[~EVIEWS
R a s genes encode low molecular weight, GTPbinding, GTP-hydrolysing proteins that are highly conserved throughout all eukaryotic species. Mutational activation of these genes can induce cancer in humans and other mammals. A number of investigators have proposed that Ras proteins in mammals function in signal transduction, coupling some second messenger to the binding of serum growth factors to cellular receptors. Although this hypothesis is appealing, the components of this postulated signal tmnsduction pathway in mammalian cells have not been clearly defined. Saccbaromyces cerevisfae contains two genes that are both structurally and functionally homologous to human ras genesl, z. Human ras genes can ameliorate the growth defects arising from inactivation of yeast RAS genes, and slightly modified versions of a yeast RAS gene can induce proliferative transformation of mouse fibroblast cells3,4. This functional conservation of Ras proteins initially offered the prospect of applying yeast genetics to defining the pathway in which metazoan Ras normally resides. This early euphoric expectation has diminished somewhat. Subsequent experiments have shown that, while yeast Ras proteins function predominantly - if not exclusively - as a modulator of adenylate cyclase, metazoan ras genes appear to exert their influence independent of cAMP5. Despite the apparent disparity of the pathways in which yeast and human ras genes reside, an understanding of the function of yeast Ras protein can provide significant insight into the detailed molecular interactions required for mammalian ras function as well as into the general principles of cellular growth control in larger eukaryotes. Here, I present our current understanding of the signals that feed into the RAS pathway in yeast and the biological processes that are directly regulated by the action of the Ras proteins. More extensive reviews of Ras structure and function in yeast and in mammalian cells and the relationships between the two systems have recently appeared6--10.
RASgenes in
Saccharomyces cerevisiae: signal transducti0n in search of a pathway JAMESR. BROACH Ras proteins in budding yeasts initially appeared to regulate inittatioa of the cell cycle in response to nutrient availability. More recent wori~ while clarifying the mechanism of Ras.mediated signal transductto~ has undermined our notttm of the signal Ras transmits. We now suspect that Ras be~ps to coordinate cellular metabolism aml mass accumulatto~ but what Ras responds to is not clear. Ras proteins and the GTP/GDPcycle The yeast Ras proteins are members of a superfamily of low molecular weight GTP-binding proteins, encompassing 52 distinct species at the last count. All of these proteins probably execute their function through a cycle of GTP/GDP exchange and GTP hydrolysis as outlined in Fig. 1 (Ref. 6). In its simplest form, the model postulates that Ras proteins shuttle back and forth between an activating agent, which provides the input signal, and an effector protein, which transmits an output signal. Interaction of the Ras proteins with an activating agent stimulates removal of bound GDP and its replacement with free GTP. In this activated, GTP-bound state, the protein can stimulate the activity of the target protein. As a direct consequence of this stimulation or simply as a stochastic process, GTP bound to the Ras protein is hydrolysed to GDP. In the GDP-bound state, the Ras protein is no longer able to activate or associate with its target and
e'e°°
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GTP
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rIGO The GDP/GTPcycle of Ras. A model for ~s-mediated signal tmnsduction is shown. Input is registered as an increase in Ras bound to GTP, accomplished either by stimulating a GTP/GDP exchange factor or by inhibiting a GTPase-activating protein (GAP). Ras in its activated, GTP-bound fonn promotes the downstream process, either alone or in conjunction with GAP.See text for full description. 'lag I~a'~Y ~991 VOL."7nO. 1 01991 ElsevierSciencePublishersLtd(UK)0168- 9479191/$02.00
I~
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Go Arrest, Sporulation
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~a18 The RAS/cAMP pathway in Saccharom.w.escerevisiae. The roles of components of the RAS/cAMPpathway, each indicated by its gene designation (see Table 1), are shown schematically.P1, P2, P3, etc. indicateprote~n targets of the A kinase, which consists of two catalyticsubunits encoded redundantly by TPKI-3and two regulatory subunits encoded by BCY1. See text for full description. Dotted line indicatesA-kinase-inducedinhibition. the protein must be reactivated by recharging with GTP. More complex variations on this theme are possible, some of which are noted in the specific discussion of the yeast RAS pathway.
The RatS~cAMPpathwayin y~st Components of the pathway The RAS/cAMP pathway as we currently understand it is shown in Fig. 2 and genes whose products are components of or impinge on this pathway are listed in Table 1. RAM and RAS2 gene products (hereafter referred to collectively as Ras) are synthesized as cytoplasmic precursors, which become extensively modified at the carboxy terminus upon maturation. The precursor proteins are farnesylated, proteolyticaily cleaved, methyl esterified and palmitoylated, probably in that orderil-14. The farnesylation step is required for localizing the mature proteins to a membrane fraction, presumed to be the inner surface of the plasma membrane, and membrane localization is essential for Ras function. The product of the RAM/ gene is required for farnesylation, probably as one subunit of a heterodimeric farnesyl transferase enzyme (the prcdtlct of the RAM2 gene may be the other subunit)n.~5. The product of the STE14 gene is a methyltransferase that is probably responsible for methyl esterification of Ras (see Table 1). Since ste14 strains are viable, methyl esterification is probably not essential for Ras activity. When charged with GTP, mature Ras is capable of stimulating adenylate cyclase, the product of the CYR1
IOCUS16,17.Activation of Ras is catalysed by the product of the CDC25 gene, a protein that promotes removal of GDP bound to Ras and its replacement with free GTPm.19 (S. Jones, M.L. Vignais and J. Broach, unpublished). Stimulation of adenylate cyclase by GTP-Ras requires the product of the SRV2 l~ocus, a 70 kDa protein that copurifies with adenylate cyclase2°.zl. This protein either serves as an intermediary in the Ras-cyclase interaction or, more likely, promotes or stabilizes the physical association of the two proteins. Hydrolysis of GTP bound to Ras could occur either as a direct result of interaction with its downstream target or simply as a spontaneous process. In the former case, the target protein and the Ras GTPaseactivating protein (GAP) would be one and the same, a situation that may apply to ras in mammalian cells and to certain ras-like gene product,s involved in secretion2Z,23. Whether adenylate cyclase stimulates the GTPase of yeast Ras is not known. However, the products of two genes, IRA I and IRA2, structural and functional homologues of mammalian GAP, do stimulate Ras GTPase activityz4. Since phenotypes associated with loss of function of these two proteins are quite distinct from and completely suppressed by loss of Ras activity, neither of these proteins is a downstream target of Ras. Rather, the IRA proteins act to down-modulate Ras activity. A plausible model is that hydrolysis of GTP bound to yeast Ras is a stochastic event, which proceeds independently of its interaction with adenylate cyclase. The rate of GTP
TIGJANUARY1991 VOL.7 NO. 1
la
~EVIEWS hydrolysis by Ras imposes a time limit on stimulation of its downstream target. This would be similar to the proposed mechanism of GTP-mediated interaction of EF-Tu with the ribosome zS. How input information enters this pathway is not clear. The relative proportion of the active, GTP-bound versus the inactive, GDP-bound forms of Ras is determined by the rates of two competing reactions: the GDP/GTP exchange reaction catalysed by CDC25 protein on the one hand and the GTPase reaction, stimulated by the IRA1 and IRA2 proteins, on the other hand. Input into the RAS/cAMP pathway need only increase the absolute amount of Ras in the activated, GTP-bound form. This could be accomplished equally well by inhibiting the GTPase-activating proteins or by stimulating the exchange factor. Thus, any or all of these modulating enzymes could serve as loci for transmitting input information (Fig. 1). The sole function of cAMP in the cell appears to be to activate the cAMP-dependent protein kinase (protein kinase A) 16. This kinase is a heterotetrame# 6, comprising two regulatory subunits encoded by the BCY1 locus and two catalytic subunits redundantly encoded by three separate genes: TPK1, TPK2 and TPK3. In the absence of cAMP, the BCY1 protein restrains the catalytic subunits by serving as a tightly associated competitive inhibitor of the enzymes. Upon binding cAMP, the BCY1 inhibitor releases the catalytic subunits, which are then freed to phosphorylate their numerous cellular targets. Targets of the A kinase include enzymes involved in metabolism of storage carbohydrates, enzymes situated at strategic points in the glycolytic/gluconeogenic pathway, enzymes required for phospholipid metabolism, transcription factors associated with expression of specific genes, proteins involved in production of cAMP itself, and, undoubtedly, a substantial number of proteins currently unidentified (see Ref. 7 for an extensive listing). In general, high A kinase activity induces breakdown of stored carbohydrates, activation of the glycolytic pathway, induction of transcription of a large number of growth-specific genes (such as those encoding ribosomal proteins and certain secreted proteins), repression of some stress-protective proteins, and down-modulation of the RAS/cAMP pathway. In the reciprocal situation, low levels of A kinase activity induce accumulation of carbohydrates, activation of gluconeogenesis, reduced transcription of growth-specific genes, and induction of various stress-related proteins. Genetics of the RAS/cAMPpathway Mutations in components of the RAS/cAMP pathway yield one of two distinct phenotypes. Strains with mutations that activate the pathway are capable of normal mitotic growth in rich medium, but exhibit a number of abnormal phenotypes. These include loss of carbohydrate reserves, increased sensitivity to heat shock and starvation, and, for diploid strains, inhibition of sporulation. Strains with mutations that diminish the activity of the pathway fail to grow on nonfermentable carbon sources, and accumulate carbohydrate reserves. In addition, strains with temperature-sensitive mutations in RAS2 (in a RAM- background), CYR1 or
CDC25 arrest as unbudded cells at the nonpermissive temperature. Diploid strains homozygous for these mutations leave the mitotic cycle when shifted to the nonpermissive temperature and undergo meiosis and sporulation. This occurs even in rich medium, which normally restricts entry into the meiotic pathway. These various phenotypes have been taken as evidence that the RAS/cAMP pathway is required, directly or indirectly, for initiation of the mitotic cycle and that it participates in the developmental switch regulating entry into meiosis 27. To what signals does the RA$/eAMP lmthway r e s p o n ~ Nutrient sufficiency Yeast cells starved for nitrogen, sulfate or phosphate eventually arrest as unbudded cells. In addition, diploid cells transferred to medium lacking both a nitrogen source and a fermentable carbon source are induced to undergo meiosis and sporulation. As noted above, inactivation of the RAS/cAMP pathway leads to cell cycle arrest and induction of meiosis, as occurs on starvation; hyperactivation of the pathway prevents cell cycle arrest or meiosis under conditions of starvation. This reciprocal relationship initially suggested that the RAS/cAMP pathway mediated nutrient response. Nutrient sufficiency was presumed to induce high cAMP levels, which in turn prompted cell cycle initiation, while nutrient starvation caused reduced cAMP levels, which imposed arrest at the beginning of the cell cycle. Little evidence in support of this model has appeared. First, nutrient availability does not correlate particularly well with intracellular cAMP levels: refeeding nitrogenous compounds to nitrogen-starved cells yields almost no detectable alterations in cAMP levels~. Second, initiation of sporulation correlates poorly with cAMP levels and much better with GTP levels: diploids can be induced to sporulate by depleting their internal GTP pools even while cAMP is maintained at a high level29. Finally, Cameron et al. observed that strains expressing low-level, constitutive A kinase activity (bcyl tpk w strains) respond normally to nutrient limitation, even in the absence of both RAS genes, CYR1 or CDC25 (Ref. 30). How can these observations be reconciled with the reciprocal relationship between the RAS/cAMP pathway and nutrient status, described above? One possibility is that the decision of the cell to enter or not to enter the cell cycle is based on input from several different sensing pathways. The cell would use some integrated average of these various input signals to judge whether to initiate a cell cycle. If the input signal from the RAS/cAMP pathway were at one extreme or the other, due to its mutational inactivation or hyperactivation, then the decision-making process could be rendered oblivious to any other signalling pathways. That is, ras- cells would have such a low signal from the cAMP pathway that signals from other pathways indicating normal nutrient levels would go unheeded and the cell would arrest. In the reverse situation, activating mutations (e.g. bcyl-) would induce such a strong positive signal from the cAMP pathway that signals from other pathways indicating insufficient nutrients would be ignored and the cell would fail to arrest. Finally, if the cAMP pathway sends a neutral signal, as
TIGJANUARY1991 VOL.7 NO. 1
m
[~EVIEWS would be the case in a bcyl tpk w background, then the cell would arrest or grow on the basis of input from the other sensing pathways. Carbon source
TAmE 1. Genes of the RAS/cAMP pathway
Gene
Other names Product/function
BCY1
SRA1
Regulatory subunit of cAMP-dependent protein kinase Ras protein activator: GTP/GDP exchange factor Adenylatecyclase
Reference
33, 34
CDC25 CYR2 18, 19 Addition of glucose or any related fermentable sugar to yeast CYR1 IAC1, SRA4, 17 cells grown on a nonfermentable CDC35 carbon source or starved for gluIRA1 PPD1 Inhibitor of/?AS function 24 cose yields a rapid and dramatic IRA2 Inhibitor of RASfunction 24 increase in intracellular cAMP PDE1 Low-aff'mity(high Km) cAMP 35 concentration, followed by a prephosphodiesterase cipitous decline to a basal value PDE2 SRA5 High-affinity(low Km) cAMP 36 only about twice the level before phosphodiesterase addition2S,30,31. The precise mechRAM/ 15, 37 DPR1, SGP2 Required for Ras protein and a mating anism by which glucose addition SUPH, STE16 factor ma~ration influences Ras activity is currently RAM GTP-binding protein modulating 16 adenylate cyclase activity unknown. One model is that addition of glucose yields increased RAS2 GTP-binding protein modulating 16 adenylate cyclase activity concentrations of intermediary Protein kinase that when overexpressed 38 metabolites, such as glucose 6$0/9 can substitute for protein kinase A phosphate, which could serve as High copy suppressor of cdc25 strains; 39 allosteric effectors of CDC25 or $0925 GDP/GTP exchange factor IRA. Consistent with this, glucoseRegulator of RAM transcription 40 stimulated cAMP accumulation $RA6 so-Pc, caP1 Suppressor of activated RAS2alleles; 70 20, 21 requires at least one functional $RV2 kDa adenylate cyclase-associatedprotein hexokinase or glucokinase, while Carboxy-terminalmethyltransferase 41 fructose-stimulated cAMP ~ccuSTE14 One of three catalytic subunits of 26 SRA3, PK-25 mulation specifically requires a TPK1 cAMP-dependent protein kinase hexokinase32. One of three catalytic subunits of 26 In alternative models, glucose TPK2 cAMP-dependent protein kinase stimulation of cAMP accumulation One of three catalytic subunits of 26 could involve alterations in intraTPK3 cAMP-dependent protein kinase cellular pH or changes in memProtein kinase whose loss suppresses the 42 brane potential. Glucose addition YAK1 growth defect of tpkl tpk2 tpk3 strains leads to internal acidification of the cell. In addition, metabolic uncouplers, such as dinitrophenol and CCCP, cause a transient increase in cAMP levels biosynthesis. Finally, rates of protein synthesis, which similar to that obtained by addition of glucose 2x. appear to affect levels of CDC25, could also influence Residence of adenylate cyclase, the SRV2 protein and the activity of the system. Ras on the plasma membrane could render them or their interactions sensitive to alterations in membrane Whatis RA$doing? Despite the abundance of information that has potential. Similarly, interaction of either of these proteins with each other or with other protein modulators ~:~:umulated about the role of RAS and cAMP in yeast, of the pathway could readily be influenced by changes we still do not have a clear appreciation of their funcin intracellular pH. Thus, the glucose signal may not tion in the cell. Any model for RAS/cAMP function must enter upstream of Ras, but may be injected into the account for the various phenotypes associated with loss or hyperactivation of the pathway. Some but by RAS/cAMP pathway at some intermediate point. no means all of the phenotypes resulting from either activation or loss of the RAS/cAMP pathway can be Other signals Since the output signal from Ras is dependent currently explained on the basis of differential activities solely on the absolute level of Ras in the GTP-bound of known targets of the A kinase. Effects on mobilform, the various steps in the biosynthesis of fully ization of carbohydrate reserves are, to a first approximodified Ras could be harnessed as a means of pro- marion, accounted for by the effects of phosphorylation viding input into the system. Thus, decreased levels of on the activities of enzymes responsible for synthesis famesyl pyrophosphate, a key intermediate in sterol versus degradation of these reserve compounds, biosynthesis, are reflected in reduced activity of the although transcriptional regulation of genes encoding RAS/cAMP pathway n. Similarly, Ras biosynthesis - and, these enzymes may also play a role7. Loss of ability to as a consequence, activity from the p a t h w a y - might grow on certain carbon sources may be attributable be modulated by availability of S-adenosyl methionine in part to transcriptional regulation of RAS genes, to (required for carboxymethyl esterification) or lipid effects of phosphorylation on key constriction points
TIGJANUARY1991 VOL.7 NO. 1
[]~EVIEWS in the glycolytic pathway, and to regulation of sugar transporters by phosphorylation. Other phenotypes of mutants altered in the RAS pathway are less readily explained. Cell cycle arrest resulting from loss of kinase activity could, perhaps, be the indirect consequence of reduced synthesis of cyclins. Thus, diminished energy metabolism and synthetic capacity associated with loss of kinase activity could be coupled to the mitotic cycle simply through reduced rates of protein syntbesis. Consistent with this hypothesis is the fact that other restrictions in glycolysis or protein synthesis yield the same terminal phenotype as ras- cells. Changes in stress sensitivity of mutants altered in the RAS pathway require other explanations. An increasing body of data suggests that yeast cells can attain a distinct physiological state outside the normal mitotic cell cycle when they are nutrient starved or reach stationary phase. This state is analogous to the GO state described for mammalian cells. Yeast cells in this GO state are more refractory to heat shock and nutrient starvation and can persist in this quiescent state for extended periods without significant loss of viability, cAMP levels can influence the decision of a cell to enter this GO state and this may be how cAMP affects the response of the cell to stress. How might RAS/cAMP be involved in the transition between GO and GI? Populations of cells growing slowly are more resistant to heat shock than are rapidly growing cells, and the percentage of cells that survive heat shock correlates well with the percentage of unbudded cells in a population. One way to account for this fact is to suggest that only cells in G1 can enter the thermoprotective GO state, but that this state is available to the cell at any point in G1 between cytokinesis and commitment to a new cell cycle. A cell traversing G1 continues to do so unless it is 'shunted' into the GO state by any one of a number of different insults, such as heat shock, ethanol exposure, nutrient depletion, carbon starvation or diminished protein synthesis. Reduced A kinase activity would serve as one of the incentives to leave the mitotic cycle and enter GO, accounting for the arrest phenotype and thermotolerance of ras- strains. The sensitivity of b c y l strains to nutrient deprivation or heat shock can be explained in this model by suggesting that they are denied access to GO as a consequence of the high A kinase activity they maintain. Just as described above, this extremely high signal input from the cAMP pathway would swamp any signals from other pathways prompting entry into GO. Accordingly, even bcyl cells traversing G1 at the time of heat shock would not be able to gain entry to GO, rendering them sensitive to the treatment. The sporulation phenotype of mutants in the RAS/cAMP pathway can also be explained in this context. This model does not address the mechanism by which the decision to enter GO is effected. One could envisage that a central processor integrates input signals and regulates the battery of genes and proteins whose changes yield the distinct GO phenotype. Alternatively and perhaps more likely, the transition to GO could simply result from the accumulated changes in a number of different metabolic, physiologic and
transcriptional processes, each sensitive to regulation by cAMP, heat shock, nutrient availability, and so on. In summary, Ras proteins, through the A kinase, appear to modulate a panoply of metabolic and transcription processes that affect the level of the metabolic activity of the cell and thereby influence the rate of mass accumulation. The activity of the RAS/cAMP pathways also influences the decision to continue the mitotic cycle or to exit into GO. However, whether Ras plays a direct role in controlling the cell cycle or whether cell cycle regulation is an indtrect consequence of the effects of Ras on metabolic activity has not been resolved. Finally, while several effectors of the Ras pathway have been identified, a clear and complete picture of the pathway in which Ras resides has yet to emerge.
Acknowledgements
I thank the current and former members of my lab for their thoughtful comments and stimulating discussions conceming the role of RAS in yeast. This work was supported by grant CA41086 from NIH.
References 1 Powers, s. et al. (1984) Cell 36, 607-612 2 DeFeoJones, D., Scolnick, E.M., Koller, R. and Dhar, R. (1983) Nature 306, 707-709 3 DeFeoJones, D. et al. (1985) Science 228, 17%184 4 Kataoka, T. et al. (1985) Cell 40, 1%26 5 Bourne, H.R. (1985) Nature 317, 16-17 6 Barbacid, M. (1987) Annu. Rev. Biocbem. 55, 779--827 7 Broach, J.R. and Deschenes, R$. (1990) Adv. CancerRes. 54, 79-139 8 Gibbs,J.B. and Marshall,M.S. (1989) Microbiol. Rev. 53, 171-185 9 Tamanoi,F. (1988) Biochim. Biopbys. Acta 948, 1-15 10 Matsumoto, K., Uno, I. and Ishikawa, T. (1985) Yeast 1, 25-28 11 Schafer,W.R. et at. (1989) Science 245, 379-385 12 Casey, P.J., Solski, P.A., Der, C.J. and Buss, J.E. (1989) Proc. Naa Acad. Sci. USA 86, 8323--8327 13 Hancock, J.F., Magee, A.I., Childs,J.E. and Marshall,CO. (1989) Cell 57, 1167-1177 14 Stimmel,J.B. et al. Biochemistry (in press) 15 Schafer, W.R. etal. (1990) Science 249, 1133-1139 16 Toda, T. et al. (1985) Cell 40, 27-36 17 Kataoka, T., Broek, D. and Wigler, M. (1985) Cell43, 493-505 18 Robinson, L.C. et al. (1987) Science 235, 1218-1221 19 Broek, D. et al. (1987) Cell 48, 78%799 20 Fedor Chaiken, M., Deschenes, R.J. and Broach, J.R. (1990) Cell 61, 32%340 21 Field,J. et al. (1990) Cell61, 319-327 22 Hall, A. (1990) Cell 61, 921-923 23 Bourne, H.R. (1988) Cell 53, 669-671 24 Tanaka, K. et al. (1990) Cell 60, 803--807 25 Thompson, R.C. (1988) Trends Biochem. Sci. 13, 91-93 26 Toda, T. et al. (1987) Cell 50, 277-287 27 Malone, R.E. (1990) CeU61, 375-378 28 Francois, J., Villanueva,M.E. and Hers, H.G. (1988) Bur. J. Biochem. 174, 551-559 29 Olempska-Beer, Z. and Freese, E. (1987) Mol. Cell. Biol. 7, 2141-2147 30 Cameron, S., Levin, L., Zoller, M. and Wigler, M. (1988) Cell 53, 555-566 M Mbonyi, K. et al. (1988) 21401.Cell. Biol. 8, 3051-3057 32 Beullens, M. et al. (1988) FEBS Lett. 172, 227-231
TIGJANUARY1991 VOL.7 NO. 1
B
~'~EVIEWS 33 Matsumoto, K., Uno, I., Oshima, Y. and Ishikawa, T. (1982) Proc. Natl Acad. Sci. USA 79, 235%2359 34 Toda, T. et al. (1987) Mol. Ceil. Biol. 7, 1371-1377 35 Nikawa,J., Sass, P. and Wigler, M. (1987) Mol. Ceil. Biol.
39 Crochet,J.B. et al. (1990) Science 248, 866-868 40 Breviario, D. et aL (1986) Proc. Nad Acad. Sci. USA 83,
7, 3629-3636 36 Sass, P. etal. (1986)Proc. NaaAcad. Sci. USA 83, 9303--9307 37 Powers, S. et al. (1986) Cell 47, 413-422 38 Toda, T., Cameron, S., Sass, P. and Wiglet, M. (1988) Genes Dev. 2, 517-527
5071-5076 42 Garrett, S. and Broach,J.R. (1989) GenesDev. 3, 1336-1348
4152--4156 41 Hrycyna, C.A. and Clarke, S. (1990) Mol. Cell. Biol. 10,
[~.R. BROACH!$ I N THE JDP.PARTMP.NTOFMOL~CUL~ BXOLOG~,I IPRINCLriDNUNIVF'g~rlY"PRINCLr~N"N~ 0854(6 USA. J
~OOKBEVIEWS I particularly liked the chapter by EW. Studier and colleagues on the use of T7 RNA polymerase to direct expression of cloned genes in vivo. In addition to providing a detailed description of these widely used vectors (induding the Gene ExpressionTechnology sequences of the major regulatory (Methods in Enzymology elements), this chapter provides Vol. 185) clear and detailed descriptions for their use. A similarly detailed and edited by D.V.Goeddel,AcademicPress, 1990. authoritative chapter on fusions to $80.00(xxxi+ 681 pages)ISBN0 12 1820866 staphylococcal protein A is provided by Nilsson and Abrahmsrn. The section on expression in In the early 1980s, when biotechyeast is perhaps the strongest in the nology companies could raise book. I especially liked the six millions of dollars of equity simply by declaring an interest in gene ex- chapters on specific yeast propression, Sydney Brenner proposed motets ( GAL4, ADH2, CUP1, PGK, GAPDH and temperature-sensitive that the Central Dogma should be promoters) and the four chapters reformulated as 'DNA makes RNA on secretion of proteins from yeast makes Money'. Alas, it has not proved to be so simple! As many of (use of homologous and heterolothose early entrepreneurs have now gous signal sequences, mannandefective mutants, etc.). Together discovered, although it is easy to these chapters provide a clone and sequence a new gene, marvellous introduction to yeast the idiosyncracies of proteins make the recovery of useful products from expression technology. Unfortunately, the section on genes a challenging undertaking. mammalian cell expression is comGene Expression Technology, paratively short and is probably the the latest volume of the excellent Methods in Enzymology series, f ,- weakest section of the book. Many important expression systems, esvides an up-to-date guide through pecially expression systems based the maze of gene expression syson viruses, are not described in any tems. The book does not attempt detail. I would have liked to have to be comprehensive. The three main sections focus on some of the seen chapters on vaccinia virus, adenovirus and retroviral vectors. The more recently developed protein most useful chapter is on selection expression systems for use in and coamplification of heteroloEscbericbia coli, yeast and mamgous genes in mammalian cells, by malian cells, and there is a short R.J. Kaufman, who describes in section on expression in Bacillus detail methods for enhancing gene subtilis. However, there are no expression by the selection of chapters devoted to the widely stable lines carrying amplified used Baculovirus system nor are genes. Many of the other chapters there chapters concerning expression in plant cells. Nonetheless, in this section lack comparable experimental detail. within the areas that are covered, As in any multi-author work, there are many outstanding some chapters are less successful contributions. TIGJANUARY1991 VOL.7 NO. 1
m
than others. For example, although the chapter on refolding of recombinant proteins, by Kohno et aL, provides two useful examples of protein-refolding schemes, much more information could have been provided about this critical problem. In general, I found the introductory and 'review' chapters to be less interesting than the technical methods papers for which the Methods in Enzymology series is justly famous. The two most successful review chapters are the thorough and scholarly discussions of the secretion of proteins from E. coli by J.A. Stader and T.J. Silhavy and of vectors used for the expression of heterologous genes in mammalian cells by R.J. Kaufman. It is a pity that they did not include some more experimental methods to illustrate the key points of their chapters. Overall, the volume is very successful. The articles have been carefully edited to avoid undue repetition and the selection of topics is interesting. Althou h one might expect that a volume devoted to gene expression would simply list the various vectors and promotets, the editor, D.V. Goeddel, has wisely chosen to view gene expression from a broader perspective and has selected several informative articles on protein processing and proteolysis. I strongly recommend this book both to novices who are embarking on a gene expression project for the first time and to frustrated veterans who are seeking the latest 'tips' about how to improve the expression of their favourite pro*.eins.
Josm MRCLaboratoryofMolecularBiologF,Hills Road, CambrfdgeCB22QH, IlK.
R a s genes encode low molecular weight, GTPbinding, GTP-hydrolysing proteins that are highly conserved throughout all eukaryotic species. Mutational activation of these genes can induce cancer in humans and other mammals. A number of investigators have proposed that Ras proteins in mammals function in signal transduction, coupling some second messenger to the binding of serum growth factors to cellular receptors. Although this hypothesis is appealing, the components of this postulated signal tmnsduction pathway in mammalian cells have not been clearly defined. Saccbaromyces cerevisfae contains two genes that are both structurally and functionally homologous to human ras genesl, z. Human ras genes can ameliorate the growth defects arising from inactivation of yeast RAS genes, and slightly modified versions of a yeast RAS gene can induce proliferative transformation of mouse fibroblast cells3,4. This functional conservation of Ras proteins initially offered the prospect of applying yeast genetics to defining the pathway in which metazoan Ras normally resides. This early euphoric expectation has diminished somewhat. Subsequent experiments have shown that, while yeast Ras proteins function predominantly - if not exclusively - as a modulator of adenylate cyclase, metazoan ras genes appear to exert their influence independent of cAMP5. Despite the apparent disparity of the pathways in which yeast and human ras genes reside, an understanding of the function of yeast Ras protein can provide significant insight into the detailed molecular interactions required for mammalian ras function as well as into the general principles of cellular growth control in larger eukaryotes. Here, I present our current understanding of the signals that feed into the RAS pathway in yeast and the biological processes that are directly regulated by the action of the Ras proteins. More extensive reviews of Ras structure and function in yeast and in mammalian cells and the relationships between the two systems have recently appeared6--10.
RASgenes in
Saccharomyces cerevisiae: signal transducti0n in search of a pathway JAMESR. BROACH Ras proteins in budding yeasts initially appeared to regulate inittatioa of the cell cycle in response to nutrient availability. More recent wori~ while clarifying the mechanism of Ras.mediated signal transductto~ has undermined our notttm of the signal Ras transmits. We now suspect that Ras be~ps to coordinate cellular metabolism aml mass accumulatto~ but what Ras responds to is not clear. Ras proteins and the GTP/GDPcycle The yeast Ras proteins are members of a superfamily of low molecular weight GTP-binding proteins, encompassing 52 distinct species at the last count. All of these proteins probably execute their function through a cycle of GTP/GDP exchange and GTP hydrolysis as outlined in Fig. 1 (Ref. 6). In its simplest form, the model postulates that Ras proteins shuttle back and forth between an activating agent, which provides the input signal, and an effector protein, which transmits an output signal. Interaction of the Ras proteins with an activating agent stimulates removal of bound GDP and its replacement with free GTP. In this activated, GTP-bound state, the protein can stimulate the activity of the target protein. As a direct consequence of this stimulation or simply as a stochastic process, GTP bound to the Ras protein is hydrolysed to GDP. In the GDP-bound state, the Ras protein is no longer able to activate or associate with its target and
e'e°°
:/
IInPut;)
•/ "
GTP
GDP
/
IRASIGTP[/'IOutPu ' "%*%leeee]
rIGO The GDP/GTPcycle of Ras. A model for ~s-mediated signal tmnsduction is shown. Input is registered as an increase in Ras bound to GTP, accomplished either by stimulating a GTP/GDP exchange factor or by inhibiting a GTPase-activating protein (GAP). Ras in its activated, GTP-bound fonn promotes the downstream process, either alone or in conjunction with GAP.See text for full description. 'lag I~a'~Y ~991 VOL."7nO. 1 01991 ElsevierSciencePublishersLtd(UK)0168- 9479191/$02.00
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Go Arrest, Sporulation
Energy Activation, AlteredTranscription, Cell Growth
~a18 The RAS/cAMP pathway in Saccharom.w.escerevisiae. The roles of components of the RAS/cAMPpathway, each indicated by its gene designation (see Table 1), are shown schematically.P1, P2, P3, etc. indicateprote~n targets of the A kinase, which consists of two catalyticsubunits encoded redundantly by TPKI-3and two regulatory subunits encoded by BCY1. See text for full description. Dotted line indicatesA-kinase-inducedinhibition. the protein must be reactivated by recharging with GTP. More complex variations on this theme are possible, some of which are noted in the specific discussion of the yeast RAS pathway.
The RatS~cAMPpathwayin y~st Components of the pathway The RAS/cAMP pathway as we currently understand it is shown in Fig. 2 and genes whose products are components of or impinge on this pathway are listed in Table 1. RAM and RAS2 gene products (hereafter referred to collectively as Ras) are synthesized as cytoplasmic precursors, which become extensively modified at the carboxy terminus upon maturation. The precursor proteins are farnesylated, proteolyticaily cleaved, methyl esterified and palmitoylated, probably in that orderil-14. The farnesylation step is required for localizing the mature proteins to a membrane fraction, presumed to be the inner surface of the plasma membrane, and membrane localization is essential for Ras function. The product of the RAM/ gene is required for farnesylation, probably as one subunit of a heterodimeric farnesyl transferase enzyme (the prcdtlct of the RAM2 gene may be the other subunit)n.~5. The product of the STE14 gene is a methyltransferase that is probably responsible for methyl esterification of Ras (see Table 1). Since ste14 strains are viable, methyl esterification is probably not essential for Ras activity. When charged with GTP, mature Ras is capable of stimulating adenylate cyclase, the product of the CYR1
IOCUS16,17.Activation of Ras is catalysed by the product of the CDC25 gene, a protein that promotes removal of GDP bound to Ras and its replacement with free GTPm.19 (S. Jones, M.L. Vignais and J. Broach, unpublished). Stimulation of adenylate cyclase by GTP-Ras requires the product of the SRV2 l~ocus, a 70 kDa protein that copurifies with adenylate cyclase2°.zl. This protein either serves as an intermediary in the Ras-cyclase interaction or, more likely, promotes or stabilizes the physical association of the two proteins. Hydrolysis of GTP bound to Ras could occur either as a direct result of interaction with its downstream target or simply as a spontaneous process. In the former case, the target protein and the Ras GTPaseactivating protein (GAP) would be one and the same, a situation that may apply to ras in mammalian cells and to certain ras-like gene product,s involved in secretion2Z,23. Whether adenylate cyclase stimulates the GTPase of yeast Ras is not known. However, the products of two genes, IRA I and IRA2, structural and functional homologues of mammalian GAP, do stimulate Ras GTPase activityz4. Since phenotypes associated with loss of function of these two proteins are quite distinct from and completely suppressed by loss of Ras activity, neither of these proteins is a downstream target of Ras. Rather, the IRA proteins act to down-modulate Ras activity. A plausible model is that hydrolysis of GTP bound to yeast Ras is a stochastic event, which proceeds independently of its interaction with adenylate cyclase. The rate of GTP
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~EVIEWS hydrolysis by Ras imposes a time limit on stimulation of its downstream target. This would be similar to the proposed mechanism of GTP-mediated interaction of EF-Tu with the ribosome zS. How input information enters this pathway is not clear. The relative proportion of the active, GTP-bound versus the inactive, GDP-bound forms of Ras is determined by the rates of two competing reactions: the GDP/GTP exchange reaction catalysed by CDC25 protein on the one hand and the GTPase reaction, stimulated by the IRA1 and IRA2 proteins, on the other hand. Input into the RAS/cAMP pathway need only increase the absolute amount of Ras in the activated, GTP-bound form. This could be accomplished equally well by inhibiting the GTPase-activating proteins or by stimulating the exchange factor. Thus, any or all of these modulating enzymes could serve as loci for transmitting input information (Fig. 1). The sole function of cAMP in the cell appears to be to activate the cAMP-dependent protein kinase (protein kinase A) 16. This kinase is a heterotetrame# 6, comprising two regulatory subunits encoded by the BCY1 locus and two catalytic subunits redundantly encoded by three separate genes: TPK1, TPK2 and TPK3. In the absence of cAMP, the BCY1 protein restrains the catalytic subunits by serving as a tightly associated competitive inhibitor of the enzymes. Upon binding cAMP, the BCY1 inhibitor releases the catalytic subunits, which are then freed to phosphorylate their numerous cellular targets. Targets of the A kinase include enzymes involved in metabolism of storage carbohydrates, enzymes situated at strategic points in the glycolytic/gluconeogenic pathway, enzymes required for phospholipid metabolism, transcription factors associated with expression of specific genes, proteins involved in production of cAMP itself, and, undoubtedly, a substantial number of proteins currently unidentified (see Ref. 7 for an extensive listing). In general, high A kinase activity induces breakdown of stored carbohydrates, activation of the glycolytic pathway, induction of transcription of a large number of growth-specific genes (such as those encoding ribosomal proteins and certain secreted proteins), repression of some stress-protective proteins, and down-modulation of the RAS/cAMP pathway. In the reciprocal situation, low levels of A kinase activity induce accumulation of carbohydrates, activation of gluconeogenesis, reduced transcription of growth-specific genes, and induction of various stress-related proteins. Genetics of the RAS/cAMPpathway Mutations in components of the RAS/cAMP pathway yield one of two distinct phenotypes. Strains with mutations that activate the pathway are capable of normal mitotic growth in rich medium, but exhibit a number of abnormal phenotypes. These include loss of carbohydrate reserves, increased sensitivity to heat shock and starvation, and, for diploid strains, inhibition of sporulation. Strains with mutations that diminish the activity of the pathway fail to grow on nonfermentable carbon sources, and accumulate carbohydrate reserves. In addition, strains with temperature-sensitive mutations in RAS2 (in a RAM- background), CYR1 or
CDC25 arrest as unbudded cells at the nonpermissive temperature. Diploid strains homozygous for these mutations leave the mitotic cycle when shifted to the nonpermissive temperature and undergo meiosis and sporulation. This occurs even in rich medium, which normally restricts entry into the meiotic pathway. These various phenotypes have been taken as evidence that the RAS/cAMP pathway is required, directly or indirectly, for initiation of the mitotic cycle and that it participates in the developmental switch regulating entry into meiosis 27. To what signals does the RA$/eAMP lmthway r e s p o n ~ Nutrient sufficiency Yeast cells starved for nitrogen, sulfate or phosphate eventually arrest as unbudded cells. In addition, diploid cells transferred to medium lacking both a nitrogen source and a fermentable carbon source are induced to undergo meiosis and sporulation. As noted above, inactivation of the RAS/cAMP pathway leads to cell cycle arrest and induction of meiosis, as occurs on starvation; hyperactivation of the pathway prevents cell cycle arrest or meiosis under conditions of starvation. This reciprocal relationship initially suggested that the RAS/cAMP pathway mediated nutrient response. Nutrient sufficiency was presumed to induce high cAMP levels, which in turn prompted cell cycle initiation, while nutrient starvation caused reduced cAMP levels, which imposed arrest at the beginning of the cell cycle. Little evidence in support of this model has appeared. First, nutrient availability does not correlate particularly well with intracellular cAMP levels: refeeding nitrogenous compounds to nitrogen-starved cells yields almost no detectable alterations in cAMP levels~. Second, initiation of sporulation correlates poorly with cAMP levels and much better with GTP levels: diploids can be induced to sporulate by depleting their internal GTP pools even while cAMP is maintained at a high level29. Finally, Cameron et al. observed that strains expressing low-level, constitutive A kinase activity (bcyl tpk w strains) respond normally to nutrient limitation, even in the absence of both RAS genes, CYR1 or CDC25 (Ref. 30). How can these observations be reconciled with the reciprocal relationship between the RAS/cAMP pathway and nutrient status, described above? One possibility is that the decision of the cell to enter or not to enter the cell cycle is based on input from several different sensing pathways. The cell would use some integrated average of these various input signals to judge whether to initiate a cell cycle. If the input signal from the RAS/cAMP pathway were at one extreme or the other, due to its mutational inactivation or hyperactivation, then the decision-making process could be rendered oblivious to any other signalling pathways. That is, ras- cells would have such a low signal from the cAMP pathway that signals from other pathways indicating normal nutrient levels would go unheeded and the cell would arrest. In the reverse situation, activating mutations (e.g. bcyl-) would induce such a strong positive signal from the cAMP pathway that signals from other pathways indicating insufficient nutrients would be ignored and the cell would fail to arrest. Finally, if the cAMP pathway sends a neutral signal, as
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[~EVIEWS would be the case in a bcyl tpk w background, then the cell would arrest or grow on the basis of input from the other sensing pathways. Carbon source
TAmE 1. Genes of the RAS/cAMP pathway
Gene
Other names Product/function
BCY1
SRA1
Regulatory subunit of cAMP-dependent protein kinase Ras protein activator: GTP/GDP exchange factor Adenylatecyclase
Reference
33, 34
CDC25 CYR2 18, 19 Addition of glucose or any related fermentable sugar to yeast CYR1 IAC1, SRA4, 17 cells grown on a nonfermentable CDC35 carbon source or starved for gluIRA1 PPD1 Inhibitor of/?AS function 24 cose yields a rapid and dramatic IRA2 Inhibitor of RASfunction 24 increase in intracellular cAMP PDE1 Low-aff'mity(high Km) cAMP 35 concentration, followed by a prephosphodiesterase cipitous decline to a basal value PDE2 SRA5 High-affinity(low Km) cAMP 36 only about twice the level before phosphodiesterase addition2S,30,31. The precise mechRAM/ 15, 37 DPR1, SGP2 Required for Ras protein and a mating anism by which glucose addition SUPH, STE16 factor ma~ration influences Ras activity is currently RAM GTP-binding protein modulating 16 adenylate cyclase activity unknown. One model is that addition of glucose yields increased RAS2 GTP-binding protein modulating 16 adenylate cyclase activity concentrations of intermediary Protein kinase that when overexpressed 38 metabolites, such as glucose 6$0/9 can substitute for protein kinase A phosphate, which could serve as High copy suppressor of cdc25 strains; 39 allosteric effectors of CDC25 or $0925 GDP/GTP exchange factor IRA. Consistent with this, glucoseRegulator of RAM transcription 40 stimulated cAMP accumulation $RA6 so-Pc, caP1 Suppressor of activated RAS2alleles; 70 20, 21 requires at least one functional $RV2 kDa adenylate cyclase-associatedprotein hexokinase or glucokinase, while Carboxy-terminalmethyltransferase 41 fructose-stimulated cAMP ~ccuSTE14 One of three catalytic subunits of 26 SRA3, PK-25 mulation specifically requires a TPK1 cAMP-dependent protein kinase hexokinase32. One of three catalytic subunits of 26 In alternative models, glucose TPK2 cAMP-dependent protein kinase stimulation of cAMP accumulation One of three catalytic subunits of 26 could involve alterations in intraTPK3 cAMP-dependent protein kinase cellular pH or changes in memProtein kinase whose loss suppresses the 42 brane potential. Glucose addition YAK1 growth defect of tpkl tpk2 tpk3 strains leads to internal acidification of the cell. In addition, metabolic uncouplers, such as dinitrophenol and CCCP, cause a transient increase in cAMP levels biosynthesis. Finally, rates of protein synthesis, which similar to that obtained by addition of glucose 2x. appear to affect levels of CDC25, could also influence Residence of adenylate cyclase, the SRV2 protein and the activity of the system. Ras on the plasma membrane could render them or their interactions sensitive to alterations in membrane Whatis RA$doing? Despite the abundance of information that has potential. Similarly, interaction of either of these proteins with each other or with other protein modulators ~:~:umulated about the role of RAS and cAMP in yeast, of the pathway could readily be influenced by changes we still do not have a clear appreciation of their funcin intracellular pH. Thus, the glucose signal may not tion in the cell. Any model for RAS/cAMP function must enter upstream of Ras, but may be injected into the account for the various phenotypes associated with loss or hyperactivation of the pathway. Some but by RAS/cAMP pathway at some intermediate point. no means all of the phenotypes resulting from either activation or loss of the RAS/cAMP pathway can be Other signals Since the output signal from Ras is dependent currently explained on the basis of differential activities solely on the absolute level of Ras in the GTP-bound of known targets of the A kinase. Effects on mobilform, the various steps in the biosynthesis of fully ization of carbohydrate reserves are, to a first approximodified Ras could be harnessed as a means of pro- marion, accounted for by the effects of phosphorylation viding input into the system. Thus, decreased levels of on the activities of enzymes responsible for synthesis famesyl pyrophosphate, a key intermediate in sterol versus degradation of these reserve compounds, biosynthesis, are reflected in reduced activity of the although transcriptional regulation of genes encoding RAS/cAMP pathway n. Similarly, Ras biosynthesis - and, these enzymes may also play a role7. Loss of ability to as a consequence, activity from the p a t h w a y - might grow on certain carbon sources may be attributable be modulated by availability of S-adenosyl methionine in part to transcriptional regulation of RAS genes, to (required for carboxymethyl esterification) or lipid effects of phosphorylation on key constriction points
TIGJANUARY1991 VOL.7 NO. 1
[]~EVIEWS in the glycolytic pathway, and to regulation of sugar transporters by phosphorylation. Other phenotypes of mutants altered in the RAS pathway are less readily explained. Cell cycle arrest resulting from loss of kinase activity could, perhaps, be the indirect consequence of reduced synthesis of cyclins. Thus, diminished energy metabolism and synthetic capacity associated with loss of kinase activity could be coupled to the mitotic cycle simply through reduced rates of protein syntbesis. Consistent with this hypothesis is the fact that other restrictions in glycolysis or protein synthesis yield the same terminal phenotype as ras- cells. Changes in stress sensitivity of mutants altered in the RAS pathway require other explanations. An increasing body of data suggests that yeast cells can attain a distinct physiological state outside the normal mitotic cell cycle when they are nutrient starved or reach stationary phase. This state is analogous to the GO state described for mammalian cells. Yeast cells in this GO state are more refractory to heat shock and nutrient starvation and can persist in this quiescent state for extended periods without significant loss of viability, cAMP levels can influence the decision of a cell to enter this GO state and this may be how cAMP affects the response of the cell to stress. How might RAS/cAMP be involved in the transition between GO and GI? Populations of cells growing slowly are more resistant to heat shock than are rapidly growing cells, and the percentage of cells that survive heat shock correlates well with the percentage of unbudded cells in a population. One way to account for this fact is to suggest that only cells in G1 can enter the thermoprotective GO state, but that this state is available to the cell at any point in G1 between cytokinesis and commitment to a new cell cycle. A cell traversing G1 continues to do so unless it is 'shunted' into the GO state by any one of a number of different insults, such as heat shock, ethanol exposure, nutrient depletion, carbon starvation or diminished protein synthesis. Reduced A kinase activity would serve as one of the incentives to leave the mitotic cycle and enter GO, accounting for the arrest phenotype and thermotolerance of ras- strains. The sensitivity of b c y l strains to nutrient deprivation or heat shock can be explained in this model by suggesting that they are denied access to GO as a consequence of the high A kinase activity they maintain. Just as described above, this extremely high signal input from the cAMP pathway would swamp any signals from other pathways prompting entry into GO. Accordingly, even bcyl cells traversing G1 at the time of heat shock would not be able to gain entry to GO, rendering them sensitive to the treatment. The sporulation phenotype of mutants in the RAS/cAMP pathway can also be explained in this context. This model does not address the mechanism by which the decision to enter GO is effected. One could envisage that a central processor integrates input signals and regulates the battery of genes and proteins whose changes yield the distinct GO phenotype. Alternatively and perhaps more likely, the transition to GO could simply result from the accumulated changes in a number of different metabolic, physiologic and
transcriptional processes, each sensitive to regulation by cAMP, heat shock, nutrient availability, and so on. In summary, Ras proteins, through the A kinase, appear to modulate a panoply of metabolic and transcription processes that affect the level of the metabolic activity of the cell and thereby influence the rate of mass accumulation. The activity of the RAS/cAMP pathways also influences the decision to continue the mitotic cycle or to exit into GO. However, whether Ras plays a direct role in controlling the cell cycle or whether cell cycle regulation is an indtrect consequence of the effects of Ras on metabolic activity has not been resolved. Finally, while several effectors of the Ras pathway have been identified, a clear and complete picture of the pathway in which Ras resides has yet to emerge.
Acknowledgements
I thank the current and former members of my lab for their thoughtful comments and stimulating discussions conceming the role of RAS in yeast. This work was supported by grant CA41086 from NIH.
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[~.R. BROACH!$ I N THE JDP.PARTMP.NTOFMOL~CUL~ BXOLOG~,I IPRINCLriDNUNIVF'g~rlY"PRINCLr~N"N~ 0854(6 USA. J
~OOKBEVIEWS I particularly liked the chapter by EW. Studier and colleagues on the use of T7 RNA polymerase to direct expression of cloned genes in vivo. In addition to providing a detailed description of these widely used vectors (induding the Gene ExpressionTechnology sequences of the major regulatory (Methods in Enzymology elements), this chapter provides Vol. 185) clear and detailed descriptions for their use. A similarly detailed and edited by D.V.Goeddel,AcademicPress, 1990. authoritative chapter on fusions to $80.00(xxxi+ 681 pages)ISBN0 12 1820866 staphylococcal protein A is provided by Nilsson and Abrahmsrn. The section on expression in In the early 1980s, when biotechyeast is perhaps the strongest in the nology companies could raise book. I especially liked the six millions of dollars of equity simply by declaring an interest in gene ex- chapters on specific yeast propression, Sydney Brenner proposed motets ( GAL4, ADH2, CUP1, PGK, GAPDH and temperature-sensitive that the Central Dogma should be promoters) and the four chapters reformulated as 'DNA makes RNA on secretion of proteins from yeast makes Money'. Alas, it has not proved to be so simple! As many of (use of homologous and heterolothose early entrepreneurs have now gous signal sequences, mannandefective mutants, etc.). Together discovered, although it is easy to these chapters provide a clone and sequence a new gene, marvellous introduction to yeast the idiosyncracies of proteins make the recovery of useful products from expression technology. Unfortunately, the section on genes a challenging undertaking. mammalian cell expression is comGene Expression Technology, paratively short and is probably the the latest volume of the excellent Methods in Enzymology series, f ,- weakest section of the book. Many important expression systems, esvides an up-to-date guide through pecially expression systems based the maze of gene expression syson viruses, are not described in any tems. The book does not attempt detail. I would have liked to have to be comprehensive. The three main sections focus on some of the seen chapters on vaccinia virus, adenovirus and retroviral vectors. The more recently developed protein most useful chapter is on selection expression systems for use in and coamplification of heteroloEscbericbia coli, yeast and mamgous genes in mammalian cells, by malian cells, and there is a short R.J. Kaufman, who describes in section on expression in Bacillus detail methods for enhancing gene subtilis. However, there are no expression by the selection of chapters devoted to the widely stable lines carrying amplified used Baculovirus system nor are genes. Many of the other chapters there chapters concerning expression in plant cells. Nonetheless, in this section lack comparable experimental detail. within the areas that are covered, As in any multi-author work, there are many outstanding some chapters are less successful contributions. TIGJANUARY1991 VOL.7 NO. 1
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than others. For example, although the chapter on refolding of recombinant proteins, by Kohno et aL, provides two useful examples of protein-refolding schemes, much more information could have been provided about this critical problem. In general, I found the introductory and 'review' chapters to be less interesting than the technical methods papers for which the Methods in Enzymology series is justly famous. The two most successful review chapters are the thorough and scholarly discussions of the secretion of proteins from E. coli by J.A. Stader and T.J. Silhavy and of vectors used for the expression of heterologous genes in mammalian cells by R.J. Kaufman. It is a pity that they did not include some more experimental methods to illustrate the key points of their chapters. Overall, the volume is very successful. The articles have been carefully edited to avoid undue repetition and the selection of topics is interesting. Althou h one might expect that a volume devoted to gene expression would simply list the various vectors and promotets, the editor, D.V. Goeddel, has wisely chosen to view gene expression from a broader perspective and has selected several informative articles on protein processing and proteolysis. I strongly recommend this book both to novices who are embarking on a gene expression project for the first time and to frustrated veterans who are seeking the latest 'tips' about how to improve the expression of their favourite pro*.eins.
Josm MRCLaboratoryofMolecularBiologF,Hills Road, CambrfdgeCB22QH, IlK.
