MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 1161-1166

Vol. 11, No. 2

0270-7306/91/021161-06$02.00/0 Copyright © 1991, American Society for Microbiology

NOTES The DAL81 Gene Product Is Required for Induced Expression of Two Differently Regulated Nitrogen Catabolic Genes in Saccharomyces cerevisiae PATRICIA A. BRICMONT, JON R. DAUGHERTY, AND TERRANCE G. COOPER* Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163 Received 5 September 1990/Accepted 25 October 1990

We demonstrate that the DAL81 gene, previously thought to be specifically required for induced expression of the allantoin pathway genes in Saccharomyces cerevisiae, functions in a more global manner. The data presented show it to be required for utilization of 4-aminobutyrate as a nitrogen source and for 4-aminobutyrate-induced increases in the steady-state levels of UGAl mRNA. The DAL81 gene encodes a 970-amino-acid protein containing sequences homologous to the Zn(ll)2Cys6 motif and two stretches of polyglutamine residues. Deletion of sequences homologous to the Zn(H)2Cys6 motif did not result in a detectable loss of function. On the other hand, loss of one of the polyglutamine stretches, but not the other, resulted in a 50% loss of DAL81 function.

Genetic and biochemical studies of Saccharomyces cerevisiae yield increasing amounts of data supporting the idea that some transcriptional regulatory factors are required for expression of multiple, unrelated genes (ABFl and RAP]), whereas others are specific to single metabolic pathways or commonly regulated cell biological processes (GAL4) (6, 15, 29). Like the GAL4 gene product, the DAL81 and DAL82 gene products have heretofore been thought to function as specific regulatory factors of the allantoin degradation pathway (5, 21, 32). Expression of several genes required for allantoin degradation has been shown to be induced by the presence of allophanate or oxalurate and to be sensitive to nitrogen catabolite repression (8, 9, 34). Cis-acting elements that mediate induction and nitrogen catabolite repression have been identified and shown to be situated upstream of the mRNA start site of the DAL5 and DAL7 genes (26, 36). Three different elements have been characterized: an upstream activation sequence (UAS) responsible for transcriptional activation of the gene, an upstream repression sequence (URS) responsible for maintaining transcription at a low level when the inducer is absent, and an upstream induction sequence (UIS) required for response to the inducer (26, 36). Point mutations at the dal81 locus, located on the right arm of chromosome IX, were shown to result in poor growth when allantoin was provided as the sole nitrogen source and in a loss of induced allantoin-degradative-enzyme production following the addition of oxalurate to the culture medium (32). Growth of the dal8J mutants on several other nitrogen sources was not affected; arginase induction was also normal. These observations suggested that the DAL8J gene product has an allantoin pathway-specific function. Complete deletions of the DAL8J gene have recently been constructed in vitro and used to demonstrate that the protein encoded by this gene functions predominantly in association *

with the UIS element of the DAL7 regulatory region (5). Neither UAS nor URS-mediated functions were significantly affected in the mutant strains. Recently, utilization of 4-aminobutyrate (GABA) as a nitrogen source by S. cerevisiae has been characterized (19a, 27, 33). GABA degradation is reported to require two enzymes encoded by the UGAl and UGA2 genes (27). Expression of UGAI and UGA2 is induced by the addition of GABA to the culture medium, and this induction has been shown to be lost in strains with mutations at the UGA3 and UGA35 loci (27, 33). Mutations at the uga35 locus have been reported to be allelic to a mutation at a locus designated DURL (33). DURL is one of two loci identified by Wiame and colleagues on the basis of poor use of urea as a nitrogen source, a characteristic shared by four mutant classes, including strains with mutations at dal8l and da182 (14, 21, 32). The phenotypic similarity of the dal81 and durL mutants raised the possibility that mutations at the two loci might be allelic. Since DURL has not been cloned and the durL mutant strains are not available for allelism tests, we determined whether dal8l mutants possessed a defect in GABA utilization. If pleiotropic effects could be demonstrated for dal81 mutants, it would suggest a more general role for this putative transcription factor. These experiments and experiments on the DAL81 structure are described below. (A preliminary report of this work has already been presented

[4].)

Culture conditions and preparative methods. Yeast and bacterial strains used in this work have been described previously (5, 21). Yeast strain PB200 is isogenic to M168219b, except that it contains a chromosomal deletion of the DAL81 gene. Culture conditions were described previously (5, 21). Total yeast RNA was isolated by the method of Carlson and Botstein (7). Poly(A)+ RNA was isolated by using oligo(dT) cellulose and resolved on 1.4% agaroseformaldehyde gels (18). The mRNA was transferred to GeneScreen Plus Nylon 66 membranes (Dupont, NEN Re-

Corresponding author. 1161

1162

MOL. CELL. BIOL.

NOTES

X_a_ 1~~~~~~~~~~~~~~~~ BamH

/ Partal DAL81

p/

PU

SaW

_ i. 2. 3. 4.

Denature DNA. Anneal with 6 indvidua sets of primers. Perform 6 poldyrae chain reactions (PCR). Lgate applreiat pairs of PCR producs toger using m emn restnicin enzyme sites. 5. Digest PCR product wSst I and Xba I and isoa the foll g fagmet: A) 1381b B) 1.42kb C) 1.39kb

pPB62I Digest pPB62 wih Sst I and Xba and iolate e 6.2 kb fragment

I

set

A) B)

XbaI I

_

Xba I

Sst m

Xba I

Sst I

C)

1. Ligate each of hse faments with te 6.2 kb Sst - Xba I fragment of pPB62. 2. IWenify the desired done using resticton

enzyme dgeston and sequence ana*sis.

II Pvu l Sa

A) 1. DigwstwiFthSal I and am iHl 2. Isolate 9.2 kb ftragment

B) C) C

Ht I Xbasam

S

SWl set qal

1w

Hl _

Xba I Bam H

Lgate each frgment wi te 92kb Sst I - Bam HI fament of pPB71

A) PVUI

FIG. 1. Strategy used to generate deletions of structural motifs of the DAL81 protein.

search Products) and hybridized to radiolabeled DNA probes according to the manufacturer's instructions. Standard cloning procedures were used, and the yeast vectors have been described elsewhere (5, 18). Plasmids containing precise deletions of portions of the DAL81 coding sequence were constructed (Fig. 1 and 2). The polymerase chain reaction was employed to amplify the DNA immediately flanking the sequences to be removed (20). The structures of all deletion plasmids were verified by DNA sequence analysis with the method of Sanger (31). DAL81 requirement for induced UGAI gene expression. Mutations at the dal8J locus result in a decreased ability to use allantoin as the sole nitrogen source (32). Deletion of the DAL81 gene also results in a diminished ability to use GABA as the sole nitrogen source (Table 1), as was previously reported for allantoin (32). To ascertain whether the loss of GABA utilization correlated with a decrease in steady-state levels of a GABA-degradative mRNA, we determined the

levels of UGAI mRNA in wild-type (M1682-19b) and dal8J deletion mutant (PB200) strains. High-level (18-fold) UGAI induction was observed in cultures grown in glucose-GABA medium compared with that in cultures grown in glucoseproline medium (Fig. 3, lanes A and B). A less-than-twofold increase in UGAI mRNA was observed, however, when this experiment was repeated with a dal8J deletion mutant (Fig. 3, lanes C and D). Nucleotide sequence analysis of the DAL81 gene. We determined the nucleotide sequence of DAL81 and its flanking regions (3,700 bp) (Fig. 4). Computer analysis resulted in identification of a single open reading frame of 2,910 bp encoding a 970-amino-acid protein with a calculated relative molecular weight of 109,119. Consistent with the observation that cellular DAL8M mRNA is present at a very low concentration (5), we found a codon usage bias index of 0.06 for DAL81, which is similar to the indices observed for the LEU3 (0.02) and PPRI (-0.9) genes (2, 13, 16).

NOTES

VOL . 1 l, 1991

J4

W. T.

GGA-AG.tfrGATCCTCcT1XCTCCCT

602

Set

+217 - .282

MG

217-+212

AAG

Lys

#10 TCAMAGtCACAGTGATGTAGGCAATTC

LOU

m

CCAAGCTTAGCTTGGATACCCTiTTC

#1 1 GTGAGTTCTCTATAAGAATACT

ilm

Sel l +1

3'

24 +W217

+272

Xba

#10 TCACzAGAGTGATGTAGGCMTTC

69+

314

Pdm#11

Prlm,s.-

Sst l

+679 .711

+1 PAI_#10

+1025 3.

PrW

GGGGTACCAGATCCCATCAGATTACC

833

st

#10 TCAGAGCTCACAGTGATGTAGGCMTTC 71 AMn go CChGAGAAACGTCAAGMCTMATGC

611 GTGAGTTCTCTATMAGATCTAGAATACT

+448 - 516

+448- +516

509

p

GGT ACC Gly Th

GGTACC Gy Th

0

+1025

IIIseXa

FIG. 2. Polymerase chain reaction primers used to generate DAL81 deletions, with their locations and the amino acid substitutions that resulted at the point of deletion.

At position -159, upstream from the open reading frame, is a TATAA sequence. Sequences with less homology to the TATAA sequence also appear at -135 and -205. A striking characteristic of the predicted protein structure was the occurrence of multiple homopolymeric residues in the amino-terminal region (underlined residues in Fig. 4). In the first stretch (amino acids 28 to 41), 9 of 12 residues were asparagine. A second stretch of asparagine residues was situated at residues 130 to 138. Polyglutamine stretches were observed at positions 73 to 94 and 227 to 237. Comparison of the predicted DAL81 amino acid sequence (residues 149 to 179) with those of other fungal regulatory proteins revealed a sequence closely resembling the Zn(II)2Cys6 structure observed in several known and postulated eucaryotic DNA-binding proteins (15, 23) (Fig. 5A). A second structure correlated with DNA binding is the helixturn-helix motif first reported for procaryotic DNA-binding proteins (22). This motif has also been reported for the MATa2 protein of S. cerevisiae (24). Figure SB depicts various proteins predicted to contain this motif and the homologous region of DAL8M (amino acids 464 to 483). The TABLE 1. Growth of wild-type and daI81 deletion mutant strains on various nitrogen sources Doubling time (min)

Allantoin y-Aminobutyrate Glutamic acid Ammonia

Asparagine Glutamine

A B

Xba I

so-' +448 +516

Pik sr#10

Nitrogen source

ii

GA+A TTC

n? GGMIrCCGTATACAATACCCCAGA #11 GTGAGTTCTCTATAAGATAGAATACT

+I

>

GAA TTC G Ph.

+879 - +711

064

EGoo d

SdtI