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    Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 26 5, No. 21, Issue of July 25, pp. 12111-12114,[email protected] 1990 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in U.S.A.Heat Shock Proteins

    Milton J. SchlesingerFrom the Department of Molecular Microbiology,Washington University School of Medicine,St. Louis, Missouri 63110

    The enhanced synthesis of a few proteins immediately aftersubjecting cells to a stress such as heat shock was firstreported for drosophila cel ls in 1974 (l), and the universa lityof the response from bacteria to human was recognized shortlythereafter (reviewed in Ref. 2). In the ensuing 16 years, a vastliterature has accumulated that describes a wide varie ty ofevents in a cells response to a heat shock. The scope of thedata ranges from x-ray crystallographic measurements andphysical chemical studies on specific heat shock proteins tothe effects of heat shock gene expression on an organismsecological niche. Recently, the emphasis in this field hasfocused on the unction of various heat shock proteins andtheir possible role as molecular chaperones (3). In thesubject matter that follows, I review recent data and otheraspects of the heat shock phenomenon. A more thorough andcomprehensive discussion of this topic can be found in anumber of recent reviews and monographs (4-10). Primaryreferences to much of the material described here are in thesereviews.Much of the initial molecular biology and biochemistry ofheat shock consis ted of cloning the genes, determining pri-mary sequences of the proteins, and probing the regulatoryfactors responsible for their induction. From these latterstudies, we learned that the DNA sequence responsible forregulating heat shock gene expression in the eukaryotic cellwas invarian t from yeast to human (reviewed in Ref. 11). Themost recent analysis of this element suggests it is an invertedrepeat of the 5-nucleotide base pair, nGAAn (12). The pres-ence of this element located about 80-150 base pairs upstreamof the start site of RNA transcr iption is the most definitiveevidence that the gene encodes a heat shock protein. However,most of these genes have other regulatory signa ls that activatethe gene when the appropriate protein factors are present.For example, there are at least four sequence motifs upstreamof the human hsp702 gene that are responsive individually toserum factors, heavy metals, and the ElA protein of adeno-virus (13). The obvious interpretation of these resul ts is thatthe hsp70 protein is synthesized for reasons other than heatshock and, in fact, this gene is activated at a specific stage(early S) in the cells mitotic cycle, during mitogenesis andupon other st ress conditions (see Table I). Most of the heatshock proteins are induced by other stress agents (a listing isin Ref. 14) and during normal development of an organism(reviewed in Ref. 15).The gene encoding the protein that binds to the heat shock-respons ive DNA sequence has been cloned from yeast (16,17)and shown to be essential for viabili ty of this organism. This

    That a protein might act as a molecular chaperone was first suggested byHugh Pelham (56) in a paper speculating on functions of the major heat shockproteins. Althoug h he did not coin the term (it first appeared in a paper by R.J. Ellis (57)) Pefh am argued that some heat shock proteins might participatein the assembly and disassembly of proteins and pro~in-containing structuresand in this manner prevent or repair stress-incurred damage to cellular struc-tures.2 The numbers do not represent precise molecular weights for these proteins.

    yeast protein is present in cells under normal growth condi-tions and binds to the DNA under non-s tress condi tions;however, its state of phosphorylation changes with an increasein the leve l of temperature stress (16). In higher eukaryotes,the factor is not normally bound to the DNA but does sorapidly after stress, and additional phosphorylation of thefactor i s detected (18). The mRNAs from heat shock geneshave structures that allow for their selective translation in astressed cell. These include a lack of introns, regions in the5-untranslated regions conferring translational e fficiency,and regions in the 3-untranslated segments providing forincreased stability.The events regulating heat shock gene expression in theprokaryote differ in several respects. First, unlike the euka-ryote where different heat shock genes are expressed non-coordinately , heat shock genes in the prokaryote (here I referto studies on ~sc~erichi~ col i) form a regulon and appearsimult~eously. Second, the heat shock transcription factoris an isomer of the u subunit, the regulatory element in thebacterial RNA polymerase. This u factor ex ists at low levelsunder normal growth conditions, but levels rise quickly afterheat shock due to much s lower degradation of the protein,enhanced translation of its mRNA, and increased transcrip-tion of its gene.How a cell senses a temperature stress is still unknown, butin vitro systems exist in which the heat shock factor (extractedfrom the cytoplasm of HeLa cel ls) binds to a heat shockelement only after heat or treatment at low pH and additionof calcium ions or non-ionic detergents (W-20). If the heatshock factor itself is the sensor, then its structure must besufficiently species-specific in order for vastly different tem-peratures to activa te it, i.e. 30 C is a heat shock for drosophilabut 43-45 C is heat shock for avian cells. In drosophila,transcription of heat shock genes is initiated even under non-stress conditions, but the RNA polymerase is blocked in itsprogression and remains poised on the DNA (21). Presumably,binding of the transcription factor allows for ins~ntaneousrelease of this block.

    Fzmctione of Heat Shock ProteinsThe initi al studies on cloned heat shock genes and purifiedproteins led to two important resul ts. Firs t, the heat shockproteins proved to be incredibly highly conserved amongwidely divergent organisms. For example, the major heatshock protein, hsp70, has about 50% of its sequence conserved

    between E. col i and human, and some domains are 96%similar. Second, several of the major heat shock proteins aremembers of gene (protein) families that include proteinsnormally present and, in most cases, essential for cell func-tion. The hsp70 of yeast is the most thoroughly studied heatshock family system (reviewed in Ref. 4); it consists of ninemembers that differ to vary ing degrees in sequence and incellular localization. Three members are localized to specificcompartments of the celi : the mitochondrion, the nucleus,and the endoplasmic reticulum. They differ also with regardto the conditions under which they are synthes ized (Table I).In fact, i t is the discovery of the function of hsp70 proteinsmade in normal cel ls that p rovided the data for the chaperonemodel. One of these studies revealed a requirement for anhsp70 in order for certain yeast proteins that are synthes ized


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    12112 Minireview: Heat Shock ProteinsTABLE I

    Inducers of heat shock protein family membersEnvi ronmenta l Pathouhvsioloeical Normalstress conditions

    HeatHeavy metalsOrganicsOxidants

    stress conditionsMicrobial infect ionsTissue t raumaGenet ic lesions

    condi t ionsCell cycleEmbryonic developmentCel l di f ferent iat ionHormonal st imulat ionMicrobial growth

    in the cytoplasm to be translocated into the mitochondrialmatrix (22). These hsp70 proteins are needed for import ofseveral proteins into other eukaryotic cell organelles includingthe endoplasmic reticulum (23), the chlorop last (24), and thelysosome (25). Other hsp70 family members act inside theseorganelles as does a protein that belongs to a different familyof heat shock proteins, the hsp60 or GroEL group (seebelow).For import into the organelle, it is proposed that an hsp70unfolds the partially folded polypeptide so that i t can betranslocated through a membrane pore. Consistent with thisrole are data showing that an hsp70-like protein can bind toand, in the presence of ATP, dissociate protein complexes.Among these are clathrin-coated vesicles, a X bacteriophageDNA replication complex, nucleolar proteins that have be-come insoluble as a result of heat shock, and immunoglobulinheavy chains formed in the absence of light chains (Table II).In the latter complex, the hsp70 protein is termed B iP and isthe member of the hsp70 family that is loca lized to the lumenof the endoplasmic reticulum. BiP binds proteins that haverecently been imported into the endoplasmic reticulum and ispostulated to prevent abnormal oligomeric protein formationas well as aid in the folding of a protein to its stable quaternarystructure (26). The yeast homologue is encoded by the KAR2gene whose protein product is essential for viabili ty andnuclear fusion.Just how hsp70 proteins manage all of these activities hasyet to be determined, although some structural informationexis ts about the molecule. The protein has two major domains:a highly conserved compact amino-terminal portion contain-ing an ATP-binding site, weak ATPase activity, and a cal-modulin-binding site (27) connected by a protease-sensitiveregion to a more diverse carboxyl domain containing sitesimportant for local ization to the nucleolus (28). The nativemolecule is a dimer but forms higher oligomeric complexeswith many structures in the cell (Table II). Long chain fattyacids bind tightly, but not covalently, indicating the presenceof a hydrophobic pocket on the hsp70 surface. This domaincould be a site for binding solvent-exposed hydrophobic re-gions of incompletely folded polypeptides.3 These would con-sist of those cytoplasmic proteins destined for import intoorganelles as well those that accumulate as a result of heatshock and other types of stress. In most stressed cells, thenewly made hsp70 localizes to the nucleus and to the nucleoluswhere it is tightly complexed in an insoluble form that ispartially solubilized by ATP. The nucleolus is the site ofribosome assembly and is unusually thermal sensitive. Theguess is that hsp70 binds to proteins that are incompletelyfolded in the preribosome assembly unit and protects themfrom irreversible denaturation. There are other structures inthe nucleus sensitive to heat shock, i.e. the splicosome andnoncondensed chromatin. Proteins in these structures mightalso be protected by hsp70.Another family of heat shock proteins (with subunit molec-

    a Recent studies indicate that hsp70 binds to nascent polypept ide chains inHeLa cel ls (58). The authors suggest that h sp70 aids in the folding of nascentchains; however, experiments in which puri f ied preparat ions of hsp70 are addedto in vi t ro protein-synthesizing systems show thatnascent chain-elon at ion isactual lv inhibi ted and folding of speci f ic nolvpent ides is uerturbe 4 (M. J.

    ular weights of about 60,000) also forms complexes withpolypeptides and has ATPase activity (Table II). But incontrast to the postulated unfolding and disassembly role ofmost forms of hsp70, hsp60s participate in the folding andassembly of polypeptides. Based on this property, they havebeen referred to as chaperonins (29). In the eukaryote, thehsp60s are localized to cy toplasmic organelles such as themitochondrion and the chloroplast (29-31). The hsp60 hom-ologue in the chloroplast is the ribulose-Ps carboxylase/oxy-genase heavy chain binding protein that i s required for assem-bly of the hexadecameric enzyme complex (24). The yeasthsp60 protein is encoded by the MIF4 gene in which muta-tions give rise to phenotypes defective in mitochondrial func-tion (32). The hsp60 bacteria l homologue is the GroEL proteinof E. coli, a major protein in this cell. It is an unusual proteinforming a tetradecameric oligomer with 7-fold symmetry andis essential for E. c oli growth. Secretion of some bacterialproteins is blocked in groEL mutants (33). During X bacteri-ophage infection, this protein participa tes in assembly of thephage. Recent elegant studies show that GroEL promotesassembly of the ribulose-Pz carboxylase/oxygenase when itsprotein subunits are expressed from a plasmid introduced intoE. coli (34). Equally impressive are data showing that theGroEL protein can suppress temperature-sensitive mutationsaffecting the structure of a variety of oligomeric proteinsincluding those in the pathway of histidine and isoleucinebiosynthesis, a Salmonella phage tail spike protein subunit,and bacterial translocation enzymes (35). These data may bethe best example of how this family of heat shock proteinsfunctions during a heat shock .A third heat shock protein family is the hsp90 group. Inthe eukaryote hsp90 is abundant in normal cells, is highlyphosphorylated on serines and threonines, and is local ized tothe cytoplasmic compartment of the cell. A small fraction ofit translocates to the nucleus after heat shock. Like the hspfamilies noted above, hsp90 complexes with a varie ty ofnormal cellular proteins (Table II). The most thoroughlystudied are the glucocorticoid receptors that are maintainedin an inact ive conformation bound to hsp90 until activatedby hormone. Several kinases are transiently complexed withthis heat shock protein; most notable are several of thetyrosine kinases encoded by oncogenes. Another kinase, onethat phosphoryla tes the eukaryotic transla tion-initia tion fac-tor, eIF-2a subunit, is activated by hsp90. The cytoskeleta lproteins, actin and tubulin, are associated noncova lently withhsp90. How these interactions are related to hsp90s role inheat shock remain a puzzle although the chaperone conceptis a good bet. Microfilaments and microtubules are not un-usually sensitive to stress although a prolonged and severeheat shock modifies both structures. In contrast, the inter-mediate filament network is very thermal sensitive. Possib ly,hsp90 protects and aids in the recovery of these cytoskeletalsystems.We can classify the three heat shock proteins (hsp70, hsp60,

    TABLE I IProteins complexed with heat shock protein family members

    hsp70 hsp90fami ly fami lv hsp60 (GroEL) fami lyClathrin-c oated vesicles Glucortico id receptor h phage col larsPrepro-a factor Tyrosine kinases Ribulo se-P p carboxylase/Nucleolar nroteins eIF-2a kinase oxvgenase heavy chainIgG heavy-chainp53 tumor ant igenDNA repl icat ion-ini t iat ion complex(pha e, plasmid)Ca lm0 u l i nfSV40 T-ant i genMicrotubules (p-internexinl

    Yeast protein kinase C Cyto&rome c; fh$n Fl-ATPaseTemperature-sen-si t ive mutants

    Schle&ger, unpubl ished experiments).

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    Minireview: Heat Shock Proteins 12113and hsp90) discussed above as those designed to protect,preserve, and recover the funct ion of various protein com-plexes. But there is another group of heat shock proteins thatpart ic ipates in the degradat ion of proteins. This act iv ity alsoserves to protect the cell for not only is proteolyt ic turnoveran essent ial act iv ity in normal cells, but accumulat ion ofdenatured protein could be toxic to a cell. All euka ryotic cellsemploy a small very highly conserved (even more so than thehsps described above) polypept ide called ubiquitin to mark aprotein for degrada tion. The ubiquitin-dependent proteo lyticsystem has been described in a previous minireview (36), andI note here only those features pert inent to the heat-shockedcell. Firs t, the re are three kinds of genes encoding a ubiquitinsequence, and one contains mult iple ubiquitin sequences con-tiguously arrayed as a polyubiquitin. This polyubiquitin genehas heat shock regulatory elements, and levels of ubiquitinrise 5-7-fold after the stres s. Secon d, in the ubiquitin degra-dation pathw ay there are three kinds of ubiquitin-spe cificenzym es, called El, E2, and E3. Free ubiquitin is act ivated inthe presence of ATP and El and then transferred via an E2to form covalent l inkages with target proteins that have beenbound by an E3. I t is the E3 that recognizes proteins dest inedfor degradat ion. One isomer of E3 is postulated to bind tounfolded proteins. Two of the yeast E2 enzym es that transferubiquit in to an E3 have been cloned and are heat shockproteins (37). The product of El, E2, and E3 act iv it ies is apolyubiquitinated protein that is degraded by a large multi-component organelle containing within i t the proteasome(38), a complex of about eight subunits with protease act iv ity.Polyubiquitinated protein complexe s increase about 2-fold inheat-shocked cells. There is no ubiquitin in the prokaryote,but one of the heat shock genes in E. coli encodes an ATP-dependent protease.

    Molecular ModelsBased on the results from structural and funct ional studies

    of four major heat shock protein famil ies i t is clear that theirrole in the folding and unfolding of polypep tides provides thebest model to date for explaining how the major heat shockproteins funct ion. There is now an atomic structure of amolecular chaperone, PapD of E. coli , based on x-ray crys-tal lography (39). PapD forms transient complexes with sev-eral proteins as it shutt les them to an assemb ly site for thebacterial pi lus. The protein contains two globular domainsseparated by a large crevice containing solvent-exposed hy-drophobic sites. The lat ter is postulated to be a binding pocketfor the protein to be transported. PapD is not a heat shockprotein nor does it contain an ATP-binding site, but i t mayprove to possess structural elements important to certain ofthe heat shock proteins described above.

    Early studies o f protein folding from extended polypept idechains indicated that the process was self-driven with noadditional cofac tors required. How ever, folding of individualpolypept ide domains to a stable conformation is dependenton other po rtions within the polypeptide as well as on inter-act ions with other polypept ides in those cases where thepolypept ide normally funct ions as part of an ol igomeric pro-tein complex. Indeed, folding that results from protein subunitinteract ions proved to be the molecular explanat ion for recov-ery of mutat ionally altered funct ions by intracistronic genet iccomplementat ion (40), a phenomenon similar to that de-scribed above for suppression of temperature-sensit ive mu-tants by overexpression of the GroEL protein. I t is onlyrelat ively recent ly that specif ic enzymes such as protein di-sulf ide isomerase and prol ine isomerase have been shown toplay an essential role in modu lating the folding of secrete dproteins. The models now in place for heat shock protein

    funct ion offer an important addit ional and much more generalrole for the concept of assisted folding in maturat ion ofprotein structure.

    Events in the Heat-shocked CellThe suggest ion that one of the major effects of a heat shockis an unfolding or an incomp lete or improper folding of

    proteins appeared early in the heat sho ck literature (41).Many recent studies have conf irmed this hypothesis (Ref. 42,for example) and, clearly, the concept that heat shock proteinsfunct ion as chaperones provides further strong support forthese ideas. An interesting extrapolation of this hypothesis isthat it is the imprope rly folded polypeptide that actuallyinit iates the induct ion of the heat shock response. In bacteria,manipulat ions of protein structure that lead to increasedamounts of badly folded proteins turn on the heat shockregulon (43). In animal cells, there is a temp erature-se nsitivemutant with a defect in the ubiquit in-dependent proteolyt icdegradat ion pathway that leads to accumulat ion of defect iveproteins (44). This mutant shows enhanced levels of heatshock proteins under non-heat shock condit ions. In contrast,the addit ion of deuterium oxide or glycerol, agents that canstabil ize proteins, inhibits and delays the induct ion of heatshock proteins (45).In addition to improper polypeptide folding, heat sho ckleads to a plethora of changes that are dependent on both theintensity of the stress and on the cell system . These includeeffects on macromolecular synthesis, on levels of cat ions, onstates of protein phosphorylat ion, on metabolic pathways, oncytoskeleton networks, etc.

    A number of other proteins have been reported to be heatshock proteins, based on their enhanced rate of synthesisafter stress. In E. coli , the heat shock regulon includes about20 genes, and there are probably twice that number in theeukaryot ic cell . Some of these have been ident if ied as glyco-lyt ic enzym es, i .e. glyceraldehyde-3-phosphate dehydrogen-ase, enolase, phosphoglycerate kinase, and others are notedby act iv it ies such as a collagen-binding protein and hemeoxygenase. We can rat ionalize the appearance of the formerset since stressed cells tend to shif t f rom aerobic to glycolyt icmetabolism for energy product ion. But others remain enig-matic. The mo st predominant in this lat ter category is a setof small molecular weight stress proteins that vaguely resem-ble each other in hydropathy prof i les but not sequence exceptfor a short region that is homologous to a sequence in themamm alian lens (Y crystal l in. These low molecular mass stressproteins (15-30 kDa) are mos t abundant in stressed plantswhere som e are found as RNA nucleoproteins and others aretranslocated to the chloroplasts and to the mitochondria. Inall eukaryot ic cells, they can form huge granular arrays. Likesome of the heat shock proteins noted above, these smallproteins are found during various stages of embryonic devel-opment and, in yeast, during sporulat ion.

    ThermotoleranceNo discussion of the heat shock phenomenon can ignore

    the topic of thermotolerance; however, there are recent re-views (46, 47) and l i t tle new to add to these. Many studieshave shown that a pre-shock treatment can render a biologicalsystem more resistant to a subsequent stress and that thisprotect ive effect is transient (48, 49). Posit ive correlationsexist between the amount of heat shock proteins present andthe degree of tolerance, yet except ions occur. Thus some heatshock proteins are necessary but may not be suff ic ient forthermotolerance. Furthermore, thermotolerance appears tobe important for survival under stress condit ions. A f ineexample of the latter is the comparison of two species of

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    12114 Minireview: Heat Shock Proteinshydra, Hydra ol igact is and Hydra attennata (50). The formeris extremely sensitive to thermal stress and incapable ofaquiring thermotolerance whereas the latter is thermoresis-tant and can become thermotolerant. After thermal stress, H .attennata makes a large amount of a 60-kDa protein, whichis not found in H. ol igact is. In nature, the H. ol igact is speciesis much more restric tive in its ecological niche; it i s not foundin shallow ponds and, in addition, shows strong seasonalvariations in population and rapidly declining populationsafter increases in surface temperatures of pond water. Consid-ering the survival value of heat shock genes, it is surprisingthat H. ol igact is persists, raising the possib ility that there arenegative effects of heat shock proteins.3 In this regard it isnoteworthy that the cel l autoregulates the level of hsp70 andtitrates the amounts of hsp70 relative to the extent of thestress (51).

    Heat Shock Proteins and the Immune ResponseGiven the very high degree of conservation in structureamong several major heat shock proteins, one would havepredicted that an individuals immune system would developtolerance to these polypeptides. In fact, just the opposite

    occurs, and a significant fraction of the immunoglobulins andcytotoxic T lymphocytes formed during an immune responseto several types of microbial infections is directed againstpeptides derived from heat shock proteins (reviewed in Refs.52 and 53). Many pathogenic microorganisms experience aheat shock during their initial invasion of a mammalian hostand thus produce heat shock proteins. In addition, host ce llsturn on these same proteins as a result of the st ress fromintracellu lar growth of the microbe. Immunoglobulins and T-cell s that recognize epitopes on these proteins will destroycell s infected with the microbes, thereby limiting the infectionand protecting the indiv idual from disease. But the uninfectedhost contains these proteins in most of its cells and thusincurs a serious risk of an autoimmune response. Actually,there are animal models showing that hsp60 peptides caninduce autoimmunity, and some indiv iduals suffering fromautoimmune diseases have antibodies to heat shock proteinsin their sera and heat shock proteins at sites of immune-mediated tissue damage.There i s specula tion that an immune response rather thantolerance to these universal proteins allows not only for animmediate protection from infections by a varie ty of patho-gens but also for immune surveillance, an activity of theimmune system that elim inates abnormal and damaged cel ls.In normal cells, heat shock proteins are at low levels andsequestered inside the cell, but stress will increase their levelsand their location in cel l compartments. In a damaged cel l,some might appear on the cel l surface. In this regard, it i s ofinterest that a major tumor-specific transplantation antigenin mice given carcinogens is identical to a member of thehsp90 family of heat shock proteins (54). How potentialdamage from autoimmunity by this immune surveillance sys -tem i s avoided remains unknown. One may expect new dis-coveries about the immune system that will resolve this partof the heat shock puzzle. Of particular interest is the recog-nition of heat shock protein peptides by T-ce lls bearing they-b subunits of the T-cell receptor and the possible involve-ment of hsp70 family members in antigen presentation (55).It i s clear that we now have substantial information aboutmolecular events associated with heat shock, a scientific phe-nomenon that has attracted legions of investigators through-

    out the world, produced a very large body of scientific litera-ture, and, as noted in this min ireview, led to solutions offeringnew information about fundamental biochemical processes.REFERENCES

    1. TissiBres, A., Mitchell, H. K., and Tracy, U. (1974) J. Mol. B iol. 84, 3892. Schlesinger, M. J., TissiBres, A., and Ashburner, M. (eds) (1982) Heat ShockProteins: from Bacteria to Man, Cold Spring Harbor Laboratory, ColdSpring Harbor, NY3. Ellis, R. J. (ed) (1990) Semin . Cell Biol. 1, l -724. Lindquist, S., and Craig, E. A. (1988) Annu. Reo. &net. 22,631-6775. Pelha m, H. R B. (1989) EMBO J. 8,3171-31766. Rothman, J. E. (1989) Cell 69,591-6017. Never, L. (ed) (1990) Heat Shock Response, CRC Press, Boca Raton, FL8. Morimoto, R., TissiGres, A., and Geor opolous, C. (eds) (1990) StressProteina in Biology and Medicine, Co1 dSpring Harbor, NY Sprmg Harbor Laboratory, Cold9. Schlesinger, M. J., Santoro, G.,, and Garaci, E. (eds) (1990) Stress Proteins:Induction and Function, Springer-Verlag. Heidelberg , in press10. Hartl, F-U., an d Neupert, W. (1990) Science 247,930-93811. Kingston, R. E. (1990) in Hormona l Control of Gene ReG., and Cohen, P., eds) Elsevier Science Publish ing E u&on (Foulkes,o., New York, inpress12. Perisic, O., Xiao, H., and Lis, J. T. (1990) Cell 69, 797-80613. Morimoto, R. I., Ahravava, K., Mosser, D., and Williams, G. T. (1990) inStress Proteins: Induction and Functton (Schlesinger, M. J., Santoro, G.,and Garaci, E., eds) S rin er-Verlag, Heidelberg , m press14. Never, L. (ed) (1984) H%t hwck Response of Euhyot ic Cells, Springer-15, Bz;lp Heidelberg , gand Schlesm er, M. J. (1988) Adu. Genet. 24, l-2916. Sorg