JOURNAL OF BACTERIOLOGY, Jan. 1991, p. 255-261

Vol. 173, No. 1

0021-9193/91/010255-07$02.00/0 Copyright © 1991, American Society for Microbiology

DAL82,

Second Gene Required for Induction of Allantoin System Gene Transcription in Saccharomyces cerevisiae

a

MICHAEL G. OLIVE, JON R. DAUGHERTY, AND TERRANCE G. COOPER*

Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163 Received 3 July 1990/Accepted 9 October 1990

Several highly inducible enzyme activities are required for the degradation of allantoin in Saccharomyces cerevisiae. Induction of these pathway enzymes has been shown to be regulated at transcription, and response to inducer is lost in dal81 and dal82/durM mutants. The similar phenotypes generated by da)81 and dal82 mutations prompted the question of whether they were allelic. We demonstrated that the DAL81 and DAL82 loci are distinct, unlinked genes situated on chromosomes IX and XIV. DAL82 gene expression did not respond to induction by the allantoin pathway inducer or to nitrogen catabolite repression. Expression was also not significantly affected by mutation of the daI80 locus. From the nucleotide sequence of the DAL82 gene, we deduced that it encodes a protein with a mass of 29,079 Da that may possess the structural motifs expected of a regulatory protein. This protein was shown to be required for the function mediated by the cis-acting upstream induction sequence situated in the 5'-flanking regions of the inducible allantoin pathway genes. Expression of several allantoin pathway genes (DAL4, DAL7, and DUR1,2) in Saccharomyces cerevisiae increases 10- to 20-fold when compounds that can be degraded to the inducer, allophanate, are added to the culture medium (4, 10, 36, 39). A similar response is caused by the gratuitous inducer oxalurate (30). In contrast, when cells are provided with a readily used nitrogen source, such as asparagine or glutamine, mRNA levels drop precipitously whether or not inducer is present, a phenomenon termed nitrogen catabolite repression (4, 10, 25, 36, 39). cis-acting elements required for basal-level gene expression, response to inducer, and nitrogen catabolite repression have been shown to be situated upstream of the inducible DAL7 transcribed sequences, suggesting that regulation of the gene occurs at transcription (38). The DAL7 cis-acting elements were found to be of three types (38). The first type of element, a nitrogen-regulated upstream activation sequence (UASNTR), was originally reported to be responsible for DALS gene expression (26). This element, when present in two or more copies, supports high-level, inducer-independent, nitrogen catabolite repression-sensitive gene expression (6, 26, 38). A functional GLN3 gene has been shown to be required for UASNTR function (5). Saturation mutagenesis of the element was completed recently (2b), and the results suggest that its core sequence, 5'-GATAA-3', is very similar in structure to chicken Eryf-1, mammalian GF-1 binding sites, a cis-acting site upstream of the Neurospora crassa qa-JF gene, and a footprinted region upstream of the rat growth hormone gene (1, 9, 18, 24, 33, 35). The second element was shown to possess the properties of a negatively acting upstream repression sequence (URS). When a DNA fragment containing the URS element was placed adjacent to a DAL UASNTR-containing fragment, it mediated complete inhibition of transcriptional activation (38). The heterologous CYCJ UAS was affected by the DAL7 URS in the same way (38). The URS-mediated inhibition of transcriptional activation was independent of inducer. A third element was shown to be required for response to inducer, the upstream induction sequence (UIS). When this element was *

placed adjacent to the UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of P-galactosidase synthesis (38). Three mutant classes with apparent defects in the induction process have been reported. We isolated a set of recessive mutations (dal8J) (34) that resulted in complete loss of response to induction at both the enzyme and steady-state mRNA levels (10, 34, 39). The dal8J locus was located on the right arm of chromosome IX by conventional genetic mapping methods (33). The DAL81 gene has been cloned and sequenced, and its expression was shown not to respond to induction by oxalurate or to nitrogen catabolite repression (2, 2a). Mutations at the dal80 locus, which result in 10- to 20-fold overproduction of allantoin pathway enzymes, were also shown not to significantly affect DAL81 expression. A dal81 null mutant, generated by deleting the gene, possessed the same physiological characteristics as the point mutant originally characterized (2). Mutation of a second locus, durMIdal82, was reported to affect urea amidolyase activity and DURl,2-specific mRNA levels in a manner similar to dal81 (15). The gene was cloned but not sequenced or characterized (16). The third class of mutants, durL, also exhibited a phenotype similar to dal8J mutants but have not been characterized (15). The similarity of phenotypes generated by the dal81 and da182 mutations raises the possibility that they are allelic. If the two loci are distinct, the lack of information about expression of the DAL82 gene, its potential regulation by induction, by nitrogen catabolite repression, or in response to mutation at the dal80 locus, and the identity of the cis-acting elements upstream of the DAL7 gene which require a wild-type DAL82 protein to function make it difficult to elucidate its role in the induction process. Therefore, we tested the potential allelism of the dal8J and da182 mutations. Finding that they were not allelic, we isolated and sequenced the DAL82 gene, characterized its expression, and identified the cis-acting elements upstream of the DAL7 gene that require its product for function. (A preliminary report of this work has already appeared

Corresponding author.

[7a].) 255

OLIVE ET AL.

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TABLE 2. Complementation of the da182-1 mutation by various plasmid-borne DNA fragmentsa

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MATERIALS AND METHODS Strains and media. The strains used in this work are listed in Table 1. Strain M02 was made by selecting for a ura3 mutant allele of strain 13H9b following treatment with 5-fluoroorotic acid. Strain M1689-13d was derived from a cross of strains 13H9b and M1-2b. Genetic manipulation and transformation were accomplished by standard procedures (12-14). Isolation of plasmids able to complement mutations in DAL82. Mutations at the da182 (durM) locus were originally isolated in an 11278b genetic background (strain 13H9b), which is not easily transformed. Therefore, strain 13H9b was crossed to strain M1-2b, which transformed at high frequency, to generate strain M1689-13d. This strain was transformed with 10 ,ug of DNA derived from strain S288C carried in shuttle vector YCp5O (28). Ura+ transformants were selected. Top agar containing these transformants was scraped from the plates and macerated, and the cell suspension was filtered. The cells were recovered, washed with sterile minimal medium devoid of a nitrogen source, and plated on YNB-allantoin plates containing tryptophan but no uracil. After 3 days of incubation at 30°C, the colonies that appeared were selected for further analysis. DNA sequence analysis. DNA sequence analysis was conducted by the dideoxy chain termination method reported earlier (2b). A combination of synthetic oligonucleotide primers and mung bean exonuclease III-generated nested TABLE 1. Strains used Relevant genotype

S. cerevisiae

11278B .............MATox 13H9b ............. MATa da182-1 M02 ............. MATa ura3 da182-1 M1-2b ............. MATax ura3-52 trpl-289 M1689-13d .........MATa ura3-52 trpl-289 da182-1 M1682-19b .........MATa ura3-52 trpl-289 PB200 . MATa ura3-52 trpl-289 /dal81::hisG M970 M 90

.

................

MA Tot lys5

MATa Iys2 M1081 . MATot M 1...............

Iys2 dal80-1

MATa lysS dal80-1 MATa his4-42 lys-23 AM100...

M02

pMO01 I

FIG. 1. Restriction map of plasmids used to test complementation of the da182-1 mutation. The circled B designates a destroyed BamHI site. Hatched areas represent vector sequences.

Strain

11278b

+

+ metl3-25 MATot + . MATa his442 lys23 gln3-1 + AM2O1 MA Tot + + gln3-1 met13-25 E. coli HB101 ........hsdR hsdM recAJ3 supE44 lacZ24 leuB proA2 thi-l Smr

UALase activity

Plasmid

None YCp5O (vector) pMO8 YEp24 (vector) pMO1l

pPB14 (DAL81)

Pro

Pro +OXLU

18 41 38 28 53 12

177 51 891 30 416 28

Plasmids YCp5O, YEp24, and pPB14 have been described previously (2). The plasmid indicated was used to transform mutant strain M02, which itself was isogenic to strain Y.1278b except at the URA3 locus. Transformants were grown to a cell density of 30 Klett units in glucose-proline (Pro) minimal medium. At that time, each culture was split into two portions. One portion received oxalurate (OXLU, 0.25 mM final concentration), while the other did not. Following growth to a cell density of 60 Klett units, each culture was sampled in duplicate and assayed for the urea amidolyase (UALase) activity it contained by previously described procedures (36). UALase activities are expressed as picomoles per minute per milliliter of culture. a

deletions were used in the sequencing strategy. Both strands were completely sequenced, and all sites were crossed. Northern (RNA) blot analysis. Strains used for Northern blot analysis were grown to mid-log phase in Wickerham's medium containing the indicated nitrogen source at a concentration of 0.1% (37). Glucose (0.6%) was provided as a carbon source. Samples (10 jLg) of polyadenylated [poly(A)+] RNA were prepared (3) and resolved on a 1.4% agarose-6% formaldehyde gel by the procedures of Sumrada and Cooper (31). Following electrophoresis, the separated species were transferred to a nylon membrane. The blot was hybridized with the 1-kb BglII-BamHI fragment from plasmid pMO8 that was radioactively labeled by random-primed labeling (Boehringer Mannheim Biochemical Inc.). The blot was then stripped and hybridized a second time to DNA from plasmid pTCM3.2, radioactively labeled by randomprimed labeling, and the 2.5-kb HindlIl fragment from plasmid pHY3 carrying the DAL7 gene.

RESULTS Plasmids containing sequences that complement dal82 mutations. The procedures described in Materials and Methods were used to isolate two plasmids (pMO8 and pMO9) which were able to complement a dal82 mutation at high frequency and contained common DNA fragments following digestion with endonucleases BamHI and EcoRI. Plasmids pMO8 and pMO9 contained 10- and 11-kb inserts, respectively. Plasmid pMO8 was selected for further analysis. To test whether plasmid pMO8 sequences complemented the biochemical phenotype of the dal82 mutation (loss of urea amidolyase inducibility), transformants of strain M02 were grown in the absence or presence of oxalurate and assayed for urea amidolyase activity (36). These transformants contained nearly four times the activity exhibited by a wild-type strain of the same genetic background (11278b) and far more activity than the recipient strain (MO2) transformed with only the cloning vector (YCp5O) (Table 2). Structure of the putative DAL82-containing plasmid. The structure of plasmid pMO8 was determined (Fig. 1). Subcloning experiments localized the dal82-complementing (growth phenotype) sequences to a 2.9-kb BglII-EagI fragment (plasmid pMOll, Fig. 1). To test whether plasmid pMOll was able to complement the biochemical phenotype

VOL. 173, 1991

ALLANTOIN SYSTEM INDUCTION IN S. CEREVISIAE

of the da182 mutation, we assayed transformants containing plasmid pMOll in the presence and absence of oxalurate. The subclone was able to restore urea amidolyase inducibility (compare strain M02 containing plasmids pMOll and YEp24, Table 2). However, the fully induced level of activity observed was only half that observed with plasmid pMO8, suggesting that a portion of the complementing sequences may have been removed. Test for allelism between the dai8) and dal82 loci. The observation that dal8J and dal82 mutants had the same phenotype raised the possibility that mutations carried in the two strains were allelic. To test this possibility, we transformed a da182 mutant strain (MO2) with plasmid pPB14, which contained the wild-type DAL81 gene, and assayed the transformants for urea amidolyase (plasmids YEp24 and pPB14 in Table 2). No complementation of the biochemical defect of strain M02 was observed. A more positive indication that the two loci were distinct was obtained by identifying the chromosomal location of the dal82-complementing sequences carried on plasmid pMO8. This was accomplished by hybridizing radioactive DNA derived from plasmid pMO12 against yeast chromosomes that had been resolved by pulsed-field electrophoresis. DNA carried on plasmid pMO12 hybridized only to one chromosome which was identified, by comparison with a standard, as chromosome XIV (data not shown). The DAL81 gene has been previously shown to be situated on the right arm of chromosome IX. These data suggested that the dal8I and da182 loci were indeed distinct even though mutations in them generated indistinguishable phenotypes. Expression of the DAL82 gene and its regulation. Regulated expression of the DAL82 gene could play a central role in control of the allantoin system structural genes. This prompted us to assess the levels of DAL82-specific mRNA derived from wild-type, dal8J, and dal80 mutant strains grown under various physiological conditions. Cultures of wild-type strain M970 grown in nonrepressing medium (glucose and proline) either without or with inducer or in glucose-asparagine medium (a condition of high nitrogen catabolite repression) exhibited similar levels of DAL82specific mRNA (Fig. 2, lanes A to C). DAL7- and TCMI-specific probes were used as controls to visualize the expected behavior of an allantoin pathway gene (DAL7) and one that had been demonstrated previously not to respond to oxalurate-mediated induction or nitrogen catabolite repression (TCMI). DAL82-specific mRNA levels increased approximately threefold (quantitation obtained by densitometric scanning of the X-ray film; data not shown) in a dal80 mutant strain compared with the wild type (Fig. 2, lanes A and E). There was also threefold less DAL82-specific mRNA in dal80 cultures grown in glucose-asparagine medium than in dal80 cultures grown in glucose-proline medium (Fig. 2, lanes D and E). We observed a 1.5- to 2-fold increase in the levels of DAL82-specific mRNA in a dal8J mutant compared with the wild type (Fig. 2, lanes F to I). Finally, mutation of the gln3 locus resulted in a 30% decrease in DAL82-specific mRNA (Fig. 2, lanes J and K). These effects, though measurable, are not nearly as marked as normally observed with the allantoin pathway genes. Nucleotide sequence analysis of the DAL82 gene. We determined the nucleotide sequence of the DAL82 gene as described in Materials and Methods. The sequence of the gene and its flanking regions (3,012 bp) is shown in Fig. 3. Computer analysis (Genetics Computer Group sequence analysis package) resulted in identification of a single open reading frame encoding a 255-amino-acid protein with a

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FIG. 2. Expression of the DAL82 gene in wild-type and mutant cultures grown under various physiological conditions. Lanes A to E, Poly(A)+ RNA prepared from wild-type (W.T., M970) and daI80 mutant (M1081) strains grown in minimal glucose-proline (PRO) or glucose-asparagine (ASN) medium. Oxalurate was provided at a final concentration of 0.25 mM where indicated (+). The RNA species were resolved on a 1.4% agarose-formaldehyde gel and transferred to a nylon membrane. They were then hybridized to the 960-bp BamHI-BglII fragment of pMO10 labeled by the randomprimed DNA-synthetic method. After generation of the autoradiographs, the blot was stripped and hybridized a second time with similarly labeled DNA containing TCMJ (plasmid TCM3.2) or DAL7 (2.5-kb HindlII fragment of plasmid pHY3 [39]). Lanes F to K, Poly(A)+ RNA prepared from wild-type (W.T., M1682-19b, lanes F and G; AM100, lane J), daI81 deletion (pB200, lanes H and I), and gln3 mutant (AM201, lane K) strains grown as described above. The Northern blots generated from these RNA preparations were analyzed as described for lanes A to E above. Sample size in each lane was 10 ,ug.

calculated mass of 29,079 Da and a predicted isoelectric point of 7.7. This information suggested that the insert in plasmid pMOll contained the entire coding sequence of the DAL82 gene but lacked all of the sequences thereafter, presumably including the transcriptional termination and poly(A) addition sequences. This information may explain the decreased level of urea amidolyase induction observed when plasmid pMOll was used to complement the da182 mutation (Table 2). Comparison of the DAL82 protein with those in the data bases did not reveal the presence of a zinc finger, cyclic AMP-dependent phosphorylation sites, a nucleotide-binding site, or obvious helix-turn-helix or helix-loop-helix motif (8, 11, 17, 22, 23). The initial 100 amino acids were found to have a net charge of + 10, while residues 12 to 61 had a net charge of +14. Residues 51 to 60, RTLKTKFRRL, exhibited some similarity to a nuclear localization signal (20, 21, 27). A serine-rich (31%) region was observed between residues 100 and 150, with a string of serine resdues (9 of 12) at positions 100 to 112. Two potential glycosylation sites were observed at positions 98 and 130, respectively. There was also a region at the carboxy terminus of the protein (residues 211 to 246) that could potentially be folded into an amphipathic helix resembling the coiled-coil motif reported by Sorger and Nelson (29). cis-acting site at which the DAL82 gene product functions. Previous studies by Yoo and Cooper (38) identified three functionally distinct cis-acting elements in the 5'-flanking region of the inducible DAL7 gene. An important issue in our understanding of the biochemical events that regulate allantoin pathway gene expression and the role of the DAL82 gene product in this control mechanism was identification of the cis-acting element(s) whose function requires the DAL82 protein. To address this question, we transformed wild-type

OLIVE ET AL.

258

J. BACTERIOL. -1910*

-1870*

-1890*

ACG GCC GTT GAT GAC CGA ATG CAT TGG TTC ACT GAT TCA GAT GTG GAA GAA CAA TCG CAL

-1850* -1830* -1810* TCT TAT AAA GGT GAC CTG CGG AAA TTT TTC TGC CAT GGA CCT TAA GTA ACA TTC AAC CTG -1770* -1790* -1750* GCC CCA TAC TTG CGG ATG TGA GTG TAT TAT GAC TTC TTC AAA ATT TCC AAG GGA TGC AAT -1710* -1730* -1690* ACC ATT TGG TAG TTG GAT TGG GCT GAT TAG ACA ATG GGT ALT GGG TAC ATA TTG CAC CGC -1670* -1650* -1630* TAT AAC TTC TAT ATC TGG CGT AAT TCT ATT AGT ATC TGC TGG AGC AGG TAA GCA TAG GGC -1610* -1590* -1570* TTT TTT GAT CAT CCT ATC ACG CAA GAG ATC ATA GGA AAA AAC TAC TTG TCC ATT GGT GGA -1550* -1530* -1510* ATT TTC CAA CGG TAC CAC TGA ATA ATC TAT ACT AGT GTC GTT CTC CAA TTG GTT AAA ACA -1470* -1490* -1450* TTG GGG GAT AGA GGC TGC TGG GAG GTA CTC AAC ATC AGA TGT TGA TTG AAA TTG TTG TAA -1410* -1430* -1390* TGC AGC TTG ATG GGA ATA CGT ACC TTT GGG ACC CAG AAA AAG AAC CCT CAA AGT CTT GCT -1350* -1370* -1330* GGC CAT AAT TGA AGG ATG ALT CGC GGA AAT ATA ATG AAG TTG CTT TTC GTA CTT TTT TAG -1270* -1290* -1310* CCT GTT CTT TCT GCG TAT TAT ATT CAT CTA CTA AAA TTT TTA TTT TCA TTT TTC ATT ATG -1250* -1230* -1210* AAA ATA CAA GAA GTC ATG CTG ATA ACT GTG TAA AGA AAC TGT ACG CTT GTC GCA GAT GAT -1170* -1150* -1190* GTA CGT ATT TTA TTT ACA GGT GTC ATT AAT ATA TAT ATA TAT ATA TAT ATA CGT ATA CGG -1130* -1110* -1090* AAG TAA TTC TTC GTT AAT TTT CCA TGG ACT GTG ATA GCG AAA TCA ATT TCT CGA CGG TGA -1050* -1070* -1030* ATC CCG GAG AGG CCT TAG AGA AAT CTC TTA ACA ACT GCA ACT CTC TGT TTA GCA ACT GGL -970* -990* -1010* GTT TCT TCA CCC ATT GCA CCA TAA AAC CTT TGC ACG TTT AGC AAT AAC AGC TGT GCA TCC -930* -910* -950* TGT ACL TTC ACG TTT GTA TCC GGT TCA ACA TGG CCA TTC ATA AAA ACA ATT CCC TTA TCC -870* -850* -890* TTG ACC AAA TCA GAA TGG AAC TGC AAG GTA GTA TGT GGC CTT GCA AAT TCC TGG TGT AGT -810* -830* -790* TTG TAT TCT GCT AGG GAA GTC ATC ATA CAA TGT GTT GTT TGG GGT CCG ACA AAC TGC CAT -770* -750* -730* TGG ATG TAG TGT AGT TCC ATT CCC TGT TCT TCG TTC GGT TTG CAT CCT CAG ATT GAA CTT -690* -670* -710* GTC TAG GAA GTG GCA ACA CGA ATA TAG GGT TAT TCC TGG CAT TTG CCA TCA TCT TAT CAT -630* -610* -650* AAA CGG AGA CAG GAA TCA CAG CGC ATA ATG TAT TAT CTT TTT GTG CCC ATC TTG CTC TCC -590* -570* -550* AAA GAA ACT CTA CCT CCT GTT TCG ATA AGT CCT TCA GTT TTC CAA CAT CCA AAA AAG AGT -510* -490* -530* CCA ATG TTT TGA AGG GAA CTT TAG GAG CAC TTG GAT CGA GAG GAC TTC TTG ATT TCG TCA -450* -430* -470* TCA AAT GCT TGG AGT TGT TAT TTT CCA TTT GAG TTT TTT GTT GGT AAT CCT CTA GTT CTT -390* -370* -410* TCA GGG GGT CGA TTT TAT TAA ATT CCC GCT TCT TAG ACT CTA TAG TCT CTT TCA AAT GAT -330* -350* -310* TTT TCA ACT CTT CTA TGC TGT TAA AAC CTT GTT TTT GAG CCT CTT CCA AAA GTT TCT TTC -270* -250* -290* TAT ACT TCT GCT CTG GGC TGG AAG ALT AAA AAA CGA AAA ACA GGA ACT GTT CCA ATT GCC -210* -190* -230* TTG TTA AAA ATG GGC GAC AGC TGA TTA GAA ATA TGG ATT CTA GTG CCG ATT TTT CTG GTT -130* -150* -170* AGT CTC CAC ATA GTT ATT CTT AAA TCT CTT TTG TTC TCT ATT TTC AAG TCT TGA TAA GTT -90* -70* -110* CTT CAC ATC CTT CGA TTA CGG CGT CAA AAT AAG CGT TAT TTC CAT TCG GGT GAG TTG AAC -30* -10* -50* CAT TGA TAA CAL CAT GGT TTA ACG ATA CTT TGA AAG GTT TAG GCG GAG CCA ACC ATA ATG met

FIG. 3. Nucleotide

sequence

(M1682-19b) and da182 mutant (MO2) strains with plasmids that contained the DAL7 UAS (plasmid pHY129), the UAS and URS (plasmid pHY135), or all three elements (plasmid pHY174). ,B-Galactosidase production was then measured in these transformants grown in the absence or presence of inducer. The detailed structure of the plasmids used in this experiment has been described previously (38). High levels of P-galactosidase production were supported by plasmid pHY129 in a wild-type strain (Fig. 4). The level of activity observed in the da182 mutant was lower by 35 to 50% (Fig. 4). Plasmid pHY135 did not support 3-galactosidase production in either strain. This was the expected result, because plasmid pHY135 was used to determine whether the URS element functions and the da182 mutant did not exhibit the phenotype predicted for loss of a negatively acting factor. A quite different result was observed when plasmid pHY174 was used in this experiment. Plasmid pHY174 supported 20- to 25-fold-greater ,B-galactosidase production in the wild-type strain grown in the presence of oxalurate than in its absence. In the da182 mutant, no induction was observed. Moreover, the uninduced level of lacZ expression was

10-fold depressed compared with that in the wild type

(Fig. 4). The observations just described are consistent with the

of the DAL82

gene.

suggestion that the DAL82 gene product is required for operation of the DAL7 gene UIS element. However, it is possible that a similar phenotype would be generated if inducer was excluded from the cell. To assess this possibility, we repeated the above experiment and used arginine as an inducer in place of oxalurate. Arginine is a more repressive nitrogen source than proline in this strain (unpublished observations), which accounts for the 3.5-fold-lower level of 3-galactosidase production supported by plasmid pHY129 in a wild-type strain grown in glucose-arginine medium than in glucose-proline (Fig. 4). A similar response was observed for the da182 mutant, i.e., the mutant and wild-type strains did not respond significantly differently. When plasmid pHY174 was used in this experiment, induction was again completely lost. In this instance, inducer (arginine degraded to allophanate within the cell) exclusion cannot be suggested as an explanation for loss of response in the da182 mutant strain. DISCUSSION The results of this work suggest that increased transcription observed in response to addition of inducer, a central characteristic of allantoin system gene regulation in S. cerevisiae, requires a wild-type DAL82 gene product as well

VOL. 173, 1991

ALLANTOIN SYSTEM INDUCTION IN S. CEREVISIAE +10*

+30*

259

+50*

GAT GAA TCG GTG GAT CCT GTG GAG CTG CTT CTA CGA CTA CTG ATA CGG CAC AAA CCT CAT Asp Glu Ser Val Asp Pro Val Glu Leu Lou Lou Arg Lou Lou Ile Arg His Lys Pro His

+70*

+90*

+110*

CTG AAA CCA TAT GCC TAC AGA CAA GAT AGC TGG CAA AGG GTG CTC GAT GAG TAC AAC AGA Lou Lys Pro Tyr Ala Tyr Arg Gln Asp Ser Trp Gln Arg Val Lou Asp Glu Tyr Asn Arg

+130*

+150*

+170*

CAG ACT GGG TCA AGA TAT AGA CAA TCA AGG ACG TTA AAA ACC AAA TTT CGT CGA CTG AAG Gln Thr Gly Ser Arg Tyr Arg Gln Ser Arg Thr Lou Lys Thr Lys Ph. Arg Arg Lou Lys

+190*

+210*

+230*

GAC CTC TTC AGC GCA GAT CGA GCC CAA TTC TCT CCT TCC CAG TTG AAG CTG ATG GGA GCA Asp Lou Ph. Ser Ala Asp Arg Ala Gln Ph. Ser Pro Ser Gln Lou Lys Lou Met Gly 'Ala

+250*

+270*

+290*

CTC TTG GAC GAA GCA CCA GAA CAT CCA AGA CCA AGA ACT AAA TTC GGA AAT GAA TCA TCT Lou Lou Asp Glu Ala Pro Glu His Pro Arg Pro Arg Thr Lys Ph. Gly Asn Glu Ser Ser

+310*

+330*

+350*

TCA TCC TTA TCA TCA TCT TCT TTC ATT AAA AGT CAT CCG GGG CCT GAT CCG TTT CAA CAA Ser Ser Lou Ser Ser Ser Ser Ph. Ile Lys Ser His Pro Gly Pro Asp Pro Ph. Gln Gln

+370*

+390*

+410*

TTA TCA TCC GCT GAA CAT CCG AAT AAC CAC AGC TCC GAC GAT GAG CAT TCA GGC TCA CAA Lou Ser Ser Ala Glu His Pro Asn Asn His Ser Ser Asp Asp Glu His Ser Gly Ser Gln

+430*

+450*

+470*

CCG CTG CCC CTG GAT TCA ATA ACG ATT GGA ATT CCG CCT ACT CTT CAC ACA ATC CCC ATG Pro Lou Pro Lou Asp Ser Ile Thr Ile Gly Ile Pro Pro Thr Lou His Thr Ile Pro Met

+490*

+510*

+530*

ATT CTG TCT AAG GAT AAC GAC GTC GGG AAA GTC ATC AAA AGC CCT AAG ATA AAC AAG GGT Ile Lou Ser Lys Asp Asn Asp Val Gly Lys Va1 Ile Lys Ser Pro Lys Ile Asn Lys Gly

+550*

+570*

+590*

ACA AAT AGG TTC AGC GAG ACA GTA CTG CCT CCA CAA ATG GCT GCT GAG CAA TCG TGG TCG Thr Asn Arg Ph. Ser Glu Thr Val Lou Pro Pro Gln Met Ala Ala Glu Gln Ser Trp Ser

+610*

+630*

+650*

GAC TCT AAT ATG GAA TTG GAA ATA TGT CTA GAT TAT CTT CAC AAC GAA CTC GAG GTG ATA Asp Ser Asn Met Glu Lou Glu Ile Cys Lou Asp Tyr Lou His Asn Glu Lou Glu Val Ile

+670*

+690*

+710*

AAG AAA AGG CAA GAA GAT TTT GAG TGT AAA GTT TTA AAC AAG CTC AAC ATA ATT GAG GCT Lys Lys Arg Gln Glu Asp Ph. Glu Cys Lys Val Lou Asn Lys Lou Asn Ile Ile Glu Ala

+730*

+750*

+770*

CTC CTT TCA CAG ATG AGA CCA CCC AGC CAA GGA GAT AAA ATA TAA AAA CTT CTA TTA GAT Lou Lou Ser Gln Met Arg Pro Pro Ser Gln Gly Asp Lys Ile End

+790*

+810*

+830*

ATG CTT GAT TCA TGT CCA TAT GTA TCT ATT TAT ACA AAC ATT ACG TAA TAT ACA CAG ATT

+850*

+870*

+890* TCT TCA TAT TTG GCT TCT TGC TTC CAC TAG CCT ATC CCA ACC AGG +930* +950* AGA GTC ATC CCA TCT GCC TTT GCC TTT ACT CAT TAG AGC TTT GAT AGT AGA TCT ACA TTT +970* +990* +1010* GTA TTT CCA GTC TTT GCA AAC CAG ACT GCC TGG CCC ATA TTT ACC TTC CAT TTC ACC AAG +1030* +1050* +1070* TGA TTC ATA GGC AGC CAC CAA ATC TTC ACA AAG CCA TCC ACT GTA TCA TTC CAA ACC TTT

ATA CAT GAA AGG TGC

+910*

+1090* TCA ACA ACT TCT

FIG. 3-Continued. as the DAL81 product documented previously (2, 33). Both of these proteins appear to participate in the regulatory function mediated by the DAL system UIS element. The dal81 and da182 mutants also share, to a greater or lesser

-229

-219 -218

-204 .209

extent, the property that they do not greatly (twofold or less) affect transcriptional activation mediated by the DAL system UASNTR element. Although dal81 and da182 mutants are quite similar pheB-GALACTOSIDASE

-202

195 -188 -183 .203

pHY129 -218

2,722

2,515

1,700

1,286

24

19

26

17

274

6,257

29

25

-183

pHY135 -228

-183

pHY174

-218

PRO

ARG

PRQ

ARG

2,718

792

1,698

421

301

1,520

17

9

.203

pHY129 -183

-228

pHY174

W.T. da182 -Q PROQOQXLU EBQ PRO OXLU

_

FIG. 4. Effect of a da182 mutation on the function of DAL7 cis-acting elements. Plasmids containing one or more of the DAL7 cis-acting elements were transformed into either wild-type (W.T.) strain M1682-19b or da182 mutant strain M02. Following growth in minimal medium (Difco yeast nitrogen base), glucose-proline medium (PRO), glucose-proline medium containing 0.25 mM oxalurate (PRO + OXLU), or glucose-arginine medium (ARG), the cells were harvested and assayed for ,-galactosidase activity (38). The plasmids used in this experiment have been described in detail earlier (38). Coordinates indicated in the figures are occupied by these sequences in the upstream region of the DAL7 gene. Solid, checkered, and wavy-lined areas indicate regions of the UASNTR, URS, and UIS elements, respectively.

260

OLIVE ET AL.

notypically, they exhibit several distinct properties suggesting that their cognate products carry out different functions in the induction process. The first distinguishing characteristic was observed in the complementation experiments performed during cloning. Complementation of daI81 mutations by a plasmid-borne gene was always found to be poor regardless of the plasmid copy number (2). In no case was the level of induced urea amidolyase activity in a dal81 mutant strain carrying a complementing plasmid ever greater than the wild-type level (2). This was true even when a dal81 deletion was used in the experiment to avoid potential complications caused by negative complementation (2a). In contrast, complementation of a da182 mutation by a plasmidborne gene resulted in induced levels of urea amidolyase that were nearly fourfold greater than observed in a wild-type strain (Table 2). The second distinct difference in the characteristics of the dal81 and da182 mutations was their effect on basal-level activity observed in transformants containing plasmid pHY174. This plasmid contains all three cis-acting elements identified in the DAL7 upstream region. The uninduced level of activity supported by this plasmid is not significantly affected by deletion of the DAL81 gene (Fig. 8 in reference 2). In contrast, uninduced levels were decreased 10- to 15-fold in a da182 mutant. The molecular basis for these differences is not known at present, but they provide a framework within which to study the functions performed by the proteins encoded by these genes. The data presented in this work and that published earlier concerning the DAL81 gene suggest that the DAL80, DAL81, and DAL82 genes are not organized in a regulatory cascade similar to that suggested for other metabolic systems. Although the two- to fourfold effects of daI80, dal8J, and gln3 mutations on expression of DAL82 cannot be categorically dismissed as insignificant, they are not of the magnitude that would be expected of a cascade regulatory circuit. Therefore, our current working hypothesis is that all four gene products regulate allantoin gene expression but function primarily by modulating expression of pathway structural genes rather than the regulatory genes. The suggestion that the DAL81 and DAL82 gene products function in parallel rather than as a cascade in no way precludes the possibility that they may interact to mediate the induction process, especially since both gene products have been shown to be required for the UIS-mediated function (Fig. 4) (also see Fig. 8 in reference 2). ACKNOWLEDGMENTS We thank Francine Messenguy and Evelyn Dubois for providing strain 13H9b, on which this work so heavily depended. We thank Thomas Cunningham, who developed the pulsed-field electrophoresis methods in our laboratory and assisted in the experiment presented in this work. We also appreciated the efforts of the University of Tennessee yeast group, who read the manuscript and offered suggestions for its improvement. The oligonucleotides used in this work were provided by the University of Tennessee Molecular Resource Center. This work was supported by Public Health Service grant GM-35642. REFERENCES 1. Baum, J. A., R. Geever, and N. Giles. 1987. Expression of qa-IF activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol. Cell. Biol. 7:1256-1266. 2. Bricmont, P. A., and T. G. Cooper. 1989. A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation. Mol. Cell.

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