Proc. Nati. Acad. Sci. USA Vol. 81, pp. 3138-3142, May 1984 Developmental Biology

Developmental control of the heat shock response in Xenopus (cDNA cloning/M13/ sequences) Laboratory of , Medical Research Council Centre, Hills Road, Cambridge, England Communicated by J. B. Gurdon, January 26, 1984

ABSTRACT Xenopus cells express two major proteins on teins-i.e., at puparium formation, cells naturally produce heat shock, designated hsp 70 and hsp 30. Several cDNA small heat shock proteins (9). Furthermore, Drosophila clones for the corresponding mRNAs were identified and se- nurse cells synthesize a subset of heat shock mRNAs (10). In quenced. Inducibility and abundance of heat shock mRNAs in mouse embryonic development, hsp 68 and hsp 70 are various cell types and developmental stages was determined by among the first detectable amanitin-sensitive embryonic nuclease Sl-mapping. The only cells found to contain hsp 70 products (11). mRNA without heat shock are the oocytes. The level of this Xenopus oocytes have evolved an unusual mechanism to stored hsp 70 mRNA is not increased by heat shock. After fer- regulate their heat shock response, based entirely on a trans- tilization, hsp 70 mRNA becomes undetectable; it appears as a lational activation of preexisting hsp 70 mRNA (12); we ten- heat-inducible mRNA for the first time at gastrulation. Alter tatively have concluded that this implies a period ofconstitu- this stage, all somatic cell types accumulate hsp 70 mRNA to tive hsp 70 gene activity during oogenesis. Analyzing somat- similar levels on heat shock, presumably by transcriptional ac- ic frog cells, I found that these cells are capable of tivation of the hsp 70 genes. In contrast, hsp 30 mRNA is not expressing two types of heat shock proteins, hsB 70 and hsp detectable, even after heat shock, in oocytes or embryos that 30 (13). In the following sections, I will describe the pattern induce hsp 70 mRNA to high levels. Heat inducibility appears of heat shock gene expression in different Xenopus cell late in development-at the tadpole stage. However, the level types and developmental stages, using complementary of induced mRNA varies considerably in different adult tis- probes to the main heat shock mRNAs. The results demon- sues. This indicates that the Xenopus heat shock genes are not strate that the expression of the two classes of heat shock coordinately controlled. A long-term developmental control genes is not entirely coordinate and provide evidence that appears to be superimposed on the temporary heat inducibility these genes are not only temporarily inducible but also sub- of the heat shock genes: stage- or cell-type-specific conditions ject to developmental regulation. can lead to constitutivt or repressed heat shock genes. MATERIAL AND METHODS Heat shock and comparable environmental stresses induce a cDNA Cloning. Poly(A)' RNA was prepared from Xeno- striking physiological reaction in cells, the heat shock re- pus tissue-cultured cells (epithelial kidney fibroblast cell sponse. This consists of a drastic change in a cell's gene line) that had been heat-shocked for 4 hr at 370C, interrupted expression program in that it ceases synthesizing its typical by a 1-hr recovery at room temperature as described (14). complex set of proteins and focuses on the efficient produc- Two sequential runs of oligo(dT)-cellulose chromatography tion of a small nUmber of proteins, the heat shock proteins. were performed in order to minimize the content of ribosom- Despite efforts to assign a specific function to these pro- al RNA. For cDNA cloning, a strategy was adapted that has teins, one is still left with the vague and collective view that been used for tobacco mosaic virus cloning and sequencing they confer protection against the present and further stress- (15). Double-stranded cDNA was synthesized essentially by es to the cell (1). There is not enough experimental evidence the method of Maniatis et al. (16) using oligo(dT) priming to support the likely assumption that the individual heat- and reverse transcriptase for the first strand and Klenow shock proteins have different functions. If one assumes this polymerase for the second strand. This mixture was then di- to be the case, one could expect to find a differential expres- gested with Sau3A and ligated into BamHI-cut M13mp7 (17). sion of the heat shock proteins according to cell type and The library was plated out, and 2000 white plaques were developmental stage. picked onto a fresh bacterial lawn. Replica filters were lifted The coordinate expression of the heat shock proteins has off the plates and probed with kinase-labeled total RNA always been stressed. The main regulatory switch operates from heat-shocked or control Xenopus tissue-cultured cells at the transcriptional level: the heat shock response was dis- (16). This screening was repeated on 57 plaques that were covered by the observation of newly induced puffs in sali- positive for heat shock RNA but negative for control RNA. vary gland chromosomes (2), a phenomenon which visual- After the second screening, 27 potential heat shock clones izes this transcriptional switch. Drosophila heat shock genes were directly sequenced by the dideoxy method (17, 18), and have been cloned and sequenced (3-6), and a short oligonu- 12 clones were identified as heat shock clones by sequence cleotide that is found in most of their control regions has comparison (19) to the Drosophila heat shock genes (4, 5). been identified to be a necessary element for heat-induced Analysis by Nuclease S1 Mapping. Two of the isolated Xen- (7, 8). Thus, the temporary heat shock activa- opus heat shock clones were subcloned into M13mp8 (17) to tion of heat shock genes appears to be understood in princi- reverse the orientation. Single-stranded probes complemen- ple. tary to RNA were prepared by using the mp8 clones: a radio- There is increasing evidence, however, that the regulation active strand was first synthesized as for sequence analysis of the heat shock response is more complex. In Drosophila, with dideoxy nucleotides omitted, then cut with Ava II (at a ecdysone has been found to induce the small heat shock pro- unique site in both vectors beyond the inserts), denatured, and run on a sequencing gel (17). The gel separated comple- The publication costs of this article were defrayed in part by page charge mentary M13 sequences from the single-stranded radioactive payment. This article must therefore be hereby marked "advertisement" probe, which was subsequently eluted. An excess of probe in accordance with 18 U.S.C. §1734 solely to indicate this fact. was hybridized overnight at 42°C to 1-50 ,ug of total LiCl- 3138 Downloaded by guest on October 2, 2021 Developmental Biology: Bienz Proc. Natl. Acad Sci. USA 81 (1984) 3139 precipitable RNA (14) in 50% formamide/400 mM NaCl/10 The use of these clones for hybrid-selecting mRNA and for mM Pipes, pH 6.5/1 mM EDTA. Subsequent nuclease S1 subsequent in vitro of the selected mRNAs con- digestion and gel analysis was done as previously described firmed that two of the three clones (X4, X5) contained se- (7). quences coding for the small Mr 30,000 heat shock protein In Vitro Translation. Total LiCi-precipitable RNA was ex- (hsp 30) and that the third one (X16) selected RNA coding tracted from oocytes, embryos, adult organs, or tissue-cul- for the large Mr 70,000 heat shock protein (hsp 70; data not tured cells and translated in a reticulocyte lysate, and the shown). translational products were analyzed by gel electrophoresis Radioactive probes complementary to these heat shock as described (12). clones were subsequently used to detect heat shock RNA by nuclease S1 mapping. An internal 120-base fragment of hsp RESULTS 70 mRNA should be protected by the X16 probe, and 205 bases of hsp 30 mRNA, by the X4 probe. When total RNA Previous results concerning the Xenopus heat shock re- from heat-shocked tissue-cultured cells was subjected to nu- sponse were obtained either by in vivo labeling of proteins or clease S1 mapping, both probes gave rise to a strong band of by RNA extraction and subsequent in vitro translation. In the expected length (Fig. 3a). An M13 clone of the opposite order to carry out a more sensitive and precise analysis of orientation (X16rev) was hybridized to the X16 probe, which heat shock gene expression, it became crucial to clone the permitted estimation of the amount of hsp 70 mRNA in a mRNAs. A fast procedure was chosen that allowed cloning given sample by comparison of the signals (Fig. 3a). Al- of cDNA fragments. Double-stranded cDNA was synthe- though this method relies on an accurate determination of sized by using polyA-containing RNA from heat-shocked tis- the M13 template concentration and assumes equal hybrid- sue-cultured cells, which accumulate high levels of heat ization efficiencies, and although different experiments gave shock mRNAs under these conditions (Fig. 1; see also ref. somewhat variable quantitative results, the order of magni- 13). This cDNA was then digested with Sau3A I, an enzyme tude could be estimated. About Q.02-0.06 ng, corresponding that cuts it into fragments of a few hundred base pairs, and to 5 x 106-1.5 x 107 copies of 16Xrev, could be detected the mixture of fragments were inserted into M13. A cDNA reliably (lane 4 in Fig. 3a and lane 1 in Fig. 3b). An hsp 70 library was obtained with an efficiency of 104 plaques per pg signal of comparable intensity was obtained by analyzing of RNA. Drosophila heat shock clones could not be used for 0.16 gg of total heat-shocked cell RNA (Fig. 3a, lane 7) identification because no cross-hybridization was found be- which represents the yield from 4 x 104 cells. By averaging tween Xenopus heat shock RNA and Drosophila heat shock different experiments, it could be concluded that the signal genes. Differential screening of 2000 plaques with radioac- produced by a single cell's RNA equals the signal produced tively labeled RNA from heat-shocked and control Xenopus by 125-375 X16rev copies. However, the fragment protected cells on replica filters defined 27 clones as potential heat by X16rev was 3 times longer than the fragment protected by shock clones. hsp 70 mRNA-i.e., X16rev detection was 3 times more sen- Direct dideoxy sequence determination of the potential sitive than was hsp 70 mRNA detection. After correction for clones led to the identification of 12 heat shock clones repre- this fact, I conclude that a single heat-shocked somatic cell senting three different fragments (one of which was isolated contains about 400-1100 copies of hsp 70 mRNA, accumu- 10 times independently), on the basis of homolo- lated in 1-3 hr of heat shock. The level of hsp 30 mRNA was gy to the Drosophila heat shock proteins (Fig. 2). Sequence similar: although the signal for hsp 30 mRNA was apparently comparison shows that X16 covers about 10% of the hsp 70 twice as strong as the signal for hsp 70, this was again due to mRNA protein coding sequence and that it is derived from the above nentioned fact of protected fragment length. the 5' end of the mRNA (starting at Drosophila amino acid Oocytes Contain Low Levels of Preexisting hsp 70 mRNA. 77). The protein sequence homology between Drosophila Previously, we concluded that oocytes contain stored inac- and Xenopus is about 70%. The two clones containing small tive hsp 70 mRNA (12). To confirm this conclusion, total heat shock protein mRNA sequences, X5 and X4, probably RNA extracted from heat-shocked or control oocytes was represent adjacent fragments and cover together about 60% subjected to nuclease S1 mapping by using probes derived of the protein coding sequence. They were derived from the from X16 and X4 (Fig. 3b). Since every oocyte contains 3' end of the mRNA. The protein sequence homology be- >1000 adhering follicle cells, which are most likely to accu- tween Drosophila and Xenopus is in this case about 35-50%. mulate hsp 70 mRNA on heat-shock and would obscure the result, oocytes were defolliculated manually or by collage- b Xc nase treatment prior to heat shock (defolliculation does not induce the heat shock response; ref. 12). The results show that indeed hsp 70 mRNA was contained in non-heat shocked oocytes and that its level was not significantly in- creased by a heat shock. No hsp 30 mRNA could be detected before or after a heat shock. Analysis of RNA from whole ovarial lobes showed a 2- to 5-fold induction of hsp 70 - _ hsp 70 mRNA and a trace of hsp 30 mRNA (Fig. 3b, lane 6), which apparently reflects mRNA from follicle and other somatic

FIG. 1. In vitro translation of cells. heat shock and control RNA. To- The amount of hsp 70 mRNA per oocyte was estimated by - - tal LiCl-precipitable RNA was ex- using X16rev as a standard, as described in the previous tracted from heat-shocked (90 min paragraph. The signal of 10 oocytes (Fig. 3b, lanes 5, 7, and at 370C, lane +) and control (lane 8) corresponded to the signal generated by about 0.04 ng or -)tissue-cultured cells (lanes Xc) 107 copies of X16rev (Fig. 3b, lanes 1 and 2). Thus, after and translated in a rabbit reticu- correcting this value by a factor of three (see previous para- - hsp 30 The locyte lysate. translational graph), I conclude that one oocyte contains about 3 X 106 products were run on a one-di- of 70 mRNA. This value is below the val- mensional NaDodSO4/12% poly- copies hsp slightly acrylamide gel. Arrows indicate ue that was previously calculated (107-10 copies per oocyte; hsp 70 and hsp 30 synthesis. No ref. 12). Since the detection of hsp 30 mRNA is twice as RNA was added in lane b. sensitive as the detection of hsp 70 mRNA (the protected Downloaded by guest on October 2, 2021 3140 Developmental Biology: Bienz Proc. Natl. Acad. Sci. USA 81 (1984)

aa77 asp pro lys ile ala glu asp met lys his trp pro phe lys val val ser asp gly gly lys pro lys ile gly val glu tyr lys gly glu ser lys arg phe ala pro glu glu ile D 70 GAC CCC AAC ATC GCA GAG GAC ATG AAG CAC TGG CCT TTC MG GTG GTA ACC GAC GGC GGA MG CCC AAG ATC GGG GTG GAG TAT MG GGT GAG TCC MC AGA TTT GCC CCT GAG GAG ATC *-UU ~U**" U U * U *U"*-U U"U U U * * *"" X 16 GAT CCT GTA GTG CAG TGT GAC TTG MG CAC TGG CCG TTC CAA GTG GTG AGC GAT GAG GGG AAM CCT MG GTC AAA GTG GAG TAT AAA GGA GM GAG AAA AGC TTC TTC CCT GAG GAG ATC asp pro val val gln cys asp leu lys his trp pro phe gln val val ser asp glu gly lys pro lys val lys val glu tyr lys gly glu glu lys ser phe phe pro glu glu i]e

aa9O asp gly phe gln val cys lie asp val ser gIn phe lys pro asn glu leu thr val lys val val asp asn thr val vai val glu gly lys his glu glu arg asp gly phe gln val cys met asp val ala gln phe lys pfo ser glu leu asn val lys val val asp ala ser lie leu val glu gly lys his glu glu arg D 22-27 asp gly phe gln val cys met asp val ser his phe glu pro ser glu leu val val gly val gln asp asn ser val leu val glu gly asn his glu glu arg asp giy tyr lys leu thr leu asp val lys asp tyr ser glu leu lys val lys val leu asp gly ser val val leu val gly gly lys ser glu gin gln GAT GGC TAC AAA CTC ACC CTG GAC GTC MG GAC TAC AGC GAG CTG MG GTC MG GTG CTC CAC GAG AGC GTT CTC CTG GTG GAG GCA AAA TCG GAG CAG CAG * * * U UU - U - U *- U U U * *U * U" X 5 GAT CAC TTT GAG CTC ACG CTG GAT GTG AGG GAC TTT TCT CCT CAT GAA CTA ACA GTG AMA ACA CAG GGA AGG AGA CTA ATT GTC ACA GGA AAA CAT GAG AGG AM asp his phe lu leu thr leu asp val arg asp phe ser pro his glu leu thr val lys thr gir gly arg arg val ile val thr gly lys his glu arg lys

aal26 gly asp gly his gly net lie gln arg his phe val arg lye tyr thr leu gin asp asp his gly his ile met arg his phe val arg arg tyr lys val D22-27 glu asp asp his gly phe lie thr arg hbs phe val arg arg tyr ala leu glu ala glu gin gly gly tyr ser ser arg his phe leu arg arg phe val leu GAG GCC GAA CM GGT GGC TAT AlC TCC AGG CAC TTC CTC CGC CGA TTC GTT CTG AGT GAC ACT AAA GAT CGA AGC TAC TTC CAT GAA TAC AlA GAG AGG AAC AGA GM CCT GAT C ser asp thr lys asp gly ser tyr phe hbs glu tyr arg glu arg lys arg glu ala asp

aa144 pro lys gly leu thr pro thr lys val val ser thr val ser ser asp gly val leu thr leu arg ala pro pro pro pro gly arg glu arg ala arg ser glu arg pro asp gly tyr lys ala glu gln val val ser gln leu ser ser asp gly val leu thr val ser ile pro lys pro gln ala val glu asp lys ser lys glu arg D 22-27 pro pro gly tyr glu ala asp lys val ala ser thr leu ser ser asp gly val leu thr ile lys val pro lys pro pro ala ile glu asp lys gly asn glu arg pro glu gly tyr glu ala asp lys val thr ser thr leu ser ser asp gly val leu thr lie spr val pro asn pro pro ply val gln glu thr leu lys glu arg CCI GAA GGA TAC GAG GCG GAC MCG TG ACC TCG ACG CTG AlC AlC GAC GGC G1Y CTG ACC ATC AGT GTG CCC MT CCT CCA GGC GTC CAG GAG ACA CTC AAG GAG CIT

X4 GAT CTG CCA GM GGT GTG MT CCT GAG CAA GTT GTC TGC TCC TTA TCC MG GAC GGC CAC CTG CAT ATT CAG GCT CCT CGG CTG GCA CTG CCT CCT GCC CCA GAG ACA ATC ATT asp leu pro glu ply val asn pro glu gln val val cys ser leu ser lys asp gly his leu his ile gln ala pro arg leu ala leu pro pro ala pro glu thr lie ile

aal80 lie val arg lie gin gin thr gly pro ala his leu ser val lys ala pro ala pro glu ala gly asp gly lys ala glu asn gly ser lie lie gin lie gin gin val gly pro ala his leu asn val lys ala ann asp ser glu val lys gly lys glu asn gly ala pro asn D22-27 lile val gin lie gin gin val gly pro ala his leu asn val lys glu ser pro lys glu ala val glu gin asp gly gly asn gly lys glu val thr lie glu gin thr ily glu pro ala lys lys ser ala glu glu pro lys asp lys thr ala ser gin GAG CTG ACC ATC GAG CAG ACT GCC GAG CCC CCA MAG MG TCC GCC GAG GAG CCA AM GAC AMA ACC GCC ACT CAG TAG X4 CCT ATC AlC ATG GM CCG ACA CCC AGA CAT GCA CAG GM ATC CIA CCC GAT GCC CAG UAC AGC MT GCA GM AAA GAG CM CAM ATC CAC pro lie ser met glu pro thr pro arg asp ala gin glu lie pro pro asp ala gin asn ser asn ala glu lys glu gin gin met asp FIG. 2. Complete sequences of three different Xenopus heat shock cDNA clones. The sequence of X16 has been aligned to the homologous part of the Drosophila major heat shock gene sequence (D)70). The sequences of X5 and X4 have been aligned to the homologous and adjacent parts of the four Drosophila small heat shock proteins (D22-27: protein sequences of hsp 27, 26, 23, and 22 and the gene sequence of hsp 27). Black squares indicate a homologous amino acid. The position of the sequences within the Drosophila heat shock proteins is given by amino acid numbers (aa77-aal80, see refs. 5 and 6).

fragment being twice as long, see previous paragraph), the ble with an embryo containing >1000 nuclei (late blastula), if absence of an oocyte hsp 30 mRNA signal must mean that one considers the rate of heat shock mRNA accumulation in this RNA was present at least at a 5-fold lower level than the somatic cells (up to 1100 transcripts Fer hour) and the detec- hsp 70 mRNA and possibly was not present at all. This is tion limit for these mRNAs (about 10 molecules per analysis consistent with the fact that hsp 30 protein cannot be detect- or 106 molecules per embryo). At a stage where gastrulae ed in heat-shocked oocytes. contain >104 nuclei, about 107-108 hsp 70 transcripts per em- Heat-Inducibility of hsp 70 and hsp 80 Genes is Achieved at bryo could be observed after heat shock. However, no hsp Different Stages in Early Development. It is striking that, be- 30 mRNA was detectable at this stage. This implies that the fore gastrulation, early embryos were extremely heat sensi- hsp 30 genes are less active by a factor of 20-200 than are the tive. Mild heat shock conditions (10 min at 33°C) led to de- hsp 70 genes at this stage and may be completely repressed. colorization of the embryos and to derangement of their pro- Inducible hsp 30 mRNA was first detectable at about stage tein synthesis pattern. This sensitivity was initiated at 42 (swimming tadpoles). In conclusion, the mRNA analysis fertilization. Although protein labeling of early stages tended supports the protein labeling data: hsp 70 gene induction is to result in unreproducible patterns, it suggested that hsp 70 first detectable in gastrulae, whereas hsp'30 genes remain synthesis is first detectable at gastrulation, whereas hsp 30 uninducible until the tadpole stage. synthesis is not detectable till early tadpoles (not shown). Adult Somatic Cells Induce Constant Amounts of hsp 70 Heat shock RNA was analyzed by nuclease S1 mapping to mRNA but Variable Amounts of hsp 30 mRNA. Tadpoles determine the stage of activation of the two types of heat contain low levels of hsp 30 mRNA and high levels of hsp 70 shock genes during development. mRNA after heat shock, whereas tissue-cultured cells in- Early cleavage stages, gastrulae, and swimming tadpoles duce similar levels of the two types of heat shock mRNAs. were subjected to a mild heat shock (30-90 mim at 33°C), The question arises as to what level hsp 30 genes are induc- which is known to induce heat shock protein synthesis in ible in an adult frog. To answer this question, a frog was somatic cells and oocytes. Total RNA was extracted, and mildly heat-shocked (heat shock periods of 10-30 min at nuclease Si-analysis was performed (Fig. 4a). No heat shock 330C interrupted by recovery periods of similar length at RNA was detectable before gastrulation in heat-shocked or room temperature), and the RNA was extracted from vari- control embryos. This suggests that the stored oocyte hsp 70 ous tissues. Subsequent nuclease S1 mapping led to the fol- mRNA has been degraded at or after fertilization and that lowing conclusion: most adult tissues show heat-induced the cleavage stage heat shock genes cannot be transcription- transcription of both hsp 70 and hsp 30 genes; however, the ally activated. It should be noted, however, that de novo ac- levels of hsp 30 RNA vary considerably between cell types tivation of a gene can only be monitored when detectable (Fig. 4b). levels of RNA have accumulated. This would first be possi- Quantitation was performed in order to compare the vari- Downloaded by guest on October 2, 2021 Developmental Biology: Bienz Proc. NatL Acad. Sci. USA 81 (1984) 3141

I a b a b pMB Ga Ta LUl Li Ki Gu Xc rev Xc rev Oo d6f _-+-_+ -+ 8 1.6 .3.06.01 .03.leD 1 6 D2.08 4 2 50 50 p 0- f p lo Isip- 0- p ]P. , w.! F W. ...: w wo r' 4 rev 0. *,T $ it

A - 30 0- * 0 UJ--Hsp30 P I Hsp A- z'' It 06 ::::

I Hsp 70 1* ; Hsp70 W 0 I 1 2 3 4 s 6 7 8

1 2 3 4 5 6 7 8 9 t1

FIG. 3. Nuclease Si-mapping analysis of somatic and oocyte FIG. 4. Nuclease S1 mapping of heat shock mRNA from embry- heat shock mRNA. Total LiCl-precipitable RNA was prepared from os and adult frog tissues. (a and b) Total LiCi-precipitable RNA was heat-shocked tissue-cultured cells or from heat-shocked (+) or con- prepared from cleavage stages before the midblastula (lanes pMB), trol (-) oocytes and subjected to Si-mapping. The heat-shock con- from gastrulae (lanes Ga) or tadpoles (lanes Ta), from lung (lanes ditions in this experiment were 3 hr at 370C, interrupted by 1 hr at Lu), liver (lanes Li), kidney (lanes Ki) and gut (lanes Gu) of an adult room temperature. For quantitation, single-stranded DNA from the frog or from tissue-cultured cells (lanes Xc) before (-) and after (+) reversely oriented X16 clone was treated equally. Arrows indicate heat shock conditions for lanes: pMB, 30 min at 330C, followed by the positions of the undigested probe (p) and of fragments protected 60 min at room temperature; Ga and Ta, 90 min at 330C; Lu, Li, Ki, by hsp 70, hsp 30 mRNA, or by X16rev DNA. The occurrence of a and Gu, a total of 3 hr at 330C (see text); Xc, 3 hr at 370C, interrupted band below the hsp 70 or hsp 30 signal (particularly obvious in Fig. by 1 hr at room temperature. For nuclease S1 analysis, 5-20 kug of 3a, lane 6) is irreproducible and probably an artefact. (a) Decreasing RNA was used. Arrows indicate the bands originating from hsp 70 amounts of X16rev (rev, 0.01-8 ng) or tissue-cultured cell RNA (Xc, or hsp 30 mRNA protection or from undigested probe (p). Bands 0.03-6 gg) were analyzed. (b) RNA from ovarial lobes (lanes Oo, 50 below the hsp 70 or hsp 30 signal or below the undigested probe Mg) or defolliculated oocytes (lanes def, 50 yg) was analyzed and appear irreproducibly (see comment in Fig. 3). compared to the signal obtained by increasing amounts of X16rev (rev, 0.02-2 ng). In this analysis, 50 ,g of control oocyte RNA was 1/30th. Tissue-cultured somatic cells (derived from kidneys) mixed with X16rev DNA as a carrier. contained the highest level of hsp 30 mRNA, about the same amount as hsp 70. Testis contained an exceptionally low lev- able levels of hsp 30 expression to hsp 70 mRNA. Absolute el of hsp mRNAs, possibly because of the transcriptionally amounts of heat shock mRNAs were estimated by compari- inactive state of sperm DNA (not shown). It should be noted son with the cloned X16rev standard as described above, that none of the somatic cell types showed any detectable and a correction factor based on RNA translatability was in- level of heat shock mRNA before heat shock. This also held troduced in order to account for the varying quality of the true when high amounts (50 Ag) of non-heat-shocked tissue RNA. It is remarkable that every adult somatic cell type con- RNA were analyzed. It means that there are less than 3 x tained the same level of hsp 70 mRNA as has been found in 105 hsp mRNA copies per ,ug of somatic cell RNA or, for embryonic or tissue-cultured cells, namely 1-3 x 108 copies tissue-cultured cells, less than one molecule per cell before a per microgram of total RNA. In contrast, the levels of hsp 30 heat shock. In conclusion, hsp 70 genes appear to be fully mRNA varied within an order of magnitude. In kidney and induced in all somatic cell-types, and hsp 70 mRNA accumu- gut, the level of hsp 30 RNA was about one-third of the level lates to similar levels, whereas hsp 30 genes appear to be of hsp 70 mRNA, whereas in all other tissues it was down to expressed to a variable extent.

/\1 / FIG. 5. Semiquantitative sum- Cultured mary of heat shock gene expres- cells sion during frog development. \-D-~ The number of heat shock mRNA molecules per microgram of total 190 + - - RNA is indicated in orders of hsp magnitudes above the detection 70 33 _ limit (3 x 105 molecules per ug of analyzed total RNA). +, 3 x 105- 3 x 106; ++, 3 x 106-3 x 107; 190 -- +++, 3 x 107-3 x 108 heat shock hsp ++ or mRNA per microgram of total 3o33 ++ +++ RNA; -, undetectable. Downloaded by guest on October 2, 2021 3142 Developmental Biology: Bienz Proc. NatL Acad Sci. USA 81 (1984)

DISCUSSION nism) or they are not recognized by the hsp 70 transcription In adult Xenopus somatic cells, heat shock genes are gener- factor. The onset of hsp 30 gene heat inducibility in tadpoles ally heat inducible; during heat shock, high levels of heat would imply either loss of the repressing system or appear- shock mRNA accumulate, most likely because of induced ance of the hsp 30 . transcription initiation. This is, however, not the case for The levels of hsp 30 transcripts vary considerably between both types of heat shock genes in all periods of development different adult tissues, whereas the hsp 70 mRNA levels are (summarized in Fig. 5). Apparently, Xenopus heat shock constant and generally higher. This implies either that hsp 30 genes can be constitutively activated; in oogenesis, hsp 70 mRNA stability is variable and lower than hsp 70 mRNA mRNA accumulates as a consequence of the developmental stability or that hsp 30 genes are not fully induced, perhaps stage rather than a heat shock. Conversely, heat shock genes as a consequence of variable levels of hsp 30 transcription can be in a repressed state; in early embryogenesis, hsp 30 factor. genes cannot be activated by a heat shock to produce detect In conclusion, it is obvious that the Xenopus heat shock able mRNA. There is apparently a developmental control su- genes are not entirely coordinately expressed and regulated. perimposed on the heat inducibility of the heat shock genes, The mechanism by which the stage- or cell-type-specific which determines whether or not these genes are to be ex- expression of heat shock genes is achieved, is not known. I pressed. have suggested gene-specific transcription factors whose ac- Oogenesis. The only cell type found to contain heat shock tivity is regulated during development. A simpler model pre- mRNA before a heat shock is the oocyte. Assuming that this dicts just one heat shock gene activation factor with a higher hsp 70 mRNA is synthesized by oocyte genes and not deliv- affinity to hsp 70 genes than to hsp 30 genes, thus ensuring ered by follicle cells, its level suggests long periods of active hsp 70 gene expression in case of limiting factor concentra- transcription, previously estimated to be 10-100 days (12). tion; even this model has to involve stage- and cell-type-spe- From the level of mRNA per oocyte (3 x 106 molecules) and cific regulation of the abundance or activity of this factor. the rate of heat shock accumulation in somatic frog cells de- The aim is to understand and perhaps dissect the two differ- termined in this work (400-1100 transcripts per hr per cell), I ent levels of gene control by either defining the controling estimate that production of this hsp 70 mRNA would require sequences of the genes or by defining the factors them- full induction of the hsp 70 genes for at least 66 days during selves. Cloning, sequence determination, and expression of oogenesis (an oocyte is "tetraploid" during this time). Al- the Xenopus heat shock genes are a first step towards this though this approximate estimate is based on the assump- aim. tions that all hsp 70 genes of an oocyte are transcribed to I would like to thank Sue Whytock for providing embryos and produce this mRNA and that the rate of mRNA accumula- cells; John Gurdon, Hugh Pelham, and Andrew Travers for helpful tion is the same as in somatic cells, it indicates that the hsp discussion of the work and the manuscript; and EMBO for support- 70 genes are likely to be active during most of oogenesis. In ing this project by providing a long-term fellowship to M.B. mRNA is undetectable in oocytes, suggest- contrast, hsp 30 1. Schlesinger, M. J., Ashburner, M. & Tissieres, A. (1982) Heat ing that the hsp 30 genes do not undergo this oogenesis-spe- Shock: From Bacteria to Man (Cold Spring Harbor Labora- cific activation. tory, Cold Spring Harbor, NY). It is conceivable that the activation of heat shock genes 2. Ritossa, F. (1962) Experientia 18, 571-573. during oogenesis is a consequence of general gene activation 3. Holmgren, R., Corces, V., Morimoto, R., Blackman, R. & Me- rather than a specific mechanism. However, this is unlikely selson, M. (1981) Proc. Natl. Acad. Sci. USA 78, 3775-3778. for two reasons: (i) only hsp 70 transcripts accumulate in 4. Karch, F., Torok, I. & Tissieres, A. (1981) J. Mol. Biol. 148, oocytes, whereas hsp 30 transcripts (and hsp 30 protein) are 219-230. undetectable; and (it) the expression of the accumulated hsp 5. Ingolia, T. D. & Craig, E. A. (1982) Proc. Natl. Acad. Sci. 70 transcripts is translationally regulated (12). This suggests USA 79, 2360-2364. 6. Hackett, R. W. & Lis, J. T. (1983) Nucleic Acids Res. 11, that oocytes are programmed to express hsp 70 but not hsp 7011-7030. 30 protein. A possible interpretation is that oocytes contain a 7. Pelham, H. R. B. (1982) Cell 30, 517-528. transcription factor that recognizes either a subset of the hsp 8. Pelham, H. R. B. & Bienz, M. (1982) EMBO J. 1, 1473-1477. 70 genes (oocyte-specific genes) or an oocyte-specific recog- 9. Sirotkin, K. & Davidson, N. (1982) Dev. Biol. 89, 196-210. nition sequence in the promoter of all hsp 70 genes but does 10. Zimmerman, L., Petri, W. & Meselson, M. (1983) Cell 32, not recognize hsp 30 genes. 1161-1170. Embryogenesis. In the earliest embryonic stages, the mode 11. Bensaude, O., Babinet, C., Morange, M. & Jacob, F. (1983) of hsp 70 gene regulation is changed. No heat shock mRNA Nature (London) 305, 331-333. can be detected at first; heat-inducible hsp 70 transcripts ap- 12. Bienz, M. & Gurdon, J. B. (1982) Cell 29, 811-819. 13. Bienz, M. (1982) in Heat Shock: From Bacteria to Man, eds. pear at gastrulation, after the midblastula transition where Schlesinger, M., Ashburner, M. & Tissieres, A. (Cold Spring embryonic transcription is first observed (20). Apparently, Harbor Laboratory, Cold Spring Harbor, NY), pp. 177-181. the oocyte-activated genes cease to be constitutively ex- 14. Bienz, M. & Pelham, H. R. B. (1982) EMBO J. 1, 1583-1588. pressed, perhaps because of loss of the postulated transcrip- 15. Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, tion factor, and the stored hsp 70 mRNA turns over. It can- M. E., Gait, M. J. & Karn, J. (1982) Proc. Natl. Acad. Sci. not be decided whether the hsp 70 genes become heat-induc- USA 79, 5818-5822. ible for the first time at gastrulation or whether they have 16. Maniatis, T., Fritsch, E. F. & Sambrook, J., eds. Molecular been heat-inducible also during the previous stages because Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, the sensitivity of detection of short-term expression requires NY). 17. Bankier, A. T. & Barrell, B. G. (1983) in Techniques in the >1000 nuclei. On the other hand, gastrula cells that are Life Sciences, ed. Flavell, R. A. (Elsevier, Ireland), Vol. B5, clearly capable of heat-inducing hsp 70 genes do not accumu- pp. 1-34. late detectable hsp 30 transcripts. Possible interpretations of 18. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H. & this fact are, in analogy to the previous paragraph, that either Roe, B. A. (1980) J. Mol. Biol. 143, 161-178. the hsp 30 genes are specifically repressed (e.g., by a repres- 19. Staden, R. (1982) Nucleic Acids Res. 10, 2951-2961. sor bound to the promotor or by a DNA-modifying mecha- 20. Newport, J. & Kirschner, M. (1982) Cell 30, 687-696. Downloaded by guest on October 2, 2021