Báo cáo khoa học: The yeast stress response Role of the Yap family of b-ZIP transcription factors The PABMB Lecture delivered on 30 June 2004 at the 29th FEBS Congress in Warsaw potx

Although specific stress conditions elicit distinct cellular responses, underlying gene Keywords Saccharomyces cerevisae; stress response; Yap Correspondence C.. Abbreviations ACR, arseni

The yeast stress response

Role of the Yap family of b-ZIP transcription factors

The PABMB Lecture delivered on 30 June 2004 at the 29th FEBS Congress in Warsaw

Claudina Rodrigues-Pousada, Tracy Nevitt and Regina Menezes

Genomics and Stress Laboratory, Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal

The capacity for adaptation to changes in intra- and

extracellular conditions is a universal prerequisite for

an organism’s survival and evolution The existence of

molecular mechanisms of response, repair and

adapta-tion, many of which are greatly conserved across

nat-ure, endows the cell with the plasticity it requires to

adjust to its ever-changing environment, a homeostatic

event that is termed the stress response Through the

sensing and transduction of the stress signal into the

nucleus, a genetic reprogramming occurs that leads, on

the one hand, to a decrease in the expression of

house-keeping genes and protein synthesis and, on the other

hand, to an enhancement of the expression of genes encoding stress proteins These include molecular chaperones responsible for maintaining protein folding, transcription factors that further modulate gene expression and a diverse network of players including membrane transporters and proteins involved in repair and detoxification pathways, nutrient metabolism, and osmolyte production, to name a few Survival and growth resumption imply successful cellular adaptation

to the new conditions as well as the repair of damage incurred to the cell Although specific stress conditions elicit distinct cellular responses, underlying gene

Keywords

Saccharomyces cerevisae; stress response;

Yap

Correspondence

C Rodrigues-Pousada, Genomics and

Stress Laboratory, Instituto de Tecnologia

Quimica e Biologica, Avenida da Republica,

EAN, Apt127, 2781-901 Oeiras, Portugal

Fax: +351 2144 11277

Tel: +351 2144 69624

E-mail: claudina@itqb.unl.pt

Website: http://www.itqb.unl.pt/Research/

Biological_Chemistry/Genomics_and_Stress/

(Received 2 February 2005, revised

22 March 2005, accepted 1 April 2005)

doi:10.1111/j.1742-4658.2005.04695.x

The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gene expression when exposed to a plethora of environmental insults Therefore, yeast cell homeostasis control is achieved through a highly coordinated mechanism of transcription regulation invol-ving several factors, each performing specific functions Here, we present our current knowledge of the function of the yeast activator protein family, formed by eight basic-leucine zipper trans-activators, which have been shown to play an important role in stress response

Abbreviations

ACR, arsenic compounds resistance cluster; b-ZIP, basic leucine zipper; CRD, cysteine-rich domain; DBD, DNA-binding domain; ESR, environmental stress response; HOG, high osmolarity glycerol; HSE, heat shock factor; HSR, heat shock element; MAP, mitogen-activated protein; NEM, N-ethylmaleimide; NES, nuclear export signal; PC, phytochelatin; PKA, protein kinase A; ROS, reactive oxygen species; STRE, stress responsive element; Ybp1, Yap 1 binding protein; YCF1, yeast cadmium factor gene.

expression programmes common to all environmental

stress responses are at play [1] Specific forms of stress

such as heat shock and some forms of oxidative stress

[2] demand the activation of the heat shock factor

(HSF), a modular protein consisting of a helix–turn–

helix class of DNA-binding domain (DBD), a leucine

zipper domain, required for trimerization, and a

C-ter-minal transcription activation domain [3] Both the

HSFs of Saccharomyces cerevisiae and that of the

clo-sely related yeast Kluveromyces lactis contain a unique

transcription activation domain N-terminal to the

DBD [4,5] The HSF is a pre-existing transcription

activator that binds to an array of a 5-bp heat shock

element (HSE; nGAAn) present upstream of all

heat-shock genes [3] It has recently been shown that HSF

targets are activated not only upon heat shock, but

also by diamide, nitrogen depletion and

stationary-phase transition However, this does not reflect a

general stress response because there is no significant

target gene induction upon treatment with hydrogen

peroxide [6] HSF-independent mechanisms also exist,

namely the environmental stress response (ESR),

lar-gely mediated by the two transcription factors Msn2

and Msn4 [7] Here, we review the response of the

budding yeast S cerevisiae to several different forms

of stress highlighting, in particular, those in which the

Yap family of basic-leucine zipper (b-ZIP)

transactiva-tors play a role

The Yap protein family

The Yap family of b-ZIP proteins comprises eight

members with a significant sequence similarity to the

true yeast AP-1 factor Gcn4 at the DNA-binding

domain [8] However, in addition and common to all

family members, are several key residues that impart

distinct binding properties to these transcription

fac-tors It has been determined that Yap1 through to

Yap5 preferentially bind to the consensus site

TTAG⁄ CTAA, which differs from the true AP-1

recognition element bound by Gcn4 (TGAG⁄ CTCA)

It is unclear how many YAP sites are required for

tar-get gene regulation Work performed by Cohen et al

[9] indicates that gene clusters enriched for Yap1- and

Yap2-depedent genes have, on average, 1.9 (P-value

8.0· 10)4) and 1.8 (P-value 2.0· 10)3) consensus Yap

sites, respectively We cannot, however, exclude the

possibility that flanking bases around this core

consen-sus are also required In the case of Yap8, it has been

shown that this protein binds the sequence TTAATAA

on target gene promoters [10] (and our own results)

The Yap family has been found to be implicated in a

variety of stress responses including oxidative, osmotic,

arsenic, drug and heat stress, among others [11] Although much is currently understood about Yap1, the major regulator of the oxidative stress response, comparatively less is known about the remaining family members

Oxidative stress The response to oxidative stress can be described as the phenomenon by which the cell responds to alterations in its redox state As a consequence of aerobic growth, cells are continuously exposed to reactive oxygen species (ROS), potent oxidants capable of extensive cellular damage at the level of DNA, protein and membrane lipid content As a result, organisms, from bacteria to humans, have developed mechanisms of maintaining cellular thiol redox homeostasis This is achieved by lim-iting the accumulation of O2-derived oxidants, control-ling iron and copper metabolism, the activation of thiol redox pathways and via damage repair [12]

Yap1 was initially characterized through the obser-vation that the deletion mutant is hypersensitive to the oxidants H2O2 and t-BOOH, and to chemicals that generate superoxide anions, including menadione, plumbagine and methylviologen as well as to cad-mium, methylglyoxal and cycloheximide Recent gen-ome-wide studies have focused on modulation of the gene expression programmes that occur following exposure to an oxidative insult in S cerevisiae Indeed, Gasch et al [7] and Causton et al [13] have demon-strated that the response to mild doses of H2O2 leads

to the immediate and transient modulation of  24%

of the genome Although approximately half of this response can be attributed to the ESR, there is an

H2O2-specific response comprising genes encoding most cellular antioxidants and components of thiol redox pathways, heat shock proteins, drug transporters and enzymes involved in carbohydrate metabolism Among these genes are TRX2 [14] and GSH1 [15], two

of the first Yap1 targets to be characterized and induced under oxidative stress imposed by H2O2, di-amide and t-BOOH Since then, several Yap1 targets involved in ROS detoxification have been identified, including those involved in the thioredoxin and gluta-thione systems, and other antioxidants such as catalase and superoxide dismutase, among others Yap1 is therefore central to the adaptive response to oxidative stress, regulating not only the response to H2O2 -induced stress, but also that to chemical oxidants (redox cycling chemicals, thiol oxidants and alkylating agents), cadmium and drug stress Purified as a

90 kDa protein [16], Yap1 has a basal expression and,

in unstressed cells, shifts to and from the cytoplasm

via interaction of the Crm1 nuclear exportin with the

Yap1 nuclear export signal (NES) [17,18] Although

YAP1mRNA basal levels are enhanced upon exposure

to an oxidative stimulus, the control of Yap1 activity

is primarily regulated through subcellular localization

Indeed, Kuge et al [14] demonstrated that Yap1

nuc-lear retention is mediated by the cysteine-rich domain

(CRD) located at the C-terminus of the protein which

contains two cysteine-rich regions designated as the

n-CRD (C303, C310 and C315) and c-CRD (C598,

C620 and C629) (Fig 1A) In response to diamide, the

c-CRD is sufficient to mediate a response However, in

the case of H2O2, both n- and c-CRD regions are

required [17,19] How does Yap1 sense oxidative

stress? It has been shown that the oxidant receptor

peroxidase Orp1 (also designated Hyr1 and Gpx3), is

the main signal sensor and that a third component of

this signal relay, Yap1 binding protein (Ybp1) is

asso-ciated with Yap1 [20] Orp1 carries a conserved

peroxi-dase-active site cysteine residue (Cys36) of the Gpx

family, whose catalytic cycle is first oxidized to a

sulf-enic acid (Cys-SOH), and then reduced by GSH [18]

Orp1, however, contributes towards H2O2 resistance

not as a peroxidase, but as a sensor of oxidative

stress Orp1 activates Yap1 by forming an

intermole-cular disulfide bond between its Cys36 and the

Yap1 Cys598, which is then converted into the

Yap1 intramolecular Cys303-Cys598 disulfide bond (Fig 1B) Veal et al [20] have shown that Ybp1 is required for the signal transduction from Orp1 to Yap1 because in its absence the intermolecular disul-fide bond does not form It has been suggested that Ybp1 could act as chaperoning the formation of disul-fide bonds through the guiding of Orp1 Cys36SOH to Yap1 Cys598, and⁄ or preventing the formation of the competing Orp1 Cys36-Cys82 disulfide bond Once activated, the Yap1 NES that lies within the c-CRD is masked leading to its retention in the nucleus and the up-regulation of target genes Ybp2⁄ Ybh1, a protein homologous to Ybp1, was found in the genome of

S cerevisiae and described as having an effect on

H2O2 tolerance, through different mechanisms [21] However, these data should be regarded with caution because most of the conclusions are derived from indirect results These sensing mechanisms appear con-served in Schizosaccharomyces pombe in which a two-cysteine-based peroxidase functions in a similar way to Orp1 in the activation of Pap1, the Yap1 orthologue [22] In addition, a second Yap1 redox centre involved

in the direct binding of N-ethylmaleimide (NEM), the quinone menadione, both an electrophile and super-oxide anion generator, was shown to operate Under conditions favouring superoxide anion generation, Yap1

is activated by H2O2formed by the dismutation of the

A

B

Fig 1 (A) Comparison of the CRD of Yap1,

Yap2 and Yap8, NES is underlined (B) The

two Yap1 redox centres Under nonoxidizing

conditions, Yap1 is cytoplasmic owing to

Crm1-dependent nuclear export Upon H2O2

exposure, the formation of an intermolecular

bond occurs between the Orp1 Cys36 and

the Cys598 of Yap1 leading to its activation.

The subsequent formation of the Yap1

Cys303-Cys598 disulfide bond masks the

NES retaining it in the nucleus where it

activates target genes Under thiol-reactive

agents, and possibly the metalloids, a

second redox centre operates involving the

Cys598, Cys620 and Cys629 of Yap1, to

which the drug binds directly.

superoxide In contrast, menadione acts as an

electro-phile in the absence of oxygen and in this case binds

directly to the c-CRD Cys598, Cys620 and Cys629 in a

manner independent of the Orp1 pathway [23]

Metalloid and metal stress

The widespread distribution of the toxic metalloid

arsenic in nature leads to the acquisition of its

resist-ance in almost all living organisms [24,25] In S

cere-visiae, resistance to arsenic is achieved through the

activation of the arsenic compounds-resistance (ACR)

cluster [26], which is composed by the positive

regula-tor Acr1 (Yap8), the arsenate-reductase Acr2 and the

plasma membrane arsenite efflux protein Acr3 [27]

The yeast cadmium factor (YCF1) gene encodes an

independent detoxification system that operates by

sequestering As(GS)3 into the vacuole [28–30]

Induc-tion of the expression of ACR2, ACR3 and also YCF1

by the transcription factor Yap8 is essential to arsenic

stress response Like Yap1, Yap8 is constitutively

expressed, and under physiological conditions shuttles

to and from the nucleus [31] This is in contrast to the

results obtained by Wysocki et al [10] and may be due

to the fact that the latter use a multicopy vector,

whereas the former look at the green fluorescent

pro-tein construct within a normal chromosomal context

Under arsenic stress conditions, Yap8 is activated at

the level of its transactivation potential as well as its

nuclear accumulation, which is triggered by the loss of

interaction with Crm1 [31] Yap8 cysteine residues

Cys132, Cys137 and Cys274 are essential to both

pro-cesses (Fig 1A) Work by Haugen et al [32] on the

integration of phenotypic and expression profiles

involved in arsenic response has revealed the array of

genes whose transcription is enriched, including those

involved in methionine metabolism and sulfur

assimil-ation, protein degradation and transcriptional

regula-tion, and by proteins that form a stress response

network, including Fhl1, Msn2 Msn4, Yap1, Cad1

(Yap2), Hsf1 and Rpn4 among others Furthermore,

results obtained in microarray analyses point towards

the existence of further Yap8-mediated arsenic

detoxifi-cation pathways (C Amaral, F Devause, R Menezes,

C Facq & C Rodrigues-Pousada, unpublished

observa-tions), highlighting the relevance of multiple

mecha-nisms of arsenic management A distinct detoxification

strategy employed by S pombe, nematodes and plants

makes use of phytochelatins (PCs) for metalloid

che-lation The observation that overexpression of the

S cerevisiae ACR3-encoded arsenite transporter not

only complements the lack of phytochelatins in

S pombe, but also confers hyper-resistance to arsenic

compounds to the levels observed in the budding yeast and prokaryotes [33] further accentuates the effective-ness of this pathway in arsenic detoxification Yap1 activation by arsenic compounds is similar to its acti-vation by thiol-reactive chemicals [23] because it is unaffected by the absence of the sensor Orp1⁄ Gpx3 and does not depend on the n-CRD cysteines (Fig 1B) In contrast to Yap8, under arsenic stress conditions YAP1 basal expression is slightly enhanced and the presence of this metalloid does not signifi-cantly modulate Yap1 transactivation function [11] Heavy metals including copper, zinc, iron and man-ganese play an important role in cellular biochemistry and physiology [34] However, when the concentration

of these metals is elevated, toxicity arises for the organ-isms Although cadmium and mercury are not essential metals they cause severe damage even in low amounts Organisms therefore possess cellular detoxification mechanisms that maintain homeostasis through the con-trol of intracellular ion levels One of these involves the activation of Yap1 and Yap2 (Cad1) [9,35,36] Yap2 overexpression confers resistance to a plethora of stress agents such as cadmium, cerulenin and 1,10-phenanthro-line among others, suggesting a role in the response to drug stress Indeed, several target genes encoding a set of proteins involved in the stabilization and folding of pro-teins in an oxidative environment have been identified by microarray analyses [9] Induced upon exposure to cad-mium stress [8], Yap2 re-localizes to the nucleus via a Crm1-dependent mechanism, where it activates the tran-scription of its target gene FRM2, encoding a protein homologous to nitroreductase, whose precise role in the metal stress response remains unclear The strong sequence homology between Yap2 and Yap1 in the C-terminal CRD (residues 570–650 in Yap1 and 330–409

in Yap2) was used to further provide an insight into the function Yap2 Domain swapping of the Yap1 c-CRD

by that of Yap2 has shown that the fusion protein is regulated by cadmium but not by H2O2 (D Azevedo

& C Rodrigues-Pousada, unpublished data) Nuclear localization of the fusion protein correlates not only with activation of FRM2 transcription, but also with growth

in increasing concentrations of cadmium but not of

H2O2 Because of the high degree of homology to Yap1, the role of the Yap2 cysteine residues may prove relevant for its activation Furthermore, it has been shown that Yap2 interacts with the cytoplasmic kinase Rck1 under conditions of oxidative stress [37], although the nature and relevance of this interaction remain elusive Given that overexpression phenotypes do not necessarily reflect

a true biological function and that no phenotype has yet been associated to the yap2 mutant, the precise role for Yap2 remains to be deciphered

Osmotic stress

Hyperosmotic stress leads to the passive efflux of water

from the cell to the exterior, resulting in a decrease in

cell volume, loss of cell turgor pressure and increased

concentration of cellular solutes Conversely, an

aque-ous hypo-osmolar environment allows the movement

of water into the cell, leading to cell swelling, high

tur-gor pressure and diluted intracellular milieu [38,39]

To counteract these effects, the cell makes use of

osmolytes, small compatible solutes such as the sugar

alcohol glycerol and trehalose, which, via active

accu-mulation or extrusion, protect the cell against the

effects of an osmotic challenge by altering the

intracel-lular osmotic pressure [40,41] Many of the changes to

gene expression upon an osmotic challenge, therefore,

are dedicated to altering the metabolism and cell

per-meability to these compounds

Upon an upshift in extracellular osmolarity, the high

osmolarity glycerol (HOG) mitogen-activated protein

(MAP) kinase pathway is activated via the action of

two membrane-bound receptors, Sho1 and Sln1 that

form two independent signal input branches

conver-ging on the MAP kinase kinase (MAPKK) Pbs2 The

increased sensitivity of the Sln1 branch, as well as its

graded response [42], disregards the notion of pathway

input redundancy suggesting a capacity of the cell for

finely sensing and adjusting to external changes Upon

Pbs2 phosphorylation, the Hog1 kinase is activated

through dual Thr⁄ Tyr phosphorylation, promoting its

rapid translocation into the nucleus and increasing its

kinase activity [43] Hog1 nuclear residence is regulated

by the two tyrosine phosphatases Ptp2 and Ptp3 [44],

three phosphoserine⁄ threonine phosphatases Ptc1–3

and by several of the transcription factors it interacts

with, namely, Msn2⁄ 4 [45], Hot1 and Msn1 [46], which

subsequently mediate signal amplification via the

tran-sient modulation of global gene expression, often with

overlapping functions [47] In all, the expression of

 10% of the yeast the genome is affected by an

osmo-tic upshift This includes most of the genes typically

induced by the ESR, many of which show

Hog1-dependent gene expression [47] and a reduced number

of osmo-specific gene responses, comprising genes of

unknown function Altogether, Hog1 has been shown

to regulate not only genes required for the immediate

response to increased osmolarity, but also for the

res-toration of gene expression upon osmo-adaptation,

controlling the extent of gene expression as well as its

duration [48] Recently, Hog1 has been found to be

located at several gene target promoters through

association with the transcription factors it interacts

with [49,50] Deletion of the HOG1 gene gives rise to a

severe cellular sensitivity to increased external osmo-tica [51], whereas HOG1 pathway hyperactivation is lethal [42,52] highlighting not only the importance of this pathway to the yeast response to increased osmo-larity, but also the absolute requirement for cellular mechanisms that accurately measure and grade the response without compromising cell viability

Msn2 and Msn4 are two zinc-finger transcription factors initially described as mediators of the yeast general stress response [53] because of their capacity to jointly modulate the expression of a large battery of unrelated genes in response to a shift to suboptimal growth conditions Regulation is mediated by the bind-ing of these factors to the stress response element (STRE) (C4T) [54,55] present on the promoter of tar-get genes Cytosolic, under normal growth conditions, Msn2⁄ 4 rapidly accumulate in the nucleus under stress conditions in a manner that can be inversely correlated

to protein kinase A (PKA) activity [56,57] The Msn5 exportin contributes towards Msn2 nuclear retention through recognition of its phosphorylation state [58] Furthermore, work by Bose et al [59] revealed that the initial burst of stress-induced STRE-driven gene expression is quickly converted into the observed tran-sient response through Msn2 nuclear-dependent degra-dation and target gene transcriptional repression by Srb10 kinase, a member of the mediator complex The magnitude of target gene induction varies greatly from gene to gene, primarily due to promoter context, whereby STRE-driven regulation can be jointly modu-lated by other transcription factors including Yap1 and Hot1 [7,47] Induction of the Msn2⁄ 4 target genes

in response to one form of stress gives rise to the phe-nomenon of cross-protection against an aggravated form of the same stress or to a different type of envi-ronmental insult altogether

That Msn2⁄ 4 form a downstream branch of the HOG MAP kinase pathway under conditions of hyper-osmolarity can be inferred from the fact that, although many Hog1-dependent genes do not show Msn2⁄ 4 dependence, virtually all genes affected by the absence

of these factors are also affected by the deletion of HOG1 [47] Indeed, it has been shown that YAP4, induced under hyperosmotic stress, is regulated by Msn2 in a Hog1-dependent way via STRE located within the upstream promoter region (Fig 2) [60] The observation that, under these conditions, YAP4 is not regulated by Msn4, further supports growing evidence that the two zinc-finger transcription factors are not entirely redundant in function [59] Yap4 and Yap6 are constitutively located in the nucleus [61] and are the Yap family members that share the greatest simi-larity at the protein level with almost 33% identity

between them [11] Although no significant sensitivity

can be observed in the yap6 null mutant, the

yap4-deleted strain displays impaired growth at moderate

concentrations of hyperosmolarity [60] Furthermore,

YAP4 overexpression can significantly relieve the

severe hog1 salt-sensitive phenotype, indicative of a

role in the yeast response to increased osmolarity

Although its precise function remains unclear,

micro-array analyses revealed that this transcription factor

contributes towards the regulation of several

osmo-induced genes Of significance, genes involved in

glycerol metabolism, GCY1 encoding a putative

glycerol dehydrogenase and GPP2, encoding a

NAD-dependent glycerol-3-phosphate phosphatase, show

decreased expression in the YAP4-deleted strain

(Fig 2) Crucial to osmo-tolerance, glycerol

metabo-lism and accumulation form a relevant part of the

yeast response to hyperosmolarity [39] Indeed, a

heat-shock-stimulated increase in the level of intracellular

glycerol is sufficient to completely abolish hog1

sensi-tivity to hyperosmotic stress [62] HXT5, encoding a

hexose transporter also partially regulated by Yap4,

shows a further decrease in gene expression in the

dou-ble yap4yap6 mutant strain, suggesting cooperation

between these two transcription factors in mediating

the stress response This is further substantiated by

computational interactome data that predict their interaction [63] Interestingly, the observation that Yap4 and Yap6 are induced by a variety of unrelated forms of environmental stress [11,64] has hinted towards a more fundamentally universal role for Yap4 and Yap6 in the yeast response to stress which is in contrast to what is currently understood for the remaining family members

Perspectives

A particularity of the yeast S cerevisiae is that is pos-sesses an extended family of Yap transcription factors

S pombe Pap1 shares a high degree of similarity to Yap1 However, multiple environmental insults in

S pombeactivate the Sty1-mediated MAP kinase path-way, itself strongly homologous to Hog1 and to mam-malian p38, making this pathway more analogous to higher eukaryotes [39,65,66] Although Yap1 and Yap8 orthologues exist in the genomes of several other Saccharomyces species [67], the remaining Yap mem-bers appear to be exclusive to this microorganism, hinting towards the possibility that this extended fam-ily arose through gene duplication events to fulfil a wider genetic programme required for its environmen-tal adaptation Experimenenvironmen-tal data support both a func-tional overlap as well as distinct biological roles for this protein family [11] endowing S cerevisiae with an added flexibility with regards to sensing and grading its stress response Furthermore, data are beginning to emerge on the cross-talk between several members of this family In particular, the double mutant yap1yap2

is more sensitive to cadmium as well as the double mutant yap1yap8 to metalloid than either single mutant, respectively Indeed, several studies are emer-ging with data supporting the condition-specific cooperation between distinct sets of transcriptional modulators in target gene regulation [32,68] As was recently shown by studies using benomyl [69], it is possible that other chemical stresses also affect the early expression of genes dependent on different tran-scription factors Also, it cannot be neglected that cells respond to different stresses, for instance, those produ-cing different oxygen species [70] using distinct mecha-nisms, pointing to the selective use of different transcription factors, different combinations or differ-ent mechanisms of their activation The fact that YAP4 is responsive to a plethora of environmental insults, allied to the richness of cis-elements in its pro-moter, suggests an important role in response to stress Given that Yap4 overexpression gives resistance to cis-platin, a chemotherapeutic drug that binds the TATA box [71], it is plausible to hypothesize that Yap4 may

Fig 2 Yap4 is under the HOG pathway Upon exposure to osmotic

stress the nuclear accumulation of Hog1 activates downstream

transcription factors YAP4 is activated via Msn2 and subsequently

the encoded factor elicits the transcription of its target genes.

play a role in the realm of the basic transcriptional

machinery The construction of a strain deleted for all

Yaps in well-defined background may prove an

invalu-able tool for the functional study of each family

mem-ber Indeed, it is being shown that the various strains

not only have different sensitivities to the stress

imposed, but also that significant differences occur at

the level of gene regulation

Acknowledgements

This study was supported by grants from Fundac¸a˜o

para a Cieˆncia e Tecnologia (FCT) to CR-P (POCTI⁄

BME34967⁄ 99), fellowships to RAM (SFR ⁄ BPD ⁄

11438⁄ 2002) and to TN (SSRH ⁄ BD ⁄ 1162 ⁄ 2000)

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