5M

melanogaster

At least 63C

16

Oligomycin

+ atractyloside

As above

hydei

All but 81 B

11

Oligomycin

+ KCN

As above

hydei

All but 81 B

11

busckii

All

3

hydei

All

7

melanogaster

All

8. 15

Recovery

from anoxia

Rotenone

Saturated

hydei

All

11

Salicylate

lO-‘M

busckii

All

1

melanogaster

At least 93D. 87A. 87C

2

Uridine

lo-2M

melanogaster

At least 93D,

17

Valinomycin

2 x 10-=M

melanogaster

All

13”

Vitamin

Injection

hydei

48C

12

B6

of 0.5 Al x 0.1 M

87A. 87C

Cell 250

Table 2. Inducer

Continued Concentration

Drosophila

Organ

Puffs Induced

Reference

hydei

All

12

hydei

None

12

6 x 10-2M

virilis

20CD

18

2 x lo-*M

hydei

Only 48C

12

Saturated

melanogaster

None

20

culture:

Species

5 x lo-2M Organ

culture:

2 x lo-2M

Vitamin

66 + oligomycin

’ Cited as unpublished data. b Puffs induced in only 50% of treated glands. ’ Isolated nuclei only. (1) Ritossa (1962). (2) Ritossa (1963). (3) Ritossa (1964a). (4) Berendes and Holt (1964). (5) Berendes (1965). (6) Berendes et al. (1965). (7) van Breugel (1966). (8) Ashburner (1970). (9) Lakhotia (1971). (IO) Ellgaard (1972). (11) Leenders and Berendes (1972). (12) Leenders et al. (1973). (13) Rensing (1973). (14) Leenders et al. (1974a). (15) Zhimulev and Grafodatskaya (1974). (16) Behnel and Rensing (1975). (17) Scalenghe and Ritossa (1976). (18) Gubenko and Barichera (1978). (19) Compton and McCarthy (1978). (20) M. Ashburner (unpublished data). (21) J. J. Bonner (unpublished data).

polypeptides or why they should be synthesized so rapidly following metabolic insult to cells. Under conditions of a “normal” heat shock (that is, transfer from 25% to a continuous 37”(Z), the induced puffs eventually regress -although synthesis of the proteins continues. This regression does not occur in the absence of protein synthesis unless the temperature is lowered. Furthermore, heat-shocked cells do not recover their normal protein synthesis pattern if production of the hsp’s was blocked by preincubation with actinomycin D (S. L. McKenzie, personal communication). This suggests that the hsp’s play a role in repressing the synthesis of their mRNAs. This could be an autoregulatory phenomenon; perhaps hsp’s compensate for the metabolic imbalance brought about by the shock, in which case conditions favorable for the induction of their genes cease. High temperature (36-38°C) or anoxia causes the developmental arrest of Drosophila pupae, perhaps as a result of the inhibition of normal protein synthesis (Lindsley and Poodry, 1977). In addition, it is well known that various environmental treatments to developing organisms result in rather specific phenotypic abnormalities (phenocopies). Heat shock is indeed one such powerful phenocopy agent for Dro-

sophila (Goldschmidt, 1929, 1935). Depending precisely on the time during development when animals are heat-shocked, very different phenocopies may result (Mitchell and Lipps, 1978). Since identical hsp’s are induced as a response to shock throughout development, it is improbable that phenocopies are a direct consequence of hsp synthesis. Far more probable is the idea that phenocopies result from the inhibition of synthesis of specific proteins normally made at these times during development (Mitchell and Lipps, 1978). The precise phenocopying of certain mutant phenotypes by heat shock may provide a very powerful tool for analyzing the molecular defects of these mutants and hence the mode of action of their normal alleles, thus justifying Goldschmidt’s (1938) optimism. In view of the probable involvement of the mitochondria in the response of the cell to stimuli that induce the heat shock puffs (see Table 2), the Nijmegen Laboratory have studied the effects of heat shock and anoxia on the levels and kinetic properties of several enzymes involved in respiratory processes. These include, for example, increases in the K, of the mitochondrial a-glycerophosphate oxidase (Vossen et al., 19771, the V ,,,aXof NADH dehydrogenase (Leen-

Heat Shock 251

Table

in Drosophila

3. Suppression

of Puffing

by Malate

and Succinate 63C

Puff stage Grace’s

1 control medium,

(not heat-shocked) no additions:

36°C for 20 min

Grace’s medium + 10 mM monosodium malate succinate (final pH 4.4); 36°C for 20 min

678

878

93D

1.01 + 0.02

0.89

f 0.02

1.08

f 0.02

1.23 f

1.65

+ 0.06

1.38

-c 0.04

2.19

+ 0.08

2.16

1.08

f 0.02

0.86

f 0.02

1.01

f 0.02

1.28 It 0.03

0.03

f 0.06

+ 10 mM monosodium

Grace’s medium + 10 mM malate 6.8; 36°C for 20 min

+ 10 mM succinate,

buffered

to pH

Grace’s

medium

+ 30 mM acetate,

buffered

to pH 4.4; 36°C

Grace’s

medium

+ 30 mM acetate,

buffered

to pH 6.8; 36°C for 20 min

1.70 f for 20 min

0.05

1.45

f 0.04

2.12

f 0.04

2.05

f 0.04

r 0.02

0.86

f 0.02

1.06

+ 0.02

1.21

* 0.03

1 .t32 f 0.04

1.48

f 0.02

2.26

+ 0.06

2.02

+ 0.05

1.06

Isolated salivary glands of D. melanogaster were cultured and heat-shocked according to Bonner and Pardue (1976). Chromosome squashes were analyzed by measurement (Ashburner, 1970) of puffs and reference bands 63C:64A3-4, 67B:66EI. 87813-l 5:87F2. 93D:94A. Malate and succinate were added according to Leenders and Berendes (1972); acetate was chosen as a pH control. Data are presented as the mean and standard deviation of the mean, from measurements of four nuclei in each of five glands. The suppression of heat shock puffing by malate and succinate apparently depends, in this species at least, on the final pH of the medium and not on their use by the cell as substrates.

ders and Becker% 1972; Koninkx, 1975; Koninkx, Leenders and Birt, 1975) and the affinity of mitochondria for isocitrate (Sin and Leenders, 1975; Koninkx et al., 1975). There is also a brief report of an increase after induction of tyrosine aminotransferase, although few details have been published (Berendes et al., 1974; Leenders et al., 1974b; see Leenders and Knoppien, 1973). No correlations have been made between changes in enzyme activity and the characterized hsp’s seen after pulse labeling. This should be done before concluding that any enzymes are products of the heat shock puffs. The fact, for example, that antimycin A induced all the puffs of D. hydei except 81 B and also failed to induce an increase in NADH dehydrogenase activity cannot be used as evidence that 81 B codes for this enzyme, as suggested by Koninkx (1975). This is because, in addition to its effects on the electron transport chain, this agent is an effective inhibitor of protein synthesis (Koninkx, 1976). In light of the synthesis of the hsp’s following puff induction, we think that the following experimental criteria should be met before proposing that a given enzyme is a product of a heat shock puff: the purification of the enzyme to show that its subunit sizes and tryptic digest pattern correspond to those of the hsp’s, and/or that immune sera raised against the enzyme will also react with the hsp’s; and genetic or cytogenetic mapping of the structural locus of the enzyme to show that it is coincident with a heat shock puff. It is perhaps intriguing that in D. melanogaster the genetic mapping of an isocitrate dehydrogenase places it near the 678 heat shock puff; this may, of course, merely be a cruel coincidence. Changes in the specific activity of an enzyme unrelated to the mitochondria have been discovered by Scalenghe and Ritossa (1976). This is the cuticular glutamine synthetase of D. melanogaster. Developmentally specific increases in GSl activity occur in

young larvae after heat shock. One subunit of this enzyme has a molecular weight close to 68,000 daltons, and there is evidence from genetic studies that the structural locus for this subunit is near, if not at, the 93D puff site. A genetic approach to the function of the heat shock puffs and their proteins may be useful. The present practical problem is to develop methods allowing the recognition of heat shock puff mutants. This is particularly difficult in view of our ignorance of the functional significance of the hsp’s. The indications that 87A and 87C are duplicated loci each containing multiple genes may make these sites unsuitable for genetic study at present. Indeed D. Ish-Horowitz (personal communication) has made flies of the genotype Df(3R)229 Df(3R)kar3J/Df(3R)kar3J; Dp(2;3)ry+“‘Oh which have one 87A region and no 87C regions. These flies are viable and develop without delay. With these and other heat shock puff sites, it would be well worth investigating whether or not their absence (for example, from a deletion heterozygote) affects the ability of Drosophila to withstand the effects of temperature shock. If so, this could form the basis for a genetic screen. General Conclusion From what we have reported so far, it is clear that the response of the genome of Drosophia to heat shock and other stimuli is remarkably specific and yet occurs at several different levels. The first level is, presumably, the interaction between the external inducer and some point(s) within the metabolic machinery of the cell. This leads to the production of signal(s) that interact with the genome both to initiate and repress the synthesis of specific RNAs, at the level of RNA processing and at the level of protein synthesis. These result in the effective and efficient translation of a small number of specific polypeptides. How these polypeptides function is unknown. It is reasonable to

Cell 252

assume that the response is homeostatic, tending to correct the defects in cellular metabolism wrought by the initial stimulus. If this were so, it would be highly improbable for such a complex response to be peculiar to Drosophila; indeed one would expect it to occur in other organisms, at the very least in poilkilotherms. It is therefore of great interest that rather analogous responses to heat shock, anoxia or inhibitors have been described in organisms as different as Zea mays (Sachs and Freeling, 19781, Tetrahymena (Yuyama and Zimmermann, 1972; Hermolin and Zimmermann, 1976) and chick fibroblasts (Kelley and Schlesinger, 1978; W. Levinson, personal communication). The heat shock response may appear to be a very abnormal situation, brought about solely by sadistic experimenters. It should not be forgotten, however, that Drosophila larvae, at least, may frequently meet situations that bring about this response in their everyday lives-for example, relative anoxia while feeding in semiliquid substrates or brief temperature shock due to inadvertant exposure to the sun. In any event, the coordination of the response and the applicability of many analytical tools should encourage the most detailed analysis of the mechanisms of the response, for there is every reason to believe that the lessons thus learned will be of far greater importance. Acknowledgments This review was largely written while M.A. was visiting the Department of Biochemistry and Biophysics, University of California, San Francisco. He thanks Professor W. J. Flutter and all of his colleagues for their generous hospitality. J.J.B. is grateful to Dr. Keith Ft. Yamamoto. whose laboratory space and discussions were invaluable during the preparation of this review. We are especially indebted to Toni King for patiently preparing myriad drafts of the manuscript. Our debt to all those who study heat shock should be obvious: not only have they freely provided us with much unpublished information, but they have by their critical, discerning comments helped us to avoid at least some pitfalls. The authors remain, of course, entirely responsible for the contents of this review. J.J.B. was supported by a postdoctoral fellowship from the Amencan Cancer Society. Received

January

repleta group ature shocks.

of the genus Drosophila. I. Gene activity Genen. Phaenen. 70. 32-41.

Berendes. H. D. (1968). Factors activity in polytene chromosomes.

involved in the expression of gene Chromosoma 24. 418-437.

Berendes, H. D. and Holt. Th. K. H. (1964). The induction of chromosomal activities by temperature shocks. Genen. Phaenen. 9. l-7. Berendes. H. D.. van Breugel, F. M. A. and Holt. Th. K. H. (1965). Experimental puffs in salivary gland chromosomes of D. hydei. Chromosoma 76. 35-46. Berendes. H. D.. Alonso, C.. Helmsing. P. J.. Leenders. H. J. and Derksen. J. (1974). Structure and function in the genome of Drosophila hydei. Cold Spring Harbor Symp. Ouant. Biol. 38. 645-654. Biessmann. transcription melanogaster

H., Levy, B. W. and McCarthy, B. J. (1978). In vitro of heat-shock specific RNA from chromatin of Drosophila cells. Proc. Nat. Acad. Sci. USA 75, 759-763.

Bonner, J. J. and Pardue, M. L. (1976). The effect RNA synthesis in Drosophila tissues. Cell 8. 43-50.

of heat shock

on

Compton. J. L. and Banner. J. J. (1978). An in vitro assay for the specific induction and regression of puffs in isolated polytene nuclei of Drosophila melanogaster. Cold Spring Harbor Symp. Ouant. Biol. 42. 835-838. Compton, J. L. and McCarthy, B. J. (1978). Induction of the Drosophila heat shock response in isolated polytene nuclei. Cell 14. 191201. Craig, E. A., McCarthy, B. J. and Wadsworth, S. C. (1979). Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster. Cell 16. 575588. Elgin. S. C. R.. Serunian. L. A. and Silver, L. M. (1978). patterns of Drosophila nonhistone chromosomal proteins. Harbor Symp. Ouant. Biol. 42, 839-850. Ellgaard. E. G. (1968). An analysis of puff formation in polytene chromosomes of Drosophila melanogaster. University of Iowa. Iowa City, Iowa.

Distribution Cold Spring

and regression Ph.D. thesis,

Ellgaard. E. G. (1972). Similarities in chromosomal puffing induced by temperature shocks and dinitrophenol in Drosophila. Chromosoma 37. 417-422. Ellgaard. induction

E. G. and Clever, U. (1971). RNA metabolism during in Drosophila melanogaster. Chromosoma 36, 60-78.

puff

Ellgaard. E. G. and Maxwell, B. L. (1975). Nucleotide metabolism in Drosophila melanogaster salivary glands during temperatureand dinitrophenol-induced puffing. Cell Differentiation 3. 379-387. Goldschmidt, R. (1929). Experimentelle Mutation und das Problem der sogenannten Parallelinduktion. Versuche an Drosophila. Biol. Zent. 49, 437-448. Goldschmidt. R. (1935). chungen an Drosophila). 38-69.

12. 1979

after temper-

Gen und Ausseneigenschaft. I. (UntersuZ. indukt. Abstamm.-u. Vererb.-Lehre. 69,

References

Goldschmidt, R. (1938). don: McGraw-Hill).

Artavanis-Tsakonas. S., Schedl, P., Mirault, M.-E.. Moran, L. and Lis. J. (1979). Genes for the 70,000 dalton heat shock protein in two cloned D. melanogaster DNA segments. Cell I 7. 9-l 8.

Greenleaf. A. L.. Plagens. U.. Jamrich, M. and Bautz. E. K. F. (1978). RNA polymerase B (or II) in heat-induced puffs on Drosophila polytene chromosomes. Chromosoma 65. 127-l 36.

Ashburner, M. (1970). Patterns of puffing chromosomes of Drosophila, V. Responses ment. Chromosoma 31. 358-376.

Gubenko. I. S. and Baricheva. E. M. (1978). Drosophila virilis puffs induced by temperature and other environmental factors. Genetika (USSR), in press.

activity in salivary to environmental

gland treat-

Ashburner, M. and Richards, G. (1976). The role of ecdysone in the control of gene activity in the polytene chromosomes of Drosophila. In Insect Development, P. A. Lawrence, ed. (New York: Wiley). pp. 203-225. Behnel, J. and Rensing. L. (1975). the induction of puffs in Drosophila 97. 119-124. Belyaeva. Drosophila

E. S. and Zhimulev, melanogaster puffs.

Berendes,

H. D. (1965).

Respiratory functions involved in salivary glands. Exp. Cell Res.

I. F. (1976). RNA synthesis Cell Differentiation 4, 415-427.

Gene homologies

in different

species

in the of the

Physiological

Genetics

(New York

Henikoff, S. and Meselson. M. (1977). Transcription shock loci in Drosophila. Cell 72. 441-451. Hermolin, J. and division-synchronized RNA. J. Protozool.

Zimmermann. A. M. (1976). tetrahymena. Macronuclear 23, 594-600.

and Lon-

at two

heat

RNA synthesis in and cytoplasmic

Holt. Th. K. H. (1970). Local protein accumulation during gene activation. Il. lnterferometric measurements of the amount of solid material in temperature induced puffs of Drosophila hydei. Chromosoma 32, 428-435. Holt. Th. K. H. and Kuijpers.

A. M. C. (1972).

Induction

of chromo-

Heat Shock

in Drosophila

253

some puffs in Drosophila synthesis by a-amanitin.

hydei salivary glands after inhibition Chromosoma 37. 423-432.

of RNA

Ish-Horowitz, Cr., Holden. J. J. and Gehring. W. J. (1977). Deletions of two heat-activated loci in Drosophila melanogaster and their effects on heat-induced protein synthesis. Cell 12. 643-652. Jacq. 6.. Jourdan, Ft. and Jourdan. B. R. (1977). Structure and processing of precursor 55 RNA in Drosophila melanogaster. J. Mol. Biol. 117. 785-795. Jamrich. M.. Greenleaf, A. L. and Bautz, E. K. F. (1977a). Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 74, 2079-2083. Jamrich. M.. Haars. R.. Wulf. E. and Bautz, E. K. F. (1977b). Correlation of RNA polymerase B and transcriptional activity in the chromosomes of Drosophila. Chromosoma 64, 319-326. Kelley. P. M. and Schlesinger, M. J. (1978). The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts. Cell 15. 1277-l 286. Koninkx. J. F. J. G. (1975). Induced transcription dependent synthesis of mitochondrial reduced nicotinamide-adenine dinucleotide dehydrogenase in Drosophila. Biochem. J. f52, 17-22. Koninkx, J. F. J. G. (1976). Protein synthesis in salivary glands of Drosophila hydei after experimental gene induction. Biochem. J. 158. 623-628.

Koninkx. J. F. J. G.. Leenders. t-l. J. and Birt, L. M. (1975). A correlation between newly induced gene activity and an enhancement of mitochondrial enzyme activity in the salivary glands of Drosophila. Exp. Cell Res. 92. 275-282. Lakhotia. S. C. (1971 )..Benzamide as a tool for gene activity studies in Drosophila. Abstracts of the Fourth Cell Biology Conference. New Delhi, 1971 Leenders, H. J. and Beckers. P. J. A. (1972). The effect of changes in the respiratory metabolism upon genome activity. A correlation between induced gene activity and an increase in activity of a respiratory enzyme. J. Cell Biol. 55. 257-265. Leenders. H. J. and Berendes, l-t. D. (1972). The effect of changes in the respiratory metabolism upon genome activity in Drosophila. I. The induction of gene activity. Chromosoma 37. 433-444. Leenders, H. J. and Knoppien, W. G. (1973). Respiration of larval salivary glands of Drosophila in relation to the activity of specific genome loci. J. Insect Physiol. 7 9, 1793-l 800.

Lubsen. (1978). 2-48BC

N. H.. Sondermeiter, P. J. A., Pages, M. and Alonso, C. In situ hybridization of nuclear and cytoplasmic RNA to locus in Drosophila hydei. Chromosoma 65. 199-212.

McCarthy, B. J., Compton. J. L.. Craig, E. A. and Wadsworth, S. C. (1978). Transcription at the heat shock loci of Drosophila. Tenth Miami Winter Symposium (New York: Academic Press). pp. 317333.

McKenzre, S. L. (1976). Protein and RNA syntheses induced by heat in Drosophila melanogaster tissue culture cells. Ph.D. Theses, Harvard University, Cambridge, Massachusetts. McKenzie, Drosophila

S. L. and heat-shock

Meselson. messages.

M. (1977). Translation in vitro J. Mol. Biol. 7 17. 279-283.

of

McKenzie. S. L.. Henikoff. S. and Meselson. M. (1975). Localization of RNA from heat-induced polysomes at puff sites in Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 72. 11 17-l 121. Mayfield. J. E.. Serunian, (1978). A protein released IS preferentially associated

L. A., Silver, L. M. and Elgin. S. C. R. by DNAase I digestion of Drosophila nuclei with puffs. Cell 74. 539-544.

Mirault, M.-E., Goldschmidt-Clermont. M., Moran, L.. Arrigo, A. P. and Ttssieres, A. (1978). The effect of heat shock on gene expression in Drosophila melanogaster. Cold Spring Harbor Symp. Quant. Biol. 42, 819-827. Mitchell, induction

H. K. and Lipps. L. S. (1978). Heat shock in Drosophila. Cell 15. 907-918.

and phenocopy

Moran, L.. Mirault, M.-E., Arrigo. A. P., Goldschmidt-Clermont. M. and Tissieres. A. (1978). Heat shock of Drosophila melanogaster induces the synthesis of new messenger RNAs and proteins. Phil. Trans. Roy. Sot. Lond. B 283, 391-406. Moran. L.. Mirault. Tsakonas, S. and melanogaster DNA 70,000 dalton heat

M.-E., Tissieres, A.. Lis. J., Schedl. P.. ArtavanisGehring, W. J. (1979). Physical map of two D. segments containing sequences coding for the shock protein. Cell 17, l-8.

Plagens. U.. Greenleaf. A. L. and Bautz, E. K. F. (1976). Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect tmmunofluorescence. Chromosoma 59. 157-165. Rensing. L. (1973). Effects of 2,4-dinitrophenol and dinactin on heatsensitive and ecdysone-specific puffs of Drosophila salivary gland chromosomes in vitro. Cell Differentiation 2, 221-228. Ritossa, F. (1962). DNP in Drosophila.

A new puffing pattern induced Experientia 18. 571-573.

by heat shock

and

Leenders. H. J.. Derkson. J.. Maas. P. M. J. M. and Berendes, H. D. (1973). Selective induction of a giant puff in Drosophila hydei by VitaIIIin B6 and derivatives. Chromosoma 4 1. 447-460.

Ritossa. F. M. (1963). New puffs induced by temperature shock, DNA and salicylate in salivary chromosomes of Drosophrla melanogaster. Drosophila Information Service 37. 122-l 23.

Leenders. H. J., Kemp. A., Koninkx. J. F. J. G. and Rosing. J. (1974a). Changes in cellular ATP, ADP. and AMP levels following treatments affecting cellular respiration and the activity of certain nuclear genes in Drosophila salivary glands. Exp. Cell. Res. 86, 25-

Ritossa. polytene

30.

Leenders, H. J.. Berendes. H. D.. Helmsmg. P. J.. Derksen. J. and Koninkx. J. F. J. G. (1974b). Nuclear-mrtochondrial interactions in the control of mitochondrial respiratory metabolism. Sub-Cell. Biothem. 3, 119-147. Lengyei. J. A. and Pardue. M. L. (1975). Analysis of hnRNA made during heat shock in Drosophila melanogaster cultured cells. J. Cell Biol. 67. 240a. Lewis. M., Helmsing. P. J. and Ashburner. M. (1975). Parallel changes in Puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc. Nat. Acad. Sci. USA 72. 3604-3608.

F. M. (1964a). chromosomes

Experimental of Drosophila.

activation of specific IOCI in Exp. Cell Res. 35. 601-607.

Ritossa. F. M. (1964b). Behavior of RNA and DNA synthesis at the puff level in salivary gland chromosomes of Drosophila. Exp. Cell Res. 36’. 515-523. Rubin. G. M. and Hogness. D. S. (1975). Effect of heat shock on the synthesis of low molecular weight RNAs in Drosophila: accumulation of a novel form of 5s RNA. Cell 6. 207-213. Russel. R. L.. Abelson, J. N., Landy. A., Gefter. M. L.. Brenner. S. and Smith. J. D. (1970). Duplicate genes for tyrosine transfer RNA in Escherichia coli. J. Mol. Biol. 47, l-l 3. Sachs, M. M. and Freeling. M. (1978). Selective synthesis of alcohol dehydrogenase during anaerobic treatment of maize. Mol. Gen. Genet.. in press.

Lindsley. D. E. and Poodry. C. A. (1977). A reversible temperatureinduced developmental arrest in Drosophila. Dev. Biol. 56. 213-218.

Scalenghe. Drosophila: Nat. Lincer

F. and Ritossa. F. (1976). Controllo dell‘attivita’ genica in II puff al locus ebony e la glutamina sintetasr 1. Atti. Acad. 13, 439-528.

Lis. J.. Prestidge, L. and Hogness. D. S. (1978). A novel arrangement of tandemly repeated genes at a major heat shock site in D. melanogaster. Cell 14. 901-919.

Scalenghe. F. and Ritossa. F. (1977). 93D is responsible for the synthesis polypeptide in Drosophila melanogaster.

Livak, K. F.. Freund. R.. Schweber. M., Wensink, P. C. and Meselson, M. (1978). Sequence organization and transcription at two heat shock loci in Drosophila. Proc. Nat. Acad. Sci. USA, in press.

Schedl. P., Artavanis-Tsakonas, S.. Steward, R., Gehring, W. J.. Mirault. M.-E., Goldschmidt-Clermont, M.. Moran, L. and Tissieres. A. (1978). Two hybrid plasmids with D. melanogaster DNA sequences

The puff inducible in region of the major “heat shock” Chromosoma 63. 317-327.

Cell 254

complementary to mRNA Cell 74, 921-929.

coding

for the major

heat shock

protein.

Silver, L. M. and Elgin. S. C. Ft. (1977). Distribution patterns of three subfractions of Drosophila nonhistone chromosomal proteins: possible correlations with gene activity. Cell 17. 971-983. Silver, L. M. and Elgin. S. C. Ft. (1979). protein distributions in Drosophila polytene Nucleus, 5, in press. Sin, Y. T. (1975). by mitochondrial

Induction factor(s).

Sin, Y. T. and Leenders, NAD-dependent isocitrate induction of gene activity 458. Sondermeijer, in Drosophila 331-339.

Immunological chromosomes.

analysis of In The Cell

of puffs in Drosophila salivary Nature 258, 159-l 60.

gland cells

f-i. J. (1975). Studies on the mitochondrial dehydrogenase of Drosophila larvae after by anaerobiosis. Insect Biochem. 5. 447-

P. J. A. and Lubsen. N. H. (1978). hydei and their in vitro synthesis.

Heat shock peptides Eur. J. Biochem. 88,

Spradling, A., Penman, S. and Pardue. M. L. (1975). Analysis Drosophila mRNA by in situ hybridization: sequences transcribed normal and heat shocked cultured cells. Cell 4, 395-404. Spradling, A., Pardue, M. L. and Penman, S. (1977). Messenger in heat-shocked Drosophila cells. J. Mol. Viol. 109. 559-587.

of in RNA

Spruill. W. A., Hurwitz. D. R.. Lucchesi. J. C. and Steiner, A. L. (1978). Association of cyclic GMP with gene expression of polytene chromosomes of Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 75, 1480- 1484. Sturtevant. Bar locus.

A. H. (1925). The effects Genetics 10. 117-147.

of unequal

crossing

Tissieres. A.. Mitchell, H. K. and Tracy, U. M. (1974). sis in salivary glands of D. melanogaster. Relation puffs. J. Mol. Biol. 84, 389-398. van Breugel, chromosomes

F. M. A. (1966). Puff induction in larval of D. hydei. Genetica 37, 17-28.

over at the

Protein syntheto chromosome salivary

gland

van Breugel, F. M. A., Ray, A. and Gloor, H. (1968). A comparison banding patterns in salivary gland chromosomes of two species Drosophila. Genetica 39, 165-l 92.

of of

Vossen. J. G. H. M.. Leenders, H. J.. Derksen, J. and Jeucken. G. (1977). Chromosomal puff induction in salivary glands from Drosophila hyder by arsenite. Exp. Cell Res. 109, 277-283. Yuyama, hymena.

S. and Zimmermann, Temperature-pressure

A. M. (1972). RNA synthesis in tetrastudies. Exp. Cell Res. 77, 193-203.

Zhimulev. I. and Grafodatskaya, V. E. (1974). A simple induction of anaerobiosis puffs in Drosophila melanogaster. ila Information Service 5 7, 96.

method of Drosoph-