MOLECULAR AND CELLULAR BIOLOGY, Oct. 1990, p. 5087-5097 Vol. 10, No. 100270-7306/90/105087-11$02.00/0Copyright © 1990, American Society for Microbiology

Multiple Positive and Negative cis-Acting Elements Mediate InducedArginase (CARl) Gene Expression in Saccharomyces cerevisiae

LADISLAU KOVARI, ROBERTA SUMRADA, IULIA KOVARI, AND TERRANCE G. COOPER*

Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163

Received 30 April 1990/Accepted 5 July 1990

Expression of the arginase (CARI) gene in Saccharomyces cerevisiae is induced by arginie or its analoghomoarginine. Induction has been previously shown to require a negatively acting upstream repressionsequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of thiswork was to identify the cis-acting elements responsible for CAR] transcriptional activation and response toinducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in aheterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas thethird functions only when inducer is present. One of the inducer-independent UAS elements exhibits significanthomology to the Spl factor-binding sites identified in simian virus 40 and various mammalian genes.

Production of arginase (encoded by CAR]) is inducedwhen a small molecule inducer, arginine or its analog ho-moarginine, is exogenously provided to cultures of Saccha-romyces cerevisiae (3, 20-23, 36). Production ceases whencells are grown in the presence of a readily metabolizednitrogen source such as glutamine or asparagine; i.e., en-zyme production is sensitive to nitrogen catabolite repres-sion (NCR) (3, 6, 21-23). Conditions of induction and NCRhave been correlated with marked increases and decreases insteady-state levels of mRNA, respectively, suggesting thatinduction and NCR occur at transcription (5, 26).Two cis-dominant mutations, which result in high-level

arginase production in the absence of inducer, have beenisolated (17). They differ in phenotype by their response toNCR. Arginase production in a CAR1-0- strain was sensi-tive to NCR, whereas in a CAR1-0h strain it was resistant.Similar phenotypes were observed for steady-state CAR]mRNA levels (26). The CARJ-O' mutation was demonstratedto be a Ty insertion immediately upstream of the CAR]TATA sequence (17, 30, 31). The second mutation (CAR1-0-) was shown to be a single-base transversion at position-153 in the CAR) 5'-flanking region (28, 30, 31). A deletionremoving CAR) DNA between positions -158 and -146generated the same phenotype as the point mutation at-153, leading to the conclusion that this sequence was anegatively acting element designated an upstream repressionsequence (URS) (28, 30, 31).This interpretation of the data was supported by demon-

strating that insertion of a 13-base-pair (bp) DNA fragment,containing positions -158 to -146 of CAR), 3' to the CYCIupstream activation sequence (UAS) resulted in inhibition ofits ability to support transcriptional activation (29). Althoughthe CAR) URS was able to inhibit transcription in bothhomologous and heterologous vectors, its apparent responseto arginine was different in the two constructions. Whensituated in its native position upstream of the CAR] codingsequence, the CAR] URS was unable to function underconditions of induction; i.e., a wild-type promoter and onewith a 13-bp URS deletion supported the same level of geneexpression in cells grown under conditions of full induction.This inducer-mediated neutralization of URS function was

* Corresponding author.

not observed when the CAR) URS-containing DNA frag-ment was cloned downstream of the CYC) UAS (29). Fromthis we concluded that the CAR) URS-mediated functionwas cis-dominantly regulated in response to the presence ofinducer. A positively acting element (UAS) was also re-ported between positions -190 and -324 upstream of CARI(6, 8, 9, 28, 31). To extend these observations and addressthe question of cis-dominant regulation of the CAR) URS,we dissected the CARI 5' region in detail.(A preliminary report of this work has appeared [L.

Kovari and T. G. Cooper, Abstr. Yeast Genet. Mol. Biol.1989 Meet., p. 159A].)

MATERIALS AND METHODSStrains and media. S. cerevisiae RH218 (MATa trpl CUP]

gal2 SUC2, Mal-) was used throughout this work. Cloningin Escherichia coli was performed with either strain HB101(hsdR recA13 supE44 lacZ24 leuB proA thi-), Sm') or SURE[recB recJ sbcC201 uvrC umuC mcrA mcrB mrr lac AhsdRM5 endA) gyrA95 thi relA) supE44 (F'proAB lacIq ZAM15, TnJO)] (Stratagene). Yeast and E. coli cells were grownfor transformation as described earlier (26). For P-galactosi-dase assays, yeast cultures were grown in Difco YNBmedium containing 0.17% yeast nitrogen base without aminoacids but with ammonium sulfate, 2% glucose, and 0.1%glutamate or 0.1% arginine.

Yeast transformation and 0-galactosidase assay. E. coli andyeast transformation procedures have been described earlier(26). Since all of the plasmids used in this work contained anautonomously replicating sequence, we took precautions toavoid problems that might result from a varying copy num-ber. All of the plasmids used in a given figure or table weretransformed into the same sample of host cells, which wereassayed at the same time. Random transformants were usedas soon as they were large enough to serve as inocula. Theseinocula were grown up and assayed immediately; transfor-mants were never subcultured or stored. Each experimentwas performed at least twice with different transformants,and all assays were performed in duplicate. Data fromrepeated experiments generally varied less than 15%, anddata from duplicate assays varied less than 5%. For 0-galac-tosidase activities less than 100 U, the variation was slightlyhigher. The effect of varying the copy number has beenevaluated in the past and found not to be an influencing

5087

5088 KOVARI ET AL.

factor when the procedures described above were followed(19, 25, 27, 38).

,-Galactosidase activities were determined by using yeasttransformants grown to Awo of 0.65 to 0.75 (Gilford Re-sponse spectrophotometer); 25 ml of culture was used forassay. Cell breakage and P-galactosidase assays were per-formed by the method of Guarente (15), and activity wasexpressed in units defined by Miller (24).

Plasmid constructions. The plasmids used in this work areshown schematically in Fig. 1. Plasmid pRS46 (29) waslinearized by digestion with BamHI at position -512 up-stream of the CAR] coding sequence, and an XhoI linker(GCTCGAC) was ligated at this position (plasmid pRS52).To generate deletion plasmids, 20 ,ug of this plasmid (pRS52)was linearized with XhoI and digested with 1.7 U of nucleaseBAL 31 at 20°C in a buffer consisting of 20 mM Trishydrochloride (pH 8.0), 1 mM EDTA, 12.5 mM MgSO4, 12.5mM CaCl2, and 0.45M NaCl. Following 2, 5, 10, and 15 minof incubation, samples were mixed with phenol and pooled.The resulting DNA was phenol extracted and resolved on an0.8% agarose gel, and high-molecular-weight DNA wasrecovered by electroelution. 32P-labeled Xhol linkers wereligated onto the ends of approximately 10 ,ug of this DNA,and the resulting ligation mixture was used to transform E.coli. DNA from the E. coli transformants was digested withBamHI and XhoI to identify the desired clones. PlasmidDNA from clones identified to potentially contain deletionsof 100 to 500 bp were digested with XhoI and Sacl. TheXhoI-SacI deletion fragments were then recloned into theoriginal vector (plasmid pRS52), which had been linearizedwith these restriction enzymes. The endpoints of each dele-tion were determined by Maxam-Gilbert DNA sequenceanalysis (30).

Oligonucleotides used in this work were synthesized on anApplied Biosystems model 380B oligonucleotide synthe-sizer. SalI and EagI sites were synthesized on the 5' and 3'ends of the inserts, respectively, to facilitate cloning of theDNA fragment in the desired, native orientation. Oligonu-cleotides were purified on polyacrylamide-urea gels, visual-ized by UV shadowing, recovered by extraction, and de-salted with Sep-Pak cartridges (Waters Associates, Inc.) byusing standard cloning methods (2). The purified single-stranded oligonucleotides were dissolved in annealingbuffer. Equal amounts (30 ,ug) of complementary oligonucle-otides dissolved in annealing buffer were combined, heatedto 95°C for 10 min, and slowly cooled to room temperature.The annealed DNA fragments were ethanol precipitated,dissolved in ligation buffer, and ligated into the expressionvector. Plasmid pNG15 was linearized by sequential diges-tion with EagI and Sall (Fig. 1B). The resulting two frag-ments were separated on agarose gels, and the large frag-ment was recovered by electroelution. Ligation wasperformed at a 1,000:1 molar ratio of insert to linearizedvector. All constructs were verified by DNA sequenceanalysis (32).

RESULTS5' deletion analysis of the CAR] 5'-flanking region. We

initiated the search for cis-acting elements in the CAR]

A

B

EcoRI

XhoI EagI Sail XbaI

C

FIG. 1. Structures of plasmids used in this work. (A) PlasmidpRS46, used for 5' deletion analysis (29). (B and C) Expressionvector plasmids used to assay CAR] sequences for their ability tosupport transcriptional activation (pNG15) or repression of tran-scriptional activation mediated by a heterologous (CYCI) UAS(pNG22).

EcoRI EcoRI

/A pr \/ URA3\\R

(pMB1 X L~~~SmaI_iori pNG22 H3

EcoRI -

lacZ\\ CYCI-UAS -"00/

+4 , Destroyed

BammHIpolylinkerin XhoI

TCGAC!TAAA CGACT GCCGA

G.CATTTCA GCAGCCGT TG

XbaI Sall EagI XhoI

CAR15' RegulatoryRegion

MOL. CELL. BIOL.

cis-ACTING ELEMENTS NEEDED FOR CAR] EXPRESSION 5089

-1550 -450 -350 -250 -150 -50 1

0 1 1 III 1 E n i T T I-516I-

B-GALACTOSIDASE

GLU ARG

i~~ T T 528

pRS124 | L I-369

rA ri T T

pRS86 LIII-344

11 [A

pRS84 p-231

MN T T

MI T T

T TpRSI122 Nu225 2-225

pRS77 L-_219

pRS123 L-190

pRS81 I Ng-185

pRS82-178

pRS80

RN T T

NM T T

T T

T T

T T

FIG. 2. 5' deletion analysis of the CAR] upstream region. The areas designated A, *, and 3 represent three sets of homologoussequences which, at the end of this report, will be concluded to be the sites of cis-acting elements mediating CAR] expression and its

regulation. The area designated I represents the CAR] URS (19, 29, 30). T's indicate the positions of TATA sequences. Numbers at the

left of each insert indicate the 5' terminus of the remaining CAR] DNA in the CARI-lacZ fusion. Coordinates are indicated relative to the

start of translation in this and all subsequent figures.

upstream region by constructing a CARI-lacZ fusion plas-mid (pRS46) and generating a set of nested 5' deletionplasmids from it as described in Materials and Methods. Thepatterns of P-galactosidase production supported by thevarious CAR]-lacZ deletion plasmids were complex (Fig. 2).The parental plasmid (pRS46), which contained the CAR)5'-flanking region to position -516, supported lacZ expres-sion that was 12-fold induced by arginine, a response similarto that observed for CARl-specific mRNA. Deletion ofDNAbetween positions -516 and -401 resulted in decreased,-galactosidase production regardless of the nitrogen source

provided (Fig. 2, plasmid pRS124). However, the percentageof activity loss was greater for the uninduced culture than forthe induced one; therefore, plasmid pRS124 supported 48-fold induction compared with 12-fold induction for plasmidpRS46. The deletion in plasmid pRS86 resulted in a smalldecrease in the induced level of ,B-galactosidase production;we could not ascertain whether this decrease was physiolog-ically significant. The next two deletions (plasmids pRS126and pRS85) resulted in 25 and 46% decreases in induced lacZexpression, respectively. Deletion of the next 74 bp ofDNA(plasmid pRS84) generated the greatest loss of activity in thisseries of plasmids. Induced P-galactosidase activity de-creased 12-fold. However, the remaining lacZ expressionwas highly inducible, indicating that DNA 3' to position-251 still contained sufficient information to support in-

duced gene expression. Plasmids pRS122 and pRS77 sup-ported similar levels of P-galactosidase production, whichwere 60 to 80% greater than observed with plasmid pRS84.Deletion of the next 6 bp ofDNA (plasmid pRS123) resultedin a 30% loss in the remaining induced activity. The nextdeletion (plasmid pRS81) resulted in loss of detectable lacZgene expression. The last two deletion plasmids (pRS82 andpRS80) yielded the same results as plasmid pRS81.The above patterns of expression are similar, in formal

terms, to those observed during analysis of the DAL7 genepromoter (38); this gene is required for growth on allantoinas the sole nitrogen source. In that case the complex patternof expression is due to sequential loss of repeated cis-actingtranscriptional regulatory elements as successive deletionsremove increasing amounts of the DAL7 promoter region.We proceeded on the premise that a similar situation mightbe occurring in CAR].Our first objective was to determine whether synthetic

DNA fragments, covering individual regions shown by thedeletion analysis to be required for gene expression, couldalone support reporter gene expression when cloned into a

heterologous expression vector lacking a UAS element(plasmid pNG15). The five fragments that we synthesizedcovered nearly all of the CAR] 5'-flanking region upstreamof the URS at position -160 (Fig. 3). Three of the fivefragments (plasmids pLK81, pLK70, and pLK40) supported

pRS46 I I I-401

_tII 6,228

pRS126 III 11 2 m m m-324

pRS85 | m NE i-251

94 4,542

87 4,157

26 3,134

18 1,682

2 145

0 232

4 261

4 184

2 0

0 9

2 8

T T

T T

VOL. 10, 1990

5090 KOVARI ET AL.

-550 -450 -350 -250 -150

Em i 1 1 M NS T |

B-GALACTOSIDASE

-516

pLK81 L mu-401 GLU ARG

338 346

-402 -360

pLK29 13 28

-369 -344

pLK1 a

-344

pLK70 ii

56 166

-251

II I-231 -1 60

pLK40 |

pNG15 (Vector Only)

FIG. 3. ,-Galactosidase production supported by plasmids containing synthetic DNA fragments covering portions of the CAR] 5'regulatory region. Coordinates above each diagrammed insert, in this and all subsequent figures, indicate positions of the CARI sequences

contained in the synthetic oligonucleotides that were cloned into the vector, pNG15 in this case.

lacZ expression that was more than ninefold over back-ground. 1-Galactosidase production in one of the transfor-mants (plasmid pLK40) was highly induced (70-fold) byarginine, whereas in the others the response to inducer wasonly 2- to 3-fold higher than observed for the vector alone orone containing the CYCJ UAS. We could not eliminate thepossibility that increased levels of P-galactosidase produc-tion in glucose-arginine medium were due to higher rates ofgrowth and, hence, synthetic capacity on arginine mediumthan on glucose-glutamate medium; therefore, we did notconsider 10-fold or less response to arginine to representphysiologically significant induction. Although the use ofsuch a stringent criterion for induction might have resulted infailure to identify sequences supporting low-level induction,it minimized the possibility of misidentifying secondaryeffects of the nitrogen source for physiologically significantinduction.

Localization of UAS elements in the CAR) upstream region.DNA fragments shown in Fig. 3 and others related to themwere analyzed to locate the UAS elements more precisely.The first region considered was between positions -516 and-401 (Fig. 4). We constructed plasmids containing 5' and 3'deletion fragments cloned into the expression vector pNG15.The parental plasmid (pLK81) supported similar ,B-galactosi-dase production in the presence and absence of inducer (Fig.4). Deletion of sequences -516 to -470 (plasmid pLK82)resulted in complete loss of activity, indicating that the 5'terminus of sequences needed for lacZ expression wasbetween these positions. Deletion of sequences -401 to-415 and -415 to -424 resulted in a modest increase inreporter gene expression under both assay conditions (Fig.4, plasmids pLK120 and pLK121). We did not investigatewhether this increase derived from loss of a negatively actingsite, because the effect was modest. The next deletion(plasmid pLK114) resulted in a ninefold loss of P-galactosi-dase production in transformants grown in glucose-argininemedium; undetectable levels of P-galactosidase productionwere observed when these transformants were grown in

glucose-glutamate medium. Deletion of 10 bp or more re-sulted in the loss of all remaining P-galactosidase produc-tion. These data suggested that the 3' terminus of sequencesnecessary for 3-galactosidase production in plasmid pLK81was situated between positions -424 and -460. In agree-

ment with these results, a DNA fragment containing se-quences between -483 and -424 supported high-level a-ga-lactosidase production (Fig. 4, plasmid pLK123).The second region analyzed (Fig. 5, plasmid pLK70)

included two regions whose deletion resulted in markedlosses of lacZ expression (Fig. 2, plasmids pRS126 andpRS85). Here, the parental plasmid (pLK70) supportedhigh-level reporter gene expression in both glucose-gluta-mate and arginine media. Deletion of 5 bp resulted in ameasurable loss of ,B-galactosidase production, which be-came even greater upon deletion of the next 5 nucleotides(Fig. SA, plasmids pLK71 and pLK72). Deletion plasmidspLK74, pLK75, pLK76, pLK77, pLK78, and pLK79 sup-ported levels of ,-galactosidase production that were rea-sonably similar to those observed with plasmid pLK72. Theshortest DNA fragment (19 bp, plasmid pLK79) still sup-ported high-level ,-galactosidase production, i.e., 5- to11-fold over background (plasmid pNG15) in both media.

3' deletion of DNA sequences -251 to -270 resulted in athree- to fourfold loss of P-galactosidase production (Fig.SB, plasmid pLK88). Deletion of the next 10 bp (plasmidpLK89) resulted in loss of lacZ expression to a backgroundlevel. Plasmids pLK91 and pLK92 yielded similar results.Deletion of sequences -314 to -324 (plasmid pLK93) re-sulted in a doubling of ,-galactosidase production. Thisproduction increased a further 1.8-fold when the next 5 bpwas deleted (plasmid pLK94).These data suggested that the 5' and 3' termini of pLK70

sequences required for UAS activity were between positions-344 and -339 and positions -251 and -270, respectively.The existence of seven 5' and three 3' deletion plasmids thatsupported roughly the same levels of P-galactosidase pro-duction was consistent with the suggestion that two sets of

360 1,187

9 650

16 60

MOL. CELL. BIOL.

cis-ACTING ELEMENTS NEEDED FOR CAR] EXPRESSION

U-470

BE

-401 GLU ARG1J338 346

I 19 1750

IN-430

pLK84-420

pLK85

-401

-415

-424-, .,. ,-. , -

460

pLK114-470

pLK1151 *-480

pLK116 1pLK117 | lIII

-483pLK123 IiLI Ii i

-424

I

pNG15 (vector only) 6 48

FIG. 4. P-Galactosidase production supported by plasmids containing synthetic DNA fragments, covering the CAR] regulatory regionbetween positions -516 and -401, that were cloned into the expression vector pNG15.

sequences were associated with the UAS activity supportedby plasmid pLK70, one set being located on either end of theDNA fragment. The results obtained with plasmids pLK94and pLK78 were consistent with this interpretation (Fig. 5).We did not investigate whether the increased P-galactosi-dase production observed with plasmids pLK93 and pLK94compared with that observed with plasmid pLK92 derivedfrom deletion of a negatively acting site, because the effectwas modest.The last region analyzed was between positions -231 and

-147. The parent plasmid (pLK39) would not support lacZgene expression (Fig. 6A). The first deletion (plasmidpLK40) removed 13 bp of DNA and resulted in the appear-ance of 72-fold inducible lacZ expression. This result wassurprising at first, because sequences deleted in plasmidpLK40 were previously shown to contain the CAR] URS, anelement shown to be required for induced CAR] expression(29). Taken together, data from plasmids pLK39 and pLK40argued that the reason why plasmid pLK39 was unable tosupport gene expression was inhibition of the UAS elementsit contained by the CAR] URS at positions -156 to -148(29). These data also showed that, in isolation, positions-160 to -231 were sufficient to mediate 72-fold inducibleexpression of the reporter gene. The next deletion (plasmidpLK41) removed 16 bp and resulted in a decrease in lacZexpression to background levels. The same results were alsoobserved for plasmids pLK42, pLK43, and pLK44. Thesedata indicated that positions -160 to -177 were required forthe lacZ expression observed with plasmid pLK40.A 5' deletion analysis of this region began with plasmid

pLK40 containing positions -231 to -160 and therebylacking the URS element (Fig. 6B). Deletion of positions-231 to -216 (plasmid pLK49) resulted in a 66% decrease in

the induced level of lacZ expression. The remaining activity,however, continued to be highly inducible, indicating thatthe sequence from -216 to -160 contained sufficient infor-mation for induction. The next deletion (plasmid pLK50)removed 17 bp of DNA and resulted in loss of the ability ofthe plasmid to support gene expression regardless of thegrowth conditions. These results, combined with those fromthe 3' analysis, indicated that positions -216 to -160 were

the minimal ones required for induced reporter gene expres-sion. These experiments did not, however, permit us toascertain whether this region contained more than one

element.Localization of CAR] sequences mediating the response to

inducer. Our next objective was to subdivide the DNAfragment carried in plasmid pLK40 if possible and to identifyan element associated with the response to inducer. Theelement mediating the response ofDAL7 gene expression toinducer had little if any ability on its own to supporttranscriptional activation of an expression vector reportergene. Rather, it enhanced transcriptional activation sup-ported by a UAS placed adjacent to it (38; J. Daugherty andT. G. Cooper, submitted for publication). Our task here wasto discover whether the CAR] 5'-flanking region containedone or more elements with these characteristics.The first array of DNA fragments we considered were

those shown in Fig. 6 (see above). These data demonstratedthat the termini of DNA sequences needed to support bothtranscriptional activation and response to the inducer were-231 to -216 and -160 to -176. If all sequences betweenthe termini were required, the cis-acting site would be 40 to71 bp long. From work reported on the binding sites ofwell-characterized transcription factors, we concluded thatthis was probably too long for a single site. Moreover, DNA

A-516

pLK81 t

B-GALACTOSIDASE

pLK82 u

-4

pLK83

16

516

B-5

pLK81

pLK1 20

pLK1 21

10

13

22

20

21 23

338 346

480 943

621 1,249

18 139

2 38

1 4

9 17

312 681

VOL. 10, 1990 5091

In

5092 KOVARI ET AL.

13-GALACTOSIDASE

-251 GLU ARG

2 360 1,187

7 222 838

-1 84 521-324

1 107 619

j 87 430

-304

pLK76 L-290

pLK77 t

I 191 690

-280

pLK78

-270

pLK79

pNG15 (vector only)B -U4

I 59 470

254

137

635

623

27 55

-251l

pLK7o Linin-270

360 1,187

pLK88

pLK89

pLK91

-I28-28093 266

45 64-304

65 55-314

pLK92-324

pLK93 Z1Ui-p329

pLK94

pNG15 (vector only)

44 99

45 214

65 382

10 65

FIG. 5. ,1-Galactosidase production supported by synthetic DNA fragments, covering the CAR] regulatory region between positions -344and -251, that were cloned into the expression vector pNG15.

fragments carried in plasmids pLK42 and pLK51 were

unable to support lacZ gene expression. However, when theDNA fragments they carried were combined (plasmidpLK40), the resulting DNA fragment supported 72-foldinducible ,-galactosidase production. From these observa-tions, we hypothesized that the DNA fragment in plasmidpLK40 contained more than one cis-acting site and that atleast one of these sites was associated with a response toinducer. We tested this hypothesis by using the array ofDNA fragments shown in Fig. 7. Plasmid pLK70 was shownto carry a UAS whose function did not require an inducer(see above). Plasmid pLK80, containing the 5' half of theDNA fragment carried in plasmid pLK40, did not supportreporter gene expression above background levels. How-ever, when the DNA fragments carried on these two plas-mids were combined, 43-fold inducible ,-galactosidase pro-duction resulted. In other words, the DNA fragment carriedon plasmid pLK80 did not support UAS activity itself, butconferred a response to inducer on the UAS activity sup-ported by the DNA fragment carried in plasmid pLK70 whenplaced adjacent to it. These were characteristics reported forthe inducer-responsive element of the DAL7 gene (38).

Similar results were observed for the DNA fragments carriedin plasmids pLK78, pLK80, and pLK105 (Fig. 7B). Fromthese experiments we concluded that positions -251 to -190were sufficient for a response to inducer and that the DNAfragment carried in plasmid pLK40 therefore did containmore than one cis-acting element.

If the data from Fig. 6B and 7 are considered together, oneconcludes that positions -251 to -190 confer a response toinducer in two different ways. In plasmid pLK40 positions-231 to -190 apparently conferred a response to inducer bymaking the putative element situated between -199 and-160 competent to activate transcription. In contrast, posi-tions -251 to -190 conferred inducibility on transcriptionalactivation supported by plasmids pLK95 and pLK105 bymediating repression of transcriptional activation when theinducer was absent. Inducibility observed with plasmidspLK95 and pLK105 was high compared with that observedwith plasmids pLK70 and pLK78 because the level of geneexpression observed in glucose-glutamate medium (absenceof induction) decreased much more in the latter plasmidsthan in the former. To determine whether this characteristicwas demonstrable in a heterologous assay system, we cloned

A-344

pLK70 t33-339

pLK71

pLK72 Lu

pLK74 L-314

pLK75 L

MOL. CELL. BIOL.

cis-ACTING ELEMENTS NEEDED FOR CAR] EXPRESSION 5093

A -231

1o_cppLK39U

pLK40 MI

pLK41

pLK42 1

pLK43 R-212

pLK44~9W

-188

-160

-176

-1-197

pNG15 (vector only)

B -231

pLK40

B-GALACTOSIDASE

-147 GLU ARGMMCIL 4<3 50

9 650

-160

-216

pLK49 |-199

pLK50 t-189

pLK51 t-1 78

pLK52-188

pLK42 3

pNG15 (vector only)

<3 21

<3 41

5 37

FIG. 6. P-Galactosidase production supported by synthetic DNA fragments, covering the CAR] regulatory region between positions -231and -147, that were cloned into the expression vector pNG15.

either of two DNA fragments (positions -231 to -199 and-216 to -199) 3' of the heterologous CYCI UAS. Wheneither of the DNA fragments mentioned above was insertedinto the 3' cloning site (plasmids pLK63 and pLK64),3-galactosidase production dropped markedly (Fig. 7D).Two conclusions may be derived from this experiment.First, positions -231 to -199 or -216 to -199 behaved likea URS; i.e., they decreased transcriptional activation medi-ated by a heterologous UAS. This result supported thesuggestion derived from the experiment described in Fig.7B. Second, the URS function mediated by positions -231to -199 did not respond to an inducer (arginine). Thisobservation indicated that response to inducer may possessspecificity not only for the inducer, but also for the UAS.

Correlation of phenotypes observed with portions of theCAR] upstream region with the nature of sequences present.Data presented above suggested that the CAR] upstreamregion contained multiple cis-acting elements. If it is as-

sumed, as has been shown for many other systems, thatsome elements consist of repeated DNA sequences that are

homologous within each functional group, the core homolo-gies should be readily identified. Furthermore, if the homol-ogous sequences possessed physiological significance, itshould be possible to correlate the phenotype generated byeach deletion plasmid or synthetic oligonucleotide construc-tion with the composition of homologous sequences present.When a search for homologous sequences was performed

for the CAR] gene upstream region, 13 sequences that couldbe divided into three groups were identified. It must beemphasized that correlations to be described and the sizes ofthe designated areas were based solely on observed homol-ogies between sequences in different portions of the CAR]upstream region. There are not, at present, any point-mutational data that would support either a precise locationof a putative cis-acting element or the significance of thehomologies noted, except with regard to the URS elementfor which saturation mutagenesis has been reported (19).The first set of homologous sequences were hexanucle-

otides situated between positions -476 and -430 (Fig. 8A).All four sequences were contained on the small DNAfragment that supported UAS activity observed with plasmidpLK123 (Fig. 4). Their physiological significance may beassessed by comparing data obtained with plasmids pLK121,pLK114, and pLK115 (Fig. 4). Plasmid pLK121 supported1,249 U of,-galactosidase activity compared with 139 U andbackground levels supported by plasmids pLK114 andpLK115, respectively. Two copies of the repeated sequencewere deleted in plasmid pLK114 compared with plasmidpLK121. In plasmid pLK115 all but one of the repeatedsequences were deleted.The second set of sequences observed to exhibit high

homology were situated between positions -233 and -160(Fig. 8B). Their potential physiological significance can beassessed by comparing data obtained with plasmids pLK40,

4 51

<3 41

<3 17

3 32

5 37

9 650

<3 212

-231

13 58

10 83

R. .,-.

I&zf.lll.R5.. 1

R~

-1k..,'m R-.;

VOL. 10, 1990

----II

5094 KOVARI ET AL.

A-34E

pLK70-251

No

pLK80-344

pLK95 LEE Eu-

-251 -190

Li z ziz I-190

M -

B-GALACTOSIDASEGLU ARG247 1,19015 73

30 1,303

B-280 -251

pLK78-251

-280

pLK8o L

pLK105 *

254-190

-101-190

r -

635

13 84

38 573

C-280

nLK1 X5

-280

-190

-189 -160

pLK51 m-160-160

38 573

24 166

pLK68 [UL 28 1,527

10 65pNG15 (vector only)

Xhol

pNG22 (CYCl UAS alone)-231

pLK63-216

pLK64 L

74 137-199

i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~'

-19911 23

30 61

FIG. 7. (A and B) Localization of the CAR1 sequences responsible for the response to inducer. (C) Potential synergistic interactionsobserved between CAR] cis-acting elements. (D) Oligonucleotides in panels A to C were cloned into plasmid pNG15. This panel showsrepression of CYCI UAS function by synthetic oligonucleotides containing CAR] sequences between positions -231 and -199.Oligonucleotides were cloned into the vector pNG22.

pLK49, and pLK50 (Fig. 6 and 7). Plasmid pLK40, whichcontained all three sequences, supported highly inducible3-galactosidase production (Fig. 6B). With plasmid pLK49,

in which the first homologous sequence was deleted, theinduced level of lacZ expression dropped by two-thirds.Plasmid pLK50, in which two of the three sequences weredeleted, supported only background levels of ,3-galactosi-dase production. Deletion of the 3'-most homologous se-

quence resulted in complete loss of reporter gene expression(Fig. 6A, compare plasmids pLK40 and pLK41).

The last set of homologous sequences we observed weresituated between positions -354 and -266; all were G+Crich (Fig. 8C). The most striking characteristic of thesesequences is their homology to the conserved areas of thesimian virus 40 sequences shown to bind the Spl transcrip-tion factor (Fig. 8D) (10-12). The spacing of the G+C-richsequences in the CAR] promoter is identical to that reportedfor simian virus 40 (10-12). The potential physiologicalsignificance of these sequences can be assessed by compar-ing data obtained with plasmids pLK70, pLK71, pLK72, and

D

pNG22

Ml-F IvsW% LOLRE

MOL. CELL. BIOL.

n OLO,0%0%

cis-ACTING ELEMENTS NEEDED FOR CARI EXPRESSION 5095

+Arginine

-350 -250 , -150

IUASC2 1 UASI IUSTA , GL T| mn i

50St 1

T CARI

-476 -471

ACATCA-471 -466

ACATCA-457 -462

TCATCC-435 -430

TCATCA-233 -223

TTGCCCTTCGC-199 -209

TTGCCATTAGC-170 -160

TTTCACTTAGC-354 -349

CCGCAC-342 -337

CCGCGA-333 -328

CCGCCC-279 -274

CCGAGA-271 -266

CCGCGAD -354 -328

CARI CCGCaCCgtcgcCCGCgatcCCCGCCC101 75

SV40 KCGCcCtaact CGCc4at CCGCCCBox VI Box V Box IV

FIG. 8. Homologous sequences observed in the CAR] upstream region and a working model of the organization and potential interactionof putative cis-acting elements in the CAR] upstream region. UASCl and UASC2 function independently of the inducer, whereasUAS,-mediated transcriptional activation is inducer dependent. The URS element operates in a negative manner when inducer is absent.UASJ also acts negatively on UASC2-mediated activation when inducer is absent. We have not ascertained whether the negative action ofthese elements also occurs when inducer is present. (A to C) Homologous, repeated sequences, shown as patterned areas in UAScl (panelA), UASC2 (panel B), and UASJ (panel C), are shown. (D) Homologous sequences observed in CAR1 UASC2 and the Spl binding site ofsimian virus 40 (34) are compared (lowercase letters indicate mismatched bases in this panel).

pLK79 (Fig. 5A) and plasmids pLK70, pLK88, pLK89, andpLK94 (Fig. 5B). Deletion of these G+C-rich areas iscorrelated with the loss of transcriptional activation. Also,plasmids containing G+C-rich areas and little else were ableto support P-galactosidase production (Fig. 5, plasmidspLK79 and pLK94).

Potentially synergistic interactions observed between cis-acting elements upstream of CAR). The existence of multipleelements prompts the question of whether they interact in asynergistic manner. Several instances were observed duringour analysis in which such interactions might have occurred.

The first possible occurrence was documented by data inFig. 6. In that case, DNA sequences from -231 to -188 and-189 to -160 were incapable of supporting reporter geneexpression alone (plasmids pLK42 and pLK51, respective-ly). However, when they were combined (plasmid pLK40),72-fold inducible lacZ expression was observed.A second possible example of interaction was observed

with the set of plasmids shown in Fig. 7C. Plasmid pLK105contained positions -280 to -190 and supported 38 and 573U of 3-galactosidase production under uninduced and in-duced conditions, respectively. Plasmid pLK51 contained

-550

A(E)

B(E)

C(E)

VOL. 10, 1990

5096 KOVARI ET AL.

positions -189 to -160 and supported lacZ expression thatwas less than threefold above background. When the twosequences were combined (plasmid pLK68), the constructsupported an induced level of P-galactosidase of 1,527 U. Ifthe DNA fragments in plasmids pLK105 and pLK51 be-haved in an additive manner, the expected induced value forplasmid pLK68 would have been 739 U.

DISCUSSION

The experiments described above suggest that as many asfour cis-acting elements mediate induced expression of theS. cerevisiae CAR] gene. All of the putative elements werefound at least 100 bp upstream of the CAR] transcribedregion (Fig. 8), supporting our earlier suggestion that induc-tion of arginase occurs at transcription (26). The DNAfragments demonstrated to support UAS activity containedrepeated sequences, a characteristic observed for UASelements of the DAL genes (7, 25, 38) and many others aswell (1, 4, 12-14, 18, 33, 37). Two of the putative CAR] UASelements (UASCl and UASC2) supported inducer-independ-ent transcriptional activation of a heterologous reportergene, whereas functioning of the third (UASI) was inducerdependent.The highly inducible lacZ expression observed with plas-

mids pLK95, pLK68, and pLK40 (which lacked the CAR]URS element) prompted us to question the role of the CAR]URS in induction. Data reported earlier for plasmids pRS45,pRS46, and pRS91 showed that either point mutation ordeletion of the URS element at position -153 resulted inCAR] expression that was largely inducer independent (30).The apparent contradictory conclusions generated by thesetwo sets of data are probably explained by the differencebetween plasmids pLK95, pLK68, and pLK40 used here andplasmids pRS45, pRS46, and pRS91 used in our earlier work(29). Plasmid pLK40, which exhibited high inducibility in theabsence of the CAR) URS, lacked the inducer-independentUASCl and UASC2 elements. We suggest that without aURS element, the CAR] UASCl and UASC2 elements wouldsupport high levels of CARl expression in the absence ofinducer. By this reasoning, the function of the CAR] URS isto maintain a low level of gene expression in the absence ofinducer. When inducer is provided, we suggest that inducer-dependent UASI-mediated activation and its synergistic en-hancement of UASC2-mediated activation overcomes theaction of the URS, thereby permitting high-level CARlexpression (Fig. 7C, compare plasmids pLK105, pLK51,and pLK68). Figure 2 contains an additional observationconsistent with the above suggestion that it is the ratio ofUAS to URS activities that ultimately determines the levelof induction observed. The difference between plasmidspRS46 and pRS124 was the loss of inducer-independentUASCl. As predicted by our hypothesis, the uninduced levelof gene expression decreased, with a commensurate increasein the observed level of induction.The overall picture of CARl induction that emerges from

this work is similar in many respects to that previouslyreported for the allantoin system genes (38). Even thoughboth DAL7 and CARl are highly inducible genes, theirexpression is mediated in part by UAS elements that func-tion independently of inducer. For both genetic systems,data have been reported in support of a negative cis-actingelement that inhibits this inducer-independent UAS activitywhen inducer is not present within the cell.A somewhat perplexing result was derived from experi-

ments with the inducer-responsive sequences situated on

DNA fragments in plasmids pLK42 and pLK50 (Fig. 6B) andplasmids pLK80 and pLK78 (Fig. 7B). At face value, thesedata suggest that the element(s) situated on plasmids pLK42and pLK80 mediates inducer responsiveness by two dif-ferent mechanisms: (i) their repression of UASC2 activity inthe absence of inducer, and (ii) their interaction with a sitesituated between positions -189 and -160 (plasmid pLK51)thereby generating a functional inducer-dependent UAS.These mechanistically opposite responses presumablycaused by a single element can be rectified by consideringthe previous report that binding a protein (LexA) to DNA inthe vicinity of a UAS element will inhibit its function, eventhough the bound protein may have no physiological func-tion in S. cerevisiae (5). This may have occurred in the casedepicted in Fig. 6B and 7B. Inhibition of CYCI UASfunction mediated by CAR] positions -231 to -199 (Fig.7D) is consistent with this explanation.An interesting characteristic of promoter structure to

emerge from these studies and those with DAL7 (38) is thehigh modularity observed in the promoter regions of thesegenes and those studied in many other laboratories (1, 4,12-14, 16, 18, 33, 35, 37). This modularity of yeast promoterelements may be the mechanistic basis for generating theenormous diversity of promoter strengths and physiologicalresponses to environmental signals observed in this organ-ism. By mixing and matching various promoter elements,high flexibility of promoter strength and response can beachieved quite economically relative to the overall numberof regulatory factors required by the organism. Mixing alimited number of regulatory sites and associated factors inpromoter regions consisting of modules would also provide aconceptual basis for the integration of multiple environmen-tal regulatory signals.

Finally, we have described correlations between nucleo-tide sequences repeated in the 5'-flanking region of CARland the formal roles they seem to fulfill in the inductionprocess. However, we have not determined their preciseroles in the transcription process. It would not be surprisingto find that at least some of the sites we have identified arealso situated upstream of other genes in S. cerevisiae orother organisms. This has already been shown to be the casefor the CARl URS (19, 29). As such, they would be goodcandidates for potential sites for the binding of pathway-specific or generalized transcription factors. Particularlyinteresting in this regard is the presence of CARI regulatorysequences homologous to the Spl-binding site reported insimian virus 40 (10-12). Whether the presence of this homol-ogous sequence forecasts the existence of a yeast transcrip-tion factor with sequence specificity similar to the Spl factoris not yet clear.

ACKNOWLEDGMENTSWe thank all of the members of the University of Tennessee yeast

group for carefully reading the manuscript and offering suggestionsfor its improvement. All of the synthetic oligonucleotides used inthis investigation were prepared by the University of TennesseeMolecular Resource Center.

Part of the cost of the Molecular Resource Center was deferredthrough a Center of Excellence Grant from the State of Tennessee.This work was supported by Public Health Service grant GM-35642from the National Institutes of Health.

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