Proc. Nati. Acad. Sci. USAVol. 91, pp. 11109-11112, November 1994Genetics

A Drosophila gene promoter is subject to glucose repression inyeast cells

(myls/ge re / Sacckaromyces cevi /clerase/Drosophi w egaster)

DONAL A. HIcKEY*, KAARINA I. BENKEL*, YING FONGt, AND BERNHARD F. BENKELt*Department of Biology, University of Ottawa, 30 Marie Curie Street, Ottawa, ON Canada KiN 6N5; and tCentre for Food and Animal Research, AgricultureCanada, Central Experimental Farm, Ottawa, ON Canada KiA 0C6

Communicated by Matthew Meselson, July 27, 1994 (received for review July 28, 1993)

ABSTRACT Previous work has shown that the a-amylasegene of Drosophila Ml ogaster is subject to reps bydietary glucose. Moreover, glucose rep of this gene ismediated by promoter elements that lie uptra of thetnsrponal dar site. In this study, we examined theactIty Of the glucose-repressible Drosophila promoter intransfomed yeast cells. We show that the amylase promoterregon cm ate glue rep of a helou re-porter gene In yeast. The m t of this result Is that theyeast euory y canm e the Drosophila pro-

moersna . This, in turn, Implies an un dl highdegre iof evolutionary conservation in the of glu-co among eukaryotes. It also shows that genes thathave a ed c p patterns of developmental regulation-e.g., the Drosophia amylase gene, can stIll retain, intact, morepimive form of ruifon, such as glucose repres .

Glucose repression is a common feature in the expression ofmicrobial genes, and this phenomenon has been the subjectof extensive genetic and molecular studies (1-3). The Dro-sophila amylase gene provides a rare example of glucoserepression in multicellular animals (4, 5). As in the microbialsystems, this Drosophila gene is required for the utilizationof nonpreferred carbohydrate resources (6), and the repres-sion is mediated by promoter sequences close to the tran-scriptional start site (7, 8). Here, we show that the Drosophilaamylase promoter region is transcriptionally active and isrepressed by glucose in transformed yeast cells.Drosophila has been used extensively as an experimental

model to study the patterns of gene expression during de-velopment. Likewise, much work has been done on themechanisms ofglucose repression in microbial systems. Theresults presented here provide a link between these twodifferent areas of molecular biology research. Specifically,our results indicate an unexpected degree of evolutionaryconservation in glucose repression mechanisms betweenyeast and Drosophila. This result is consistent, however,with the recent findings on the structural and functionalconservation of basal transcription factors among yeast,Drosophila, and mammals (9-11). The results presented hereextend this conservation one step up the regulatory hierar-chy.

MATERIALS AND METHODSPlid Constcon. A HindlIl genomic DNA fragment

encompassing the proximal amylase gene from the Oregon Rstrain of Drosophila melanogaster (12) was cloned into theyeast shuttle vector pBTI-1 (Boehringer Mannheim). Thisinsert includes the entire Drosophila amylase coding region,as well as 478 bp of sequence upstream of the ATG start

codon and 1.6 kbp ofsequence downstream ofthe stop codon(see Fig. 1A).The recombinant gene Amy/Luc (Fig. 1B) was made by

replacing the amylase coding sequences with sequences thatcode for firefly luciferase. First we constructed an expressioncassette containing amylase promoter sequences (-468 toATG) and downstream noncoding sequences (550 bp paststop codon). The promoter and terminator regions are sep-arated by introduced Nco I and HindlI sites, into which areporter coding sequence could be cloned. This cassette wasbounded by Not I and BamHI sites at the upstream anddownstream ends, respectively. PCR amplification was usedto amplify luciferase sequences (14) for insertion into thiscassette; Nco I and HindIII cloning sites were introduced inthe PCR primers. Amplified fragments were routinely clonedinto pCR vectors (Invitrogen) before final assembly intohybrid constructs using a modified pIBI24 vector known asAphid (15). This approach facilitated the introduction of therestriction enzyme sites that were used for assembly of thegene components, as well as the transfer of hybrid genesbetween vectors.The Act/Luc plasmid (Fig. 1 C) was constructed by delet-

ing the amylase promoter of the Amy/Luc plasmid andreplacing it with a 427-bp firgment containing the yeast actinpromoter (13). First, a fragment of the yeast actin gene wasamplified from plasmid pTZAct34 (obtained from A. Wilde-man, University of Guelph) by using oligomers that con-verted the sequence beyond -427 into a Not I site and theATG environment into an Nco I site. Then, the fly amylasepromoter was removed by digestion ofthe Amy/Luc plasmidwith Not I and Nco I followed by agarose gel fractionation.Finally, the recovered, promoterless fragment was ligatedwith the actin promoter fragment to give the Act/Luc plas-mid.

Yeast Transformation and Expression Assays. Yeast cellsfrom the AH22 cir+ strain of Saccharomyces cerivisiae (16)were transformed with recombinant shuttle vectors by usingstandard techniques (17, 18). Cells were plated on selectivemedium (leu-dropout, Bio 101), and leu prototrophs weregrown at 30TC in rich liquid medium (yeast extract/peptone)with the carbon source [5% (vol/vol) glycerol or 5% (wt/vol)glucose] as indicated.Amylase activity is secreted from transformed yeast cells.

For the enzyme assays, cells were removed by centrifuga-tion, and aliquots ofthe extracellular medium were separatedon polyacrylamide gels under nondenatring conditions. Gelswere processed and stained for amylase activity as described(5).

Luciferase is not secreted from the transformed cells. Toassay for this enzyme, transformed cells were cultured for 24hr. Cell densities of the cultures were estimated by ODINmeasurements. Cells were harvested by centrifugation, re-suspended in cell lysis buffer (Promega), and frozen for atleast 24 hr at -70° C. Cells grown on glycerol-containingmedium were resuspended in a volume of cell lysis buffer

11109

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Proc. Natl. Acad. Sci. USA 91 (1994)

'4 A IFGH v7

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FIG. 1. Structure of genes used in yeast transformation experiments. (A) The amylase gene construct contained a genomic DNA fragmentencompassing the proximal amylase gene from the Oregon R strain of D. melanogaster (12). It includes the entire Drosophila amylase codingregion, as well as 478 bp of sequence upstream of the ATG start codon and 1.6 kbp of sequence downstream of the stop codon. (B) Therecombinant gene Amy/Luc was made by replacing the amylase coding sequences with sequences that code for firefly luciferase. (C) TheAct/Luc plasmid was constructed by deleting the amylase promoter of the Amy/Luc plasmid and replacing it with a 427-bp fragment containingyeast actin promoter (13). In the recombinant gene constructs, the switch between promoter and reporter sequences was made at the translationstart site. Thus, in each case, the 5' untranslated sequences and the transcriptional start site originated from the same gene as the promotersequences.

equal to that of the culture medium. To check the efficiencyof cell lysis, some assays were done on yeast cells that werepretreated with zymolyase enzyme to digest the cell walls;there was no significant difference in the luciferase activitiesmeasured by the two methods. Cells grown on glucosemedium grew more quickly and to a higher density; thereforesmaller inocula were used, and cell densities were adjusted tomatch those of the glycerol-grown samples by dilution withcell lysis buffer. Luciferase activity measurements were doneon an LKB 1251 luminometer with the Promega luciferaseassay kit.To produce photographic images, lysates of yeast cells

were mixed with Promega luciferase reagent in single wells ofa 96-well microtiter plate. Ten microliters of cell lysate for theAct/Luc clone and 20 p1 of lysate for the Amy/Luc clonewere mixed with 100 Al ofPromega reagent. The 96-well platewas exposed to Polaroid 57 film (3000 ASA) for =10 sec forAct/Luc extract and 40 sec for Amy/Luc extract.

RESULTSS. cerevisiae cells were transformed with a plasmid contain-ing a wild-type amylase gene from D. melanogaster (Fig. LA).This gene contained 478 bp ofupstream noncoding sequence,1.5 kbp of amylase coding sequence, and 1.6 kbp of down-stream noncoding sequence. Because the gene did not con-tain any yeast promoter sequences, we did not expect to seeexpression in yeast cells; in fact, the experiment was initiallydone to provide a negative control in a study of amylaseexpression controlled by yeast promoters. To our surprise,the transformed yeast cells produced enzymatically activeamylase that was secreted into the culture medium. As a firststep toward confirming that this enzymatic activity was,indeed, due to expression of the Drosophila gene, we com-pared the electrophoretic mobility of the enzyme producedby the yeast cells with that produced by Drosophila larvae.The two enzymes showed identical mobilities on nativepolyacrylamide gels stained for amylase activity (Fig. 2A,lanes 1 and 3). Although this comigration of the two enzymesis suggestive of identity, it might merely be due to chance. Toget a more conclusive answer, we repeated the experimentusing a Drosophila amylase clone that comes from a straincharacterized by a different electrophoretic mobility. In this

case, the yeast expressed a different electromorph, but onethat was again identical to the enzyme in the source Dro-sophila strain (Fig. 2A, lanes 2 and 4). This result confirmsthat the amylase being produced by the yeast cells is encodedby the introduced Drosophila gene.We then asked if the expression of the amylase gene was

glucose repressible in yeast cells. To do this, cells weregrown in either a glucose-rich [5% (wt/vol) dextrose] orglycerol-supplemented medium. The results indicated a verylarge reduction in amylase expression (<10% of controllevels) when the cells were grown in the glucose medium (Fig.2B).Glucose repression of the intact Drosophila gene in yeast

cells suggests that the expression is controlled by the Dro-sophila promoter. We tested this assumption in several ways.(i) We used shuttle vectors in which the gene was inserted inthe opposite orientation; this eliminated the possibility thatthe expression was controlled by vector sequences upstreamofthe amylase promoter. We found that the orientation oftheinsert did not affect the response to glucose. (ii) Next wemade new constructs in which the amylase promoter waslinked to a heterologous reporter gene, the firefly luciferase(14). Transformation with this recombinant construct re-sulted in the expression of high levels ofluciferase activity bythe transformed yeast cells, and this activity was also glucoserepressible (Fig. 3 A and C and Table 1). (iii) Finally, to testthe possible effects of the downstream noncoding sequences,we replaced the amylase promoter by a yeast actin promoter.These constructs gave luciferase expression that was notglucose repressible (Fig. 3B). In summary, these results showthat the presence of the Drosophila amylase promoter regionis both necessary and sufficient for glucose repression of thetransforming gene in yeast cells.

DISCUSSIONOur results show that the introduction ofan intactDrosophilagene into yeast cells results in glucose repressible expressionand extracellular secretion of an enzymatically active geneproduct. Because our particular focus is on glucose repres-sion, we wished to separate this effect from other aspects ofgene expression. First, we showed that the glucose effect didnot depend on the particular coding sequence because the

11110 Genetics: Hickey et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 11111

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AMY" -*

FIG. 2. Expression and glucose repression ofDrosophila amylasegenes in transformed yeast cells. (A). Expression of fly amylase genesin yeast. The amylase proteins produced by transformed yeast cellswere compared with those produced by adults ofthe source fly strains.Lanes 1 and 2 contain samples ofDrosophila homogenates from eachoftwo strains (genotypes Amy' and Amy6, respectively) that producedistinguishable electrophoretic patterns (8). Lanes 3 and 4 containsamples of extracellular medium from yeast strains transformed withshuttle vectors containing amylase clones from each of these twogenotypes. (B) Glucose repression of a fly amylase gene in yeast.Amylase activity was compared for cells growing on rich mediumcontaining either 5% (wt/vol) glucose (+) or5% (vol/vol) glycerol (-)as carbon source. Cells were transformed with the shuttle vectorcarrying theAmy' gene described in Fig. 1. These cells were grown foreither 24 hr (lanes 1) or 48 hr (lanes 2) before harvesting. Lanes 3represent a 10-fold dilution (0.1x) of the samples in lanes 1.

luciferase reporter gene was also glucose repressible. Theintroduction of the luciferase sequences also helped to elim-inate a possible complication due to the interaction ofglucosewith the yeast secretion mechanism. Luciferase does nothave a signal sequence and is not secreted from yeast cells.Thus, the Drosophila amylase promoter can mediate glucoserepression of both secreted and nonsecreted proteins inyeast. The replacement of the Drosophila promoter with theyeast actin promoter and the concomitant loss of glucoserepression confirm that the effect is mediated by sequencesupstream of the translational start site. To obtain furtherconfirmation that the Drosophila promoter sequences arebeing recognized by the yeast transcriptional machinery, wehave also tested the expression of the Drosophila amylasegene in a yeast strain that carries the ssn6 mutation; Ssn6 ispart of the general transcriptional repression complex inyeast (19). In this genetic background, the Drosophila pro-moter was not glucose repressible.

Transcription of yeast genes usually initiates at a greaterdistance from the TATA box motif than the 30 bp typical ofmammalian and Drosophila promoters (20-23). Despite thisdifference, there is now ample evidence that the basic tran-scription machinery in all eukaryotes is highly conserved(9-11). It is worth noting that the sequence of the TATAelement in the Drosophila amylase promoter (24),

DLi

P4.

FIG. 3. Drosophila amylase promoter mediates glucose repressionof luciferase activity in transformed yeast cells. (A) Expression ofluciferase activity by yeast cells transformed with the Amy/Luc clone(Fig. 1B). This clone contains the Drosophila amylase promoter. (B)Expression of luciferase activity by yeast cells transformed with theAct/Luc clone (Fig. 1C). This clone contains the yeast actin promoter.Arrows indicate the wells containing the samples. Parallel yeastcultures were grown on glycerol (Gly) or glucose (Glu) medium. A10-fold dilution of the glycerol-grown samples is included for com-parison. The samples were placed in the central well (under thearrow); the photograph shows the light radiatingfrom this well throughthe clear plastic of the microtiter plate. (C) Relative luciferase activ-ities produced by the amylase promoter construct (Amy/Luc) grownin glycerol or glucose medium. These values were calculated fromluminometer readings (Table 1).

TATATAA, is a perfect match to the fully functional yeastTATA elements defined by Chen and Struhl (25). In addition,a repeated sequence motif, 5'-GTGGGG-3', has been iden-tified in the Drosophila promoter (26) that resembles thebinding site of the MIG1 repressor of yeast, 5'-SYGGRG-3'(27, 28). This transcription factor is thought to act through ageneral repression complex in yeast (19). MIG1 is the com-ponent that is specific to genes subject to catabolite repres-sion, and its binding site has been shown to be important inthe glucose repression of yeast GAL genes (29).

Genetics: Hickey et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

Table 1. Effect of glucose on the expression of luciferaseactivity in transformed yeast cells

Glycerol Glucose

Amylase promoter (Amy/Luc) 1027 47Actin promoter (Act/Luc) 1129 1681Vector control (pBTI-1) 0.5 1.1

Transformed yeast cells were grown in glycerol or glucose mediumfor 24 hr. Cells were harvested by centrifugation, and samples wereequilibrated for cell density. Cell lysates, prepared as described forFig. 3, were diluted 10-fold for Amy/Luc and 100-fold for Act/Lucin cell lysis reagent before measurement with the luminometer; thevector control was not diluted. The transforming DNAs containingthe amylase and actin promoters, respectively, are diagrammed inFig. 1. Values shown are in arbitrary units and are the average for sixindependent experiments. The vector control represents samples ofyeast cells that were transformed with the plasmid vector containingno insert.

In addition to the basic transcription signals, such asTATA, and the glucose repression elements inferred here,the Drosophila amylase promoter also contains signals fortissue-specific expression in flies (30-32). These signals musthave been added to the promoter relatively recently in thecourse of evolution-i.e., after the divergence of metazoansfrom other primitive eukaryotic lineages. These facts, takentogether, suggest a model for the evolution of complexpromoters in higher eukaryotes-i.e., that they evolved bythe accretion of new regulatory layers to a preexisting,conserved promoter. The most primitive promoters wouldhave been capable only of constitutive expression; later,some genes evolved the ability to respond to simple envi-ronmental stimuli; and, finally, genes in multicellular eukary-otes evolved complex patterns of developmental regulation.The Drosophila amylase gene illustrates the fact that theacquisition of new layers of regulatory complexity is com-patible with the retention of more primitive forms of generegulation. A question that remains to be answered is howthese various levels of regulation interact with one another atthe molecular level.

We thank Alan Wildeman for providing the yeast actin clone andVirginia Walbot for the luciferase clone. Alexander Johnson pro-vided the yeast ssn6 mutant strain. We are grateful to C. J. Andrewsfor use of the luminometer. This research was supported by a grantfrom the Canadian Medical Research Council to D.A.H. Salarysupport was provided by Agriculture Canada (Y.F. and B.F.B.) andby a Fellowship from the Canadian Institute for Advanced Research(D.A.H.).

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