MOLECULAR AND CELLULAR BIOLOGY, Dec. 1991, p. 6216-6228

Vol. 11, No. 12

0270-7306/91/126216-13$02.00/0 Copyright © 1991, American Society for Microbiology

Sequence and Expression of GLN3, a Positive Nitrogen Regulatory Gene of Saccharomyces cerevisiae Encoding a Protein with a Putative Zinc Finger DNA-Binding Domain PATRICIA L. MINEHART AND BORIS MAGASANIK* Department ofBiology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 31 May 1991/Accepted 7 September 1991

The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergilus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLNl, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine

availability. single finger of NIT2, a nitrogen regulatory transcription factor of Neurospora crassa which has been shown to bind DNA (16, 17). Immunoprecipitation experiments with a GLN3 polyclonal antibody indicated that the GLN3 protein binds the nitrogen upstream activation sequence (UAS) of GLNI, the structural gene for GS.

In Saccharomyces cerevisiae, a number of proteins involved in nitrogen metabolism are coordinately regulated in response to the nitrogen source. In particular, the activities of glutamine synthetase (GS), NAD-linked glutamate dehydrogenase (NAD-GDH), and the general amino acid permease are greatly increased upon shifting cells from a medium containing glutamine to one containing glutamate as the sole nitrogen source (11, 38). The increased synthesis of these proteins during growth on glutamate is due to increased transcription of the corresponding genes and is dependent on the product of the GLN3 gene (1, 8, 35, 40, 52). Recent work has shown that GLN3 is also required for transcriptional activation of genes involved in the metabolism of allantoin (7). Genetic analysis has shown that, in addition to GLN3, another gene, URE2, is involved in the glutamine-glutamate regulation. While mutations in GLN3 lead to low levels of GS, NAD-GDH, and the general amino acid permease (10, 40), mutations in URE2 lead to high constitutive levels of these proteins (11, 13, 18, 57). Thus, GLN3 is a positive regulator and URE2 is a negative regulator. Since a gln3 ure2 double mutant has a gln3 phenotype, the URE2 product is thought to repress GLN3 or to inactivate the GLN3 product (8, 11). In this study, we cloned the GLN3 gene as a preliminary step in the further examination of its role in the nitrogen regulatory response. Sequencing of GLN3 showed that the N-terminal domain of the protein is highly acidic and that the central portion of the protein contains a single putative zinc finger highly homologous to the two zinc fingers in the transcription factor GATA-1 (14, 44, 55, 56) which binds to GATA consensus elements in regulatory regions of erythroid cell-specific genes (32, 60). The zinc finger of GLN3 also shows homology to the single zinc finger of AREA (25), an Aspergillus nidulans nitrogen regulatory factor, and to the *

MATERIALS AND METHODS

Strains and genetic methods. The S. cerevisiae strains used in these studies were derived from the Belgian strain 11278, the American strain S288C, or from hybrids of these two strains. 736 strains are 11278 derivatives; DBY2062 is an S288C derivative. SM14-6B and 179-2D strains are derived from one 1.1278 parent and one S288C parent. The remaining strains, including P5-SD, are three-fourths S288C background and one-fourth 1.1278 background (Table 1). Standard methods for mating, sporulation, and tetrad analysis were used (42, 50). Media. All media were made as described previously (39, 50). The synthetic complete media lacking either uracil (SC-ura) or leucine (SC-leu) are as described previously (50), except that either 0.128% glutamate or 0.3% glutamine was substituted for ammonium sulfate as the nitrogen source. Transformations. Yeast cells were transformed by the lithium acetate method (23). Directed integration of plasmids into the yeast chromosome was as described by Orr-Weaver et al. (43). Escherichia coli transformations were as previously described (30). Preparation and analysis of DNA and RNA. Yeast plasmid preparations were as described previously (58). E. coli plasmid minipreparations used the boiling method (22). 32P-labeled probes for Northern (RNA) experiments were prepared by nick translation according to the manufacturer's instructions (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Total yeast RNA was isolated as described by Carlson and Botstein (4), using 10-ml cultures grown to a

Corresponding author. 6216

VOL. 11, 1991

GLN3, A POSITIVE NITROGEN REGULATORY GENE

6217

TABLE 1. Yeast strains and plasmids Strain or plasmid

Yeast strains 179-2D PM6 PM7 PM28 PM31 P5-5D DBY2062 SM14-6B PM34 PM35 PM36 PM38 PM39 PM43

736-liD 736-12A PM48 PM49

Strains isogenic to PM38 PM63 PM64 PM65 PM66 PH2 PM71 PM73 PM76 PM77 PM79 PM80 PM81

Plasmids pPM1 pPM2 pPM4 pPM7 pPM8 pPM10 pPM11 pPM35 pPM46 pPM49 pPM60 pPM62 a All strains and plasmids for which

Genotype

MATa ade2-102 ura3-52 gln3-1 gdhl-6 179-2D(pPM1) 179-2D(pPM2)

179-2D(YCp5O) 179-2D(pPM8) = gln3-1::GLN3 MATa ura3-52 ade2-102 gdhl-6 ure2-1 MATa leu2-3,112 his4-619 MATa ade2-102 ura3-52 gdhl-6 DBY2062 x SM14-6b (parent to PM35, PM36, PM38) MATa leu2-3,112 ade2-102 gdhl-6 MATot his4-619 ura3-52 gdhl-6 MATa ura3-52 leu2-3,112 PM35 x PM36 (parent to PM43) MATa ura3-52 ade2-102 gdhl-6 gln3A4::LEU2 MATa ade2-102 ura3-52 MATa ade2-102 ura3-52 gln3-1 cani 736-12A(YCp5O) 736-12A(pPM4)

PM38(YCp5O) PM38(pPM4) PM38(YEp24) PM38(pPM7) MATTa ura3-52 leu2-3,112 ure2Al2:: URA3 MATa ura3-52 leu2-3,112 gln3A5::LEU2 MATa ura3-52 leu2-3,112 gln3A4::LEU2 PM71(pKP15) PM71(pPM49) PM38(pSLF178K) PM38(pPM46) MATTa ura3-52 leu2-3,112 gln3A6::URA3

W. Courchesne

8 D. Botstein 34

1

8

25-kb yeast genomic fragment containing the GLN3 gene in YCp5O 17-kb yeast genomic fragment containing the GLN3 gene in YCp5O 5-kb AatII GLN3-containing fragment from pPM2 in YCp5O 5-kb AatII GLN3-containing fragment in YEp24 5-kb AatII GLN3-containing fragment in YIp5 5-kb AatII GLN3-containing fragment in pBR322 2.2-kb SalI-XhoI fragment containing LEU2 (from YEp13) replacing the 3-kb XhoI-NcoI fragment in pPM10 472-bp ApaI-XhoI fragment deleted from pPM11 2[tm GLN3-1acZ fusion plasmid 2pm GALJO-CYC1-GLN3 fusion plasmid trpE-GLN3 fusion plasmid 1.2-kb HindIIl fragment containing URA3 replacing the 76-bp XhoI-XbaI fragment of pPM10 no source

is listed

were

constructed for this study in this laboratory.

density of 107 cells per ml. RNA blot hybridization analysis previously described (5). Plasmids. All subclones to test for GLN3-complementing ability were made in the low-copy vector YCp5O (26). The 5-kb AatII restriction fragment carrying the GLN3 gene was also subcloned into the high-copy vector YEp24 (3), creating pPM7, and into the integrating plasmid YIp5 (53), creating pPM8. Subclones for sequencing were cloned into Bluescript vectors (Stratagene, La Jolla, Calif.). Plasmids used to isolate fragments for probes used in Northern analysis were as follows: for pyruvate kinase, pFR2, a clone containing the pyruvate kinase gene PYKI (5); for P-tubulin, pJT71, a clone containing the TUB2 gene (kindly provided by Jim Thomas); and for GLN3, pPM10, the pBR322 (2) clone containing the 5-kb AatII GLN3 fragment. The 5-kb AatII fragment and was as

Source or referencea

various internal probes including a Sau3A fragment from nucleotide 2221 to nucleotide 2603 were used as probes for GLN3. Plasmids used in antibody preparation and in the immunoprecipitation experiments are described in those sections of the Materials and Methods. Cloning of GLN3. A YCp5O-based clone bank derived from genomic yeast DNA partially digested with Sau3AI (gift from Karl Pfeiffer) was transformed into the ade2-102 ura3-52 gln3-1 gdhl-6 strain 179-2D, and Ura+ transformants were selected on plates containing glutamine as the nitrogen source. A total of 24,000 Ura+ transformants were replica plated to minimal glucose-ammonia-adenine plates to select for colonies that could grow with ammonia as the sole nitrogen source. In 10 of 14 candidates selected for their ability to grow on ammonia, simultaneous loss for this ability

6218

MOL. CELL. BIOL.

MINEHART AND MAGASANIK TABLE 2. Complementation of gln3 phenotypea

Strain

Relevant genotype

GS transferase (p.mol/min/mg) Glt + Ade

736-liD PM28 PM6 PM7 PM31 PM48 PM49

Wild type gln3-1 gdhl-6(YCpSO) gln3-1 gdhl-6(pPM1) gln3-1 gdh1-6pPM2) gln3-1::GLN3 gdhl-6 gln3-l(YCp5O) gln3-1(pPM4)

2.5 0.3 3.4 3.4 3.6 0.4 2.6

± 0.1 ± 0.03 ± 0.1

0.1 ± 0.1 ± 0.1 ± 0.3

NAD-GDH (nmol/min/mg)

(3h + Ade

Glt + Ade

.3+0.01 b.b2 ± 0.01 0.06 ± 0.02 0.06 ± 0.04 0.05 ± 0.01 0.03 ± 0.02 0.09 ± 0.02

47 3 29 35 43 7 98

± ± ± ± ± ± ±

4 2 8 2 5 3 3

Gln + Ade

4 5 4 4 5 3 3

± ± ± ± ± ± ±

2 2 2 2 2 4 3

NADP-GDH (nmol/min/mg) Glt + Ade

105 2 ± 5± 5 5 ±

5 1 3 3 3

NDb ND

Gln + Ade

144 ± 5 4 ± 3 4± 3 4±4 4± 2 ND ND

a Midexponential cultures growing in the indicated minimal media were harvested, and GS transferase, NAD-GDH, and NADP-GDH specific activities were determined. Adenine (Ade) was added to a final concentration of 0.15 mM in both glutamate (Glt) and glutamine (Gln) media. Values indicated are the average of at least three determinations. b ND, not determined.

and the Ura+ phenotype was observed, indicating that the complementing activity was carried on the recombinant plasmid. These 10 candidates were then grown under conditions derepressing for NAD-GDH and GS, that is, with glutamate as the major nitrogen source, and assayed for NADP-GDH, NAD-GDH, and GS activities. Putative GDHI clones were distinguished from GLN3 clones by enzyme assays for NADP-GDH, NAD-GDH, and GS. In the gln3-1 gdhl-6 double-mutant strain, all three enzyme activities are low. A GDHI clone should restore NADP-GDH activity, whereas a GLN3 clone should restore both NAD-GDH activity and GS activity. Four transformants had wild-type levels of NADPGDH activity and low levels of NAD-GDH and GS activities and presumably carried GDHI clones. Two transformants, PM6 and PM7, had wild-type levels of NAD-GDH and GS activity and low levels of NADP-GDH activity when grown with glutamate as the source of nitrogen and thus carried candidate GLN3 clones designated pPM1 and pPM2, respectively (Table 2). These two candidate clones were passaged through E. coli HB101, and the plasmids obtained were used for further experiments. Construction of GLN3 null alleles. To make the first deletion allele, we replaced the XhoI-NcoI fragment in pPM10 with a 2-kb SaI-XhoI restriction fragment carrying LEU2, creating pPM11. To make the second deletion allele, we deleted the ApaI-XhoI fragment carrying the remaining 5' GLN3 coding region in pPM11, creating pPM35. Since the NcoI site was outside of the GLN3 gene, a third deletion allele which was completely internal to GLN3 was constructed by replacing the XhoI-XbaI fragment of pPM10 with a 1.2-kb HindIII fragment carrying the URA3 gene, creating pPM62. The AatII fragment of all three constructs was then used to substitutively transform either the diploid strain PM39 or the haploid strain PM38 (48). Either URA+ or LEU+ transformants were selected on SC-ura or SC-leu plates. GS and NAD-linked GDH were assayed to score for the GLN3 phenotype. Construction of GLN3-lacZ fusion. To construct the GLN3lacZ fusion plasmid pPM46, we treated the 2,um vector pLGA312 (19) with the restriction enzymes SmaI and BamI to delete the CYCI-carrying fragment. The resulting vector was then treated with the large fragment of DNA polymerase I so that the DNA ends would be flush and with alkaline phosphatase to prevent religation of the vector. A 976-bp AatII-PvuII fragment was then isolated from pPM10. This fragment was treated with bacteriophage T4 DNA polymerase to render the DNA ends flush and then ligated into the pLGA312 vector. The resulting plasmid contains 730 nucle-

otides upstream from the GLN3 translational start site and the first 82 amino acids of GLN3. lacZ is fused in frame at amino acid residue 83, which contains one nucleotide from GLN3 and two nucleotides from lacZ. The junction was verified by sequence analysis. pSLFA178K, which was used as the control plasmid in the experiments with the GLN3lacZ fusion, is a derivative of pLGA312 in which the SmaIXhoI fragment carrying the CYCI UAS has been deleted

(15).

Construction of GAL1O-CYCI-GLN3 fusion. To construct pPM49, in which the GLN3 structural gene is under control of the GAL1O-CYCI hybrid promoter, we used pKP15 (kindly provided by Karl Pfeiffer) as the vector. pKP15 is a derivative of pLGSD5 (20), in which the lacZ gene has been deleted and in which a SmaI linker has been inserted downstream of the GALIO UAS and CYCI promoter. An approximately 4-kb ApaI GLN3-containing fragment was isolated from pPM32, a Bluescript plasmid containing the GLN3 gene. This ApaI fragment, which contains the GLN3 structural gene and 26 nucleotides upstream of the translational site, was treated with bacteriophage T4 DNA polymerase to render the DNA ends flush and was then subcloned into the SmaI site of pKP15. DNA sequencing. DNA was sequenced by the dideoxychain termination method (49) with [a-35S]dATP and a modified T7 bacteriophage DNA polymerase, Sequenase (United States Biochemical Corp., Cleveland, Ohio). Double-stranded plasmid DNA templates were prepared by purification over a CsCl gradient (30). All plasmids used for sequencing GLN3 were in the Bluescript KS+ vector (Stratagene). Primers used for sequencing GLN3 hybridized to the KS polylinker and were purchased from Stratagene. Both strands of the 3-kb AatII-DdeI fragment were sequenced by using overlapping subclones. The GLN3-lacZ fusion junction of pPM46 was sequenced by using a primer which hybridized 17 to 36 nucleotides downstream from the BamHI cloning site and was a gift from L. Guarente. Computer methods. Analysis and translation of sequences were done with Macintosh software. Protein homology searches were conducted courtesy of Daphne Preuss using FastP (29) against the data base at Genentech, which contains NBRF (Georgetown University Medical Center, Washington, D.C.) sequences and the translation of GenBank nucleic acid sequences (Los Alamos National Laboratory, Los Alamos, N.M.). Enzyme assays. For all enzyme assays, extracts were prepared as by Mitchell and Ludmerer (37) and used immediately. NAD-GDH and NADPH-GDH were assayed as described previously (34), and GS transferase was assayed

VOL . 1 l, 1991

by the method of Mitchell and Magasanik (38), with the modification that time points were taken at 2 and 14 min. 3-Galactosidase assays were done as described by Rose and Botstein (47), and the specific activity (nanomoles per minute per milligram of protein) was calculated as described by Miller (33). Assays were done with 30 and 60 plI of extract and normalized to total protein. Protein concentrations for all assays were calculated by using the Bradford protein assay kit (Bio-Rad Laboratories, Richmond, Calif.). Generation of antibody. To begin to identify the GLN3 protein, a portion of the GLN3 gene was fused in frame to the E. coli trpE gene, which codes for anthranilate synthase (59). An HpaII fragment encoding amino acid 43 to amino acid 435 of GLN3, which includes the putative zinc finger, was fused at the ClaI site of pATH3, an E. coli plasmid that carries the trp operon promoter and most of the trpE gene (24, 51). Upon expression in E. coli, the resulting plasmid, pPM60, should produce a hybrid protein containing the amino-terminal 323 residues of anthranilate synthase and 393 residues of GLN3. Plasmid pPM60 was transformed into E. coli RR1, and expression of the hybrid protein was induced by tryptophan starvation as described by Koerner et al. (24). As initial analysis indicated that the fusion protein was not fractionated into the insoluble portion but remained fairly equally distributed between the insoluble and soluble fractions, whole-cell lysates were used for the preparation of protein samples. An approximately 90-kDa protein seen only in samples from cells carrying the hybrid plasmid pPM60 was electroeluted by using the Bio-Rad system. Antibody to GLN3 was raised in two New Zealand White rabbits against the electroeluted protein. The specificity of the antibody was tested by Western blotting (immunoblotting) (54). Cell extracts for Western blotting and immunoprecipitation assay. For strains carrying galactose-inducible plasmids, 25-ml cultures were grown in minimal medium containing 2% raffinose as the carbon source and 0.128% glutamate as the nitrogen source. Adenine was also added to a final concentration of 0.15 mM. When the cells reached Klett 85, galactose was added to a final concentration of 3% and the cells were grown for two more hours. Strains which did not carry a galactose-inducible plasmid were grown to Klett 90 to 100. Extracts were prepared by the method of Mitchell and Ludmerer (37) by disruption with glass beads. DNA probes for immunoprecipitation assay of DNA binding. Three DNA probes were tested for the ability to bind GLN3. The first probe was a 108-bp SalI-AvaII fragment which contains the nitrogen UAS of GLNJ (36). This fragment was isolated from pIB16, a Bluescript KS plasmid containing the 484-bp SalI-EcoRI fragment of GLNJ (36). The second probe was a 224-bp SmaI-XhoI fragment consisting of a 32-bp sequence repeated seven times. This 32-bp oligomer contains the repeated GATAA motif thought to be the GLN3-binding site. This fragment was isolated from plasmid pPM190#4, a pSLFA178K derivative (36). The third probe, used as a nonspecific control, was a 222-bp AvaIl fragment from the Bluescript KS plasmid. The first probe was end labeled with [a-32P]dCTP, [a-32P]dGTP, and [a-32P]dTTP by fill-in reaction with Klenow fragment. The second probe was end labeled with [a-32P]dCTP and [a-32P]dTTP, while the third probe was end labeled with [o-32P]dATP and [a-32P]dGTP by the same fill-in reaction. All probes were purified by acrylamide gel electrophoresis. Immunoprecipitation of GLN3 protein-GLNI DNA complexes. The 40-,ld binding reaction mixtures contained 4 mM

GLN3, A POSITIVE NITROGEN REGULATORY GENE

6219

Tris-HCl (pH 8.0), 40 mM NaCl, 4 mM MgCl2, 5% glycerol, and 40 pug of protein from total cell extracts. DNA was added to 10,000 cpm. Reaction mixtures were incubated for 30 mi at room temperature. A 0.5-pI portion of either preimmune serum or GLN3 antiserum was added to the mixtures, which were then incubated on ice for 1 h. Next, 10 RI of a 10%o solution of fixed Staphylococcus aureus Cowan I in the gel binding buffer was added to the mixtures, and incubation on ice continued for 30 min. Cells were pelleted by a 1-min spin in an Eppendorf centrifuge. Pellets were washed twice in the gel binding buffer, and Cerenkov counts were determined. Nucleotide sequence accession number. The sequence reported here has been deposited in the GenBank data base and has been given number M35267.

RESULTS GLN3-carrying plasmids. Two candidate clones, pPM1 and pPM2, were obtained which conferred wild-type levels of NAD-GDH and GS activity during growth with glutamate as the source of nitrogen (see Materials and Methods and Table 2). When transformants carrying these clones were grown with glutamine as the major nitrogen source, they had low levels of GS and NAD-GDH characteristic of the wild type. These two clones did not restore NADP-GDH activity (Table 2). The two GLN3 candidate clones, pPM1 and pPM2, were also shown to complement the growth defect of gln3-2 gdhl-6 and gln3-3 gdhl-6 strains. Subcloning localized this complementing activity to the approximately 3.5-kb region between AatII and NruI, a region which was common to the two clones. and is shown in Fig. 1. The complementing activity of pPM4, a YCp5O derivative carrying the 5-kb AatII-AatII fragment, was measured by GS and NAD-GDH enzyme assays in strain 736-12A, a gln3-1 GDHI+ strain (Table 2), in addition to determining its ability to confer growth in the gln3-1 gdhl-6 double-mutant strain. To confirm that this AatII fragment contained the GLN3 gene, we cloned the 5-kb fragment into the yeast integrating vector YIp5 (53). The resulting plasmid, pPM8, was cut with XbaI to direct integration and was transformed into the gln3-1-carrying strain 179-2D. The integrated clone restored NAD-GDH and GS activity (Table 2, see PM31). The site of integration was then mapped by crossing PM31 (179-2D integrant 1) with the GLN3+ ura3-52 strain PM30. Twenty tetrads were grown on glutamate and assayed for NADGDH and GS activity. All spores segregated 2:2 for Ura+: Ura-, and all had wild-type activity for NAD-GDH and GS, indicating that the map distance between the GLN3 locus and the site of integration is less than 2.5 map units. We concluded that this AatII fragment contains the GLN3 gene. Null alleles. Although one of the gin3 alleles, gin3-1, was known to be a nonsense allele (40), we wanted to construct a definitive null allele. Three deletion alleles were constructed (see Materials and Methods) and are diagrammed in Fig. 1. The first allele (Fig. 1, pPM11, gin3A4::LEU2), which by sequence analysis shown below carries the first 147 amino acids of the GLN3 protein, was introduced into PM39, a GLN3 wild-type diploid, by substitutive transformation (48). Tetrad dissection of a sporulated transformant resulted in 2Leu+:2Leu- spores, indicating that GLN3 is not essential for growth. GLN3 activity, as measured by GS and NADGDH enzyme activity levels, also segregated 2:2. In every case, the GLN3 activity segregated with the Leu- phenotype. This gln3::LEU2 allele was recessive and failed to

6220

MINEHART AND MAGASANIK

MOL. CELL. BIOL.

Plasmid

Complemntation

pPM4 pPM7 pPM8 pPM10

pIBlO

IH

i

I

I 4

m

I

II1 H H

11

I I

I

a^

-1

I

0

H

+

M M

m m m

04 0

4i

it z0

A

F

pPM3A

i//

pPM3B

ff/

+

pPM12

pPM11 (gln3A4: :LEU2)

pPM35 (gln3Av5:: LEU2)

LEU 2

pPM62 (gln3A6: :URA3)

I-

LEU2

-L

URA3 I1.0 kb

I

FIG. 1. Restriction map of clones containing GLN3 sequences. Plasmids are described in Materials and Methods. Only the cloned DNA segment is indicated. Restriction sites within the LEU2 and URA3 fragments are not shown. The top line represents GLN3 DNA and its restriction sites. The ability of the low-copy episomal plasmids to complement the growth defect of a gln3 gdhl double mutant is indicated. pPM4 was also tested by enzyme assays for its ability to complement a gln3 single mutation. The arrow indicates the approximate position

and direction of transcription of the GLN3 RNA established by sequencing and analysis of a partially deleted GLN3 nonlinearity in scale; *, not a unique site.

complement the other three gln3 alleles, further indicating that the gene cloned was the wild-type GLN3 gene. A second deletion allele (Fig. 1, pPM35, gln3AS5::LEU2), which had no coding sequences and no detectable message by Northern analysis (see Fig. 5), and a third deletion allele (Fig. 1, pPM62, gln3A6::URA3), which is completely internal to the coding sequence, were both introduced by substitutive transformation directly into the wild-type haploid

gene

(see below). ll,

strain PM38. Again, neither was lethal and both conferred the same phenotype as the first deletion allele (Table 3). Purine control of GLNI remains intact in gln3 disruption. It had been shown previously that the gln3-1 mutation does not entirely eliminate the ability of the cell to increase the level of GS in response to growth on glutamate rather than glutamine as the source of nitrogen (41). The results presented in Table 3 show that mutants with deletions of the

TABLE 3. Effect of disruptions of GLN3 on GS and NAD-GDHa Strain

GS transferase

Relevant

genotype

Glt

(>mol/min/mg)

NAD-GDH (nmollmin/mg)

Glt + Ade

Gln

Glt

Glt +

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

Ade

PM38 Wild type 2.3 0.1 71 10 2.0 0.2 0.05 0.01 48 10 PM73 gln3A4::LEU2 0.9 ± 0.1 0.3 0.1 0.05 0.01 8 2 3 1 PM71 gln3A5::LEU2 0.9 + 0.1 0.3 0.1 0.05 0.01 3 2 10 3 PM81 1.0 ± 0.1 gIn3A6::URA3 0.3 0.1 0.05 0.01 12 4 4 3 a Midexponential cultures growing in the indicated media with either glutamate (Glt) or glutamine (Gln) as the sole nitrogen source were harvested, +

Gln 1± 2± 2+ 2+

1 1 2 2

and GS transferase and NAD-GDH specific activities were determined. Where indicated, adenine (Ade) was added to a final concentration of 0.15 mM. Values indicated are the average of at least three determinations.

VOL. 1 l, 1991

GLN3, A POSITIVE NITROGEN REGULATORY GENE 2

2.4kbb

6221

IA IS 2A 2B 3A 3B

GLN 3 PYK2

0.4kb_..

FIG. 2. Northern analysis of GLN3-specific RNA in wild-type strain PM35 (lane 1) and in the gln3A4::LEU2 strain PM43 (lane 2). Cells were grown in minimal medium with glutamate as the sole nitrogen source. Total RNA was isolated, and 10 ,ug of each sample was run per lane. The GLN3 probe was the 5-kb AatII fragment isolated from pPM10. Transcript sizes were determined by using ethidium bromide-stained RNA size markers purchased from Bethesda Research Laboratories, Inc. (Gaithersburg, Md.).

GLN3 gene also have higher levels of GS when grown with glutamate as opposed to glutamine as source of nitrogen. Therefore, GLN3-independent stimulation of GLNI expression can occur. It can also be seen that in gln3 deletion cells, as was shown for gln3-1 cells, the level of GS in glutamategrown cells is reduced by the addition of adenine to the growth medium. The increased expression of GLN1 in gIn3 ADE+ strains apparently results in part from a deficiency of adenine, whose synthesis requires glutamine. It had also been shown that in both wild-type and gln3-1 mutant cells grown on glutamine, starvation for histidine, whose synthesis also requires glutamine, increases GLNJ expression and that this increase in GLNI expression is dependent on the product of the GCN4 gene (21, 41). The fact that the level of GS in cells of the gln3 deletion strains grown in adeninesupplemented glutamate medium is still higher than that in corresponding cells grown in glutamine-containing medium presumably reflects a partial amino acid deficiency which leads to activation of GLNI expression by the product of GCN4. Addition of adenine also slightly decreased the levels of NAD-GDH in both the wild type and the gln3 strains grown on glutamate (Table 3). High-copy GLN3 phenotype. The AatII fragment carrying the GLN3 gene was cloned into the high-copy vector YEp24 (3). This construct, pPM7, was transformed into the wildtype strain PM38, and transformants were plated on synthetic complete medium (50) lacking uracil to maintain selection for the plasmid. On this complete medium, transformants carrying the high-copy GLN3 clone grew with a doubling time similar to that of the wild-type strain carrying only the vector YEp24. However, in standard minimal medium containing either glutamine or glutamate as the nitrogen source, cells carrying the GLN3 high-copy clone grew slowly, with a doubling time of well over 10 h. Similarly, increasing the transcription of GLN3 by placing the GLN3 gene under the control of the galactose-inducible GALIO-CYCI hybrid promoter from pLGSD5 (20) (see Fig. 5) caused cells to grow extremely slowly on galactose. Identification of GLN3 mRNA. The GLN3 mRNA was identified by Northern blot hybridization of total yeast RNA. In a GLN3+ strain, a single transcript was detected when using either the 5-kb AatII-AatII fragment (Fig. 2) or an internal Sau3A fragment as the probe (see Fig. 5). In a strain carrying the first deletion allele, the AatII probe hybridized only a truncated message of approximately 0.4 kb (Fig. 2).

FIG. 3. Northern analysis of GLN3-specific RNA in wild-type (lanes 1A and 1B), gln3 (lanes 2A and 2B), and ure2 (lanes 3A and 3B) strains grown on either glutamate (lanes A) or glutamine (lanes B) as the sole nitrogen source. Total RNA was isolated, and 10 ,ug of each sample was run per lane. The GLN3 probe was the 5-kb AatII fragment isolated from pPM10, and the PYKI probe was a 6.7-kb HindIII fragnent isolated from pFR2. Strains represented are PM35 (lanes 1), PM43 (lanes 2), and P5-SD (lanes 3).

Assuming that this message represented transcription from the GLN3 promoter and not from the LEU2 promoter, the RNA start site would be approximately 0.4 kb upstream of the XhoI site, in the direction shown in Fig. 1. Sequence analysis confirmed this direction of transcription and approximate start site. Expression of GLN3. To investigate how the GLN3 gene product activates transcription in glutamate-grown but not in glutamine-grown cells, we examined the regulation of the GLN3 gene itself. One possibility was that GLN3 could be transcriptionally controlled in such a way that it would be transcribed in glutamate-grown but not in glutamine-grown cells. However, the data show that GLN3 message levels are not strongly regulated. Northern analysis of a wild-type strain grown in minimal medium showed that the GLN3 message level was slightly higher in cells grown on glutamate than in cells grown on glutamine (Fig. 3). Examination by densitometry showed that the difference was less than 2-fold and is therefore unlikely to account for the approximately 20-fold difference in enzyme levels of GS and NAD-GDH. Mutations in the URE2 gene product, which result in a great increase in GS and NAD-GDH levels in glutamine-grown cells (8, 11), did not significantly increase the GLN3 transcript in minimal medium (Fig. 3). Finally, although it was not possible to grow cells carrying GLN3 on a high-copy plasmid in minimal media, cells carrying increased copies of GLN3 were grown in synthetic complete media. In these cells, GLN3 transcription was increased, but the effect on GS and NAD-GDH was not quite as strong as the effect of the ure2 mutation, which did not increase the GLN3 transcript level in these media (Fig. 4 and Table 4). These results indicate that control exerted through GLN3 is not due to increased transcription of the GLN3 gene. A second possibility was that GLN3 expression, like that of GCN4, could be translationally controlled. To address this possibility, we constructed pPM46, a GLN3-lacZ fusion. The first 82 amino acids of GLN3 and 730 bp upstream of the GLN3 translational start site (see Fig. 6) were fused to the lacZ gene in the 2,um vector pLGA312 (19). This construct was transformed into the wild-type strain PM38, and 3-galactosidase activity was measured in both glutamate-grown and glutamine-grown cells. Since this fusion was on a high-copy vector, GS activity was also measured as a control for the activity of the chromosomally encoded GLN3 product, to guard against the possibility that the high number of GLN3 promoters in the cell was titrating out any regulatory factors. GS was normally regulated, indicating that the chromosomal copy of GLN3 was normally controlled. The levels of 1-galactosidase were only marginally higher in

6222

MOL. CELL. BIOL.

MINEHART AND MAGASANIK 12

3

4

5

GLN 3 *-

TUB2

FIG. 4. Northern analysis of GLN3-specific RNA in a wild-type background carrying multiple copies of GLN3 and in a ure2 deletion strain. RNA for lane 1 was isolated from PM63, a wild-type strain carrying the low-copy vector YCp5O. RNA for lane 2 was isolated from PM64, a wild-type strain carrying pPM4, a low-copy GLN3carrying plasmid. RNA for lane 3 was isolated from PM65, a wild-type strain carrying the high-copy vector YEp24. RNA for lane 4 was isolated from PM66, a wild-type strain carrying pPM7, a high-copy GLN3-carrying plasmid. RNA for lane 5 was isolated from PH2, a ure2 deletion strain. Cells were grown in SC-ura medium with glutamine as the nitrogen source. Total RNA was isolated, and 10 ,ug of each sample was run per lane. The 5-kb AatII fragment isolated from pPM4 was used as a probe for GLN3, and a 0.9-kb Safl-Asp 718 fragment isolated from pJT71 was used as a probe for TUB2. The band running just below GLN3 in lanes 3 and 4 is specific to the YEp24 vector and was shown in later experiments to be cross-hybridizing with sequences between PvuII and BglII within the GLN3 gene. Northern analysis of the same strains grown in SC-ura with glutamate as the nitrogen source gave the same results.

glutamate-grown cells than in glutamine-grown cells, indicating that the regulation of initiation of translation of GLN3 was not affected by the glutamate/glutamine ratio (Table 5). Thus, regulation of the translation of GLN3-specific RNA does not account for the regulation of GS levels. Finally, to confirm that neither regulation of transcription nor initiation of translation is required for normal activity of the GLN3 product, we put the GLN3 gene under the control of the galactose-inducible GALIO-CYCI hybrid promoter in pKP15, a derivative of pLGSD5 (20). The resulting construct, pPM49, contains only 26 bp upstream from the GLN3 translational start site (see Fig. 6), so it is unlikely to contain any elements involved in the regulation of GLN3 transcription or translation. This construct was transformed into the gln3 deletion strain PM71 and assayed for GS in cells grown in raffinose-glutamate and in raffinose-glutamine. NADGDH levels were not measured, because this enzyme is also regulated in response to the carbon source (9). Leaky expression from the galactose promoter in raffinose media allowed GLN3 to be transcribed at a level higher than that of wild type (Fig. 5) and to confer normal regulation of GS (Table 5).

Strain

PM63 PM64 PM65 PM66 PH2

GLN3 sequence analysis. Both strands of the 3-kb AatIIDdeI fragment were sequenced, revealing one open reading frame which encodes a polypeptide of 730 amino acids, with a molecular weight of 79,256 (Fig. 6). It has an unusually large number of asparagine and serine residues, composing 25% of the amino acid residues. Overall, the protein is fairly basic, with a predicted net charge of + 17 at pH 7.0 and a pI of 8.2. The N terminus, however, is quite acidic, having a net charge of -20, with the first 140 amino acids containing 26 of the protein's 49 aspartates and glutamates. The central portion (residues 306 to 330) contains a potential zinc finger, which begins and ends with the motif Cys-x-x-Cys and contains 17 amino acids between the cysteine cores. This cysteine finger falls in a region of about 100 amino acids which shows strong homology to each of the two zinc fingers of the erythroid-specific transcription factor GATA-1 (14, 31, 55, 56). This region and the highly basic region to the carboxy side of the zinc finger motif also show striking homology to both NIT2, the major positive-acting nitrogen regulatory protein of N. crassa (16), and AREA, a positiveacting nitrogen regulatory protein of A. nidulans (25) (Fig. 7). The zinc finger domain of GLN3, homologous to the zinc finger of other transcriptional activators, indicated that GLN3 was likely to be a DNA-binding protein. Furthermore, upstream analysis of genes under GLN3 control, including GLNJ, GDH2, and DALM (35, 36, 46), has delineated sequences involved in nitrogen regulation. Within these sequences is the motif GATAA, which is a subset of the consensus sequence (A/T)GAT(A/T)(A/G) defined for the binding of the GATA-1 factor that has zinc fingers similar to the GLN3 zinc finger. This binding site homology further indicated that GLN3 was likely to bind these upstream nitrogen UAS sequences. As gel binding has so far been unsuccessful in showing binding of GLN3 to these sequences, immunoprecipitation experiments using GLN3 antibody to precipitate putative GLN3-GLNJ protein-DNA complexes were attempted. Isolation of polyclonal GLN3 antibody. Polyclonal antibodies against GLN3 were raised against a hybrid E. coli TrpE-GLN3 protein, consisting of the amino terminal 323 residues of anthranilate synthase and 393 residues of GLN3 (see Fig. 6 for indication of GLN3 residues). Western blot analysis (27) showed that the resulting polyclonal antibody hybridizes to an approximately 90-kDa polypeptide present in cell extracts made from a galactose-induced gln3 strain carrying pPM49, the GALJO-CYCI-GLN3 plasmid. Western analysis with proteins extracted from the same gln3 strain carrying pKP15, the GALIO-CYCI promoter vector, did not show any antibody binding to this 90-kDa polypeptide.

TABLE 4. Effect of ure2 deletion and of multiple copies of GLN3 on GS and NAD-GDHa NAD-GDH (nmollmin/mg) Relevant GS transferase (RmoI/min/mg) chromosomal Plasmid Gln Glt Glt Gln genotype

YCp5O pPM4 YEp24 pPM7 None

Wild type Wildtype Wild type Wild type

ure2A&12::URA3

0.3 ± 0.1 0.5 0.1 0.4 0.1 1.0 0.1 1.3 ± 0.1

0.07 0.2 0.06 0.5 0.8

± 0.03 0.1

±0.01 ±0.2 ± 0.2

2± 2 2 1 3 1 48 ± 4 138 ± 14

2 3 2 27 111

± ± ± ± ±

2 1 2 8 10

a Midexponential cultures growing in synthetic complete medium lacking uracil with either glutamate (Glt) or glutamine (Gln) as the nitrogen source were harvested, and GS transferase and NAD-GDH specific activities were determined. pPM4 is a low-copy YCp5O GLN3 clone; pPM7 is a high-copy YEp24 GLN3 clone. Values indicated are the average of at least three determinations. Overall reduction in values for the wild-type strain is due to the completeness of the medium.

VOL. 11, 1991

GLN3, A POSITIVE NITROGEN REGULATORY GENE

6223

TABLE 5. Regulation exerted through GLN3Y Strain

Plasmid

PM79 PM80 PM76 PM77

pSLF178K (lacZ vector) pPM46 (GLN3-lacZ) pKP15 (GALIO-CYCI) pPM49 (GAL1O-CYCI-GLN3)

Relevant chromosomal genotype

Glt + Ade

Gln

Glt + Ade

Gln

Wild type Wild type gln3A5::LEU2 gln3A5::LEU2

2.3 ± 0.2 2.2 ± 0.2 0.4 t 0.2 2.3 + 0.1

0.05 ± 0.01 0.04 ± 0.01 0.02 t 0.03 0.03 t 0.01

6 ± 0.2 274 ± 20

8 ± 0.1 222 ± 10 ND ND

GS transferase (>mol/min/mg)

0-galactosidase (nmol/min/mg)

NDb ND

a Midexponential cultures growing in the indicated minimal media with either glutamate (Glt) or glutamine (Gln) as the nitrogen source were harvested, and GS transferase and ,-galactosidase specific activities were determined. Adenine (Ade) was added to a final concentration of 0.15 mM to the glutamate medium. Strains PM76 and PM77 were grown on raffinose as the sole carbon source to allow leaky expression off the GALIO promoter. Note that wild-type regulation of GS in strain PM80 indicates that the chromosomal copy of GLN3 was still normally regulated in the presence of multiple copies of the GLN3 promoter. Values indicated are the average of at least three determinations. b ND, not done.

Further, when preimmune serum was substituted for the antiserum, no antibody binding to this band could be demonstrated (Fig. 8). Thus, this 90-kDa polypeptide must be the GLN3 protein. The estimated molecular weight of the GLN3 protein correlates reasonably well with the predicted weight of 79,256. When extracts were prepared from either wildtype or ure2 strains, a faint GLN3 band could be visualized. This band was not affected by the nitrogen source (data not shown). A GLN3-dependent band running at approximately 200 kDa appeared with both the GLN3 serum and the preimmune serum (Fig. 8). This band also appeared if no serum was added in the Western protocol. This band did not appear if extracts were grown with glutamine instead of glutamate as the nitrogen source. Presumably this band corresponds to a protein under GLN3 control. Such a protein, called P200, based on its size, had been previously detected in glutamategrown GLN3+ cells (40). Most likely this is the urea carboxylase-allophanate hydrolase complex, an enzyme whose expression is thought to be dependent on GLN3 and whose activity requires biotin as a cofactor (6, 11). Presumably, biotin stays associated with urea carboxylase-allophanate hydrolase and then becomes conjugated to the biotinylated horseradish peroxidase through an avidin bridge during the Western procedure. Immunoprecipitation of GLN3 protein-GLNI DNA complexes. Total cell lysates were incubated with radioactively labeled GLNI DNA. Two GLNI DNA fragments were used in these experiments. The first fragment was a 108-bp SalI-AvaII restriction fragment which has been shown to contain the GLNJ nitrogen UAS (36). The second 224-bp fragment contains a 32-bp oligonucleotide sequence repeated seven times. This repeated oligonucleotide has also been shown to confer nitrogen-regulated, GLN3-dependent UAS activity and contains the repeated GATAA motif (36). The results in Table 6 show that after binding reactions were performed with radioactively labeled GLNJ DNA and cell extracts from a galactose-induced strain carrying pPM49, the GAL1O-CYCI-GLN3 plasmid, addition of GLN3 antibody and staphylococcus. A protein led to the precipitation of radioactive label. Substituting gln3 extracts, preimmune serum, or radioactively labeled nonspecific DNA in this procedure gave no precipitation of radioactivity. In data not shown, substituting wild-type extracts or ure2 extracts also gave no precipitation of radioactivity. Equimolar addition of the unlabeled multiple-oligomer DNA to the binding reaction using the radioactive Sall-AvaII GLNJ fragment significantly reduced the amount of radioactive counts immunoprecipitated with the GLN3 antibody. Equimolar addition of the SalI-AvaIl fragment to the radioactive multiple-

oligomer fragment did not reduce the amount of radioactivity immunoprecipitated with the GLN3 antibody. In a separate experiment, it was shown that the addition of cold DNA carrying the region upstream of the GLNI gene in 50-fold excess over the radioactive multiple-oligomer fragment reduced the amount of radioactivity immunoprecipitated to the background level. DISCUSSION We cloned the GLN3 gene and showed by constructing various deletions that it is not essential for cell viability. We also verified that purine control of GLNJ, the structural gene for GS, does not require the GLN3 product by showing that the addition of adenine to a glutamate minimal medium decreases the level of GS in a gln3 deletion strain. This effect is masked in a GLN3+ strain, presumably because GLN3 activation of GLNJ leads to sufficient amounts of glutamine for purine biosynthesis without the need for addition of adenine to the medium. Interestingly, ADE+ gln3 strains grew much more slowly in the absence of adenine than in its presence. This shows that activation of GLNJ by purine control is barely sufficient to allow growth in the absence of glutamine. Sequencing the GLN3 gene has shown that its amino acid 2

3 4

*

-GLN 3

_-TUB 2

FIG. 5. Northern analysis of GLN3-specific RNA transcribed from the GALIO-CYCI promoter. RNA for lane 1 was isolated from the wild-type strain PM38 grown on galactose. RNA for lane 2 was isolated from PM76, a gln3A5::LEU2 strain which carries pKP15, the GALIO-CYCI promoter vector. PM76 was grown on raffinose and shifted to galactose for 2 h. RNA for lane 3 was isolated from raffinose-grown PM77, a gln3A5::LEU2 strain carrying the GALIOCYCI-GLN3 fusion. RNA for lane 4 was isolated from PM77 grown on raffinose and shifted to galactose for 2 h. Total RNA was isolated for each sample, and 10 ,ug was run per lane. The GLN3 probe was a 382-bp Sau3A fragment, and the TUB2 probe was the 0.9-kb Sall-Asp 718 fragment. Note that the GAL promoter uses a different start site on galactose than it does on raffinose.

6224

MINEHART AND MAGASANIK

MOL. CELL. BIOL.

CCATAGAAGT GACTTTTCCG CCAAAGAAGA GGACCTCGCC ATAAGCAATG AGAATGATCG TCAGATTCTT 80 TAGATGGGCA CGGCAAGGTA TTGTAAGCTC TTTGACGACG TATAAATCAT CAATACGAGC AGCAAAGAAA 160 GTTTTTTACA TCTGTCCTGT TCAAAGATCA AAAATTAGCA ACGCCTACAA TTCGTAGGAT ACATAGCGTC 240 CCAGTGATTG TACAAACAAC ATCACAAAGT TCATGTTAAA GTTGTCCAGG TTAACCACGA ATTTGTTCGT 320 AAAATCGAGG ACGCGCAGTA AGATAAGATT GAAGCCGGCC CAGAGTTGGC CACTGATTCC GTCCATTCAT 400 GCTCATAATT ACCACACCTT CTTGATCTCT TTACAGCTTT TCAACCTTCC ATTCTTGTAC TCTATCTCTA 480 TAAACATTCT TAATATGAI& TAZZCACATT TTTTGCTCTA TTACCCGGCG GACAGGTTCC CGAAAGAAAG 560 ATGCTGAGAG AGTGGAAAGA GTCATCTTGC AAGACAGAGA AAGATGTTCA AGAGTGGTAA GCTAATGTCA 640 CCATCCCACA ATAACAGAGT GTGTAAGAAA GAGAGACGAG AGAGAGCACA GGG.CCCCCTT TTCCCCCACC 720 Q D D P E N S K L Y D L L N S H L D V H G R S 24 TGCAAGACGA CCCCGAAAAT TCGAAGCTGT ACGACCTGCT GAATAGTCAT CTGGACGTGC ATGGTCGAAG 800 T G E P R Q T G D S R S Q S S G N N E E D I A F 51 CCGAGACAAA CTGGTGACAG TAGGAGCCAG AGTAGTGGCA ACACCGGTGA AAACGAGGAG GATATAGCAT 880 L N G G T F D S M L E A L P D D L Y F T D F V 77 ATTAAACGGC GGCACATTCG ACTCAATGCT GGAGGCACTG CCCGATGATT TATATTTTAC GGACTTCGTG 960 A A A T T S V T T K T V K D T T P A T N H M D 104 CAGCAGCTGC CACGACCAGC GTGACTACTA AGACGGTCAA GGACACCACA CCAGCTACCA ATCATATGGA 1040 S L A T T Q P I D I A A S N Q Q N G E I A M F D 131 GCGATGTTTG ATTCACTTGC CACAACTCAG CCCATCGACA TAGCCGCATC CAACCAACAA AATGGTGAAA 1120 W D F N V D Q F N M T P S N S S G S A T I S A 157 TTGGGACTTT AACGTGGACC AATTCAACAT GACGCCCAGC AACTCGAGCG GTTCAGCTAC TATTAGTGCT 1200 S K S S L F P I PQ Y N H C S L G N S V T S D 184 TTACTTCCGA CATACCGCAA TACAACCACG GTTCCCTCGG CAACAGCGTC TCCAAATCCT CACTGTTCCC 1280 S I N N S N I N Q P N S N T N A Q S H H S T S N 211 GTATAATTCC AGCACGTCCA ACAGCAACAT CAACCAGCCA TCTATCAATA ACAACTCAAA TACTAATGCG CAGTCCCACC 1360 L Q N N N S S S S A M N I T N N N I Y K N S N 237 S F N ATTCCTTCAA CATCTACAAA CTACAAAACA ACAACTCATC TTCATCCGCT ATGAACATTA CCAATAATAA TAATAGCAAC 1440 F L K K S D S Q H P I G L S S S N T T N S V R 264 N S N I AATAGTAATA TCCAGCATCC TTTTCTGAAG AAGAGCGATT CGATAGGATT ATCTTCATCC AACACAACAA ATTCTGTAAG 1520 T S L S S I S A N F K R A A S V S 291 M S S L I K P K N S AAAAAACTCA CTTATCAAGC CAATGTCGTC CACGTCCCTG GCCAATTTCA AAAGAGCTGC CTCAGTATCT TCCAGTATAT 1600 Q C F N C K T F K T P L W Q N K K P L I 317 P S G N M E CCAATATGGA ACCATCAGGA CAAAATAAAA AACCTCTGAT ACAATGTTTC AATTGTAAAA CTTTCAAGACA CCGCTTTGG 1680 LFQ N A C G G T M R P L S 344 E G N T L C K L H R R S P AGGAGAAGCC CAGAGGGGAA TACTCTTTGC AATGCCTGCG GTCTTTTCCA GAAATTACAT GGTACCATGAG GCCATTATC 1760 K R I S K K R A K Q T D P N I A Q N T P D V I K 371 L K S CTTAAAATCG GACGTTATCA AAAAGAGGAT TTCAAAGAAG AGAGCCAAAC AAACGGACCC AAACATTGCA CAAAATACTC 1840 S T S V T T T N A K P I R S R K K S L Q 397 A T A S A P CAAGTGCACC TGCAACTGCC TCAACTTCAG TAACCACTAC AAATGCTAAA CCCATACGAT CGAGGAAAAA ATCACTACAA 1920 I P E E I I R D N I G N T N N I L N V N S R V Q N S L 424 CAAAACTCTT TATCTAGAGT GATACCTGAA GAAATCATTA GAGACAACAT CGGTAATACT AATAATATCC TTAATGTAAA 2000 Q S Y L M N S N S S S V P N A N F 451 S P V Y N F N R G G TAGGGGAGGC TATAACTTCA ACTCAGTCCC CTCCCCGGTC CTCATGAACA GCCAATCGTA TAATAGTAGT AACGCAAATT 2080 N L N S N N L- M R H N S N T V T G N F R 477 S N A N G A TTAATGGAGC AAGCAATGCA AATTTGAATT CTAATAACTT AATGCGTCAC AATTCGAACA CTGTTACTGG TAATTTTAGA 2160 N T S S S S K S S S R S V V P I L 504 T S S R S S R S S R AGGTCTTCAA GACGAAGTAG TACTTCATCG AACACCTCAA GTTCCAGTAA ATCTTCATCC AGATCTGTTG TTCCGATATT 2240 Q Q F N M N M N L M N T T N N V S A N S 531 S P N S P K P ACCAAAACCT TCACCTAATA GCGCTAATTC ACAGCAGTTC AACATGAACA TGAACCTAAT GAACACAACA AATAATGTAA 2320 S S P R S A N F N S N S P L Q Q N 557 S V A A G N I I S GTGCAGGAAA TAGTGTCGCA TCCTCACCAA GAATTATATC GTCCZCAAAC TTTAACTCAA ATAGTCCTCT ACAGCAGAAT 2400 M N I P R R K N A S Y S S S R Q G M S R 584 S F Q L L S N CTATTATCAA ATTCTTTCCA ACGTCAAGGA ATGAATATAC CAAGAAGAAA GATGTCGCGC AATGCATCGT ACTCCTCATC 2480 Q V D V N S N T N T N S N R Q L H 611 A S L Q E Q Q F M A GTTTATGGCT GCGTCTTTGC AACAACTGCA CGAACAGCAA CAAGTGGACG TGAATTCCAA CACAAACACG AATTCGAATA 2560 Q K P R S S N F V S N F D T N S 637 N S V S N S S Q N W GACAGAATTG GAATTCAAGC AATAGCGTTT CAACAAATTC AAGATCATCA AATTTTGTCT CTCAAAAGCC AAATTTTGAT 2640 S S R K S H V S R P T S L L S Q Q 664 S P S P V D I F N T ATTTTTAATA CTCCTGTAGA TTCACCGAGT GTCTCAAGAC CTTCTTCAAG AAAATCACAT ACCTCATTGT TATCACAACA 2720 K F N N R L S S D S T S P I S N H 691 S F I S E S N L Q N ATTGCAGAAC TCGGAGTCGA ATTCGTTTAT CTCAAATCAC AAATTTAACA ATAGATTATC AAGTGACTCT ACTTCACCTA 2800 K I S S T K G S S K E S S 717 S A G G E D N A D V K Y E TAAAATATGA AGCAGATGTG AGTGCAGGCG GAAAGATCAG TGAGGATAAT TCCACAAAAG GATCTTCTAA AGAAAGTTCA 2880 730 F G I * W L K A I A D E L D GCAATTGCTG ACGAATTGGA TTGGTTAAAA TTTGGTATAT GACCGCGTAT TATCATTATC ATTATTCTTA TTATGTTAAT 2960 3021 AATTACTGAA CGGTTGCATT GATAGATTTT CATTACCTCT GACCACAATC CTGAGCATTG G FIG. 6. Nucleotide sequence of GLN3 and its upstream region. The translated amino acid sequence which encodes a protein of 730 amino acids is shown above the DNA sequence. Nucleotide 704, the first nucleotide of the fragment fused to the GALIO-CYCI hybrid promoter, is underlined, as is amino acid residue 82, the last GLN3 amino acid in the lacZ fusion. The first and last residues of the trpE fusion, residues 43 and 435, are outlined. A potential TATA box and the putative cysteine zinc finger domain are underlined. GACGTCAACT GAAAATTGTG TTGGAAACCA ACAGTGCACA TACTGTCATC GCTTATGCTT CCTGGCCCTT TGACATGGCA GCGCAGTAGC M AACAAACAAA N E E TAATGAAGAG A S G TTGCCAGTGG S P F T TCTCCTTTTA D D I TGATGATATT A Q L TTGCACAACT P iS F CCTAACAGCT Y N S

VOL.

GLN3, A POSITIVE NITROGEN REGULATORY GENE

11, 1991 NIT2 aa 734

GNSTDTPTTC TNCFTOTTPL WRRNPDGOPL CNACGLFLKL HGVVRPLSLK TDVIKKRNRG

GLN3 aa 297

GQNKKPLIOC FNCKTFKTPL WRRSPEGNTL CNACGLFQKL HGTMRPLSLK SDVIKKRISK

1- .

6225

1111111 11 11 *111111 *111111 11 II-1s@1 III III

11111111 11 II III-I 1-1 IIIIII II.I LVSKRAGTVC SNCOTSTTTL WRRSPMGDPV CNACGLYYKL HQVNRPLTMR KDGIQTRNRK FIG. 7. Comparison of GLN3 zinc finger and downstream basic region with the N. crassa single-finger protein NIT2, and one of the two zinc fingers of the chicken erythroid-specific transcription factor GATA-1. Lines between amino acids (aa) designate identities, and dots designate conservative amino acid changes. Conservative amino acid changes are defined by the following groups of amino acids: (L, I, V, M), (A, G, P, S, T), (Q, D, E, N), (R, K, H), (F, Y, W) (12). The putative zinc finger is underlined. Note the additional homology between GLN3 and NIT2 in the downstream region as well as the homology in the zinc finger region. This additional downstream homology is also seen with the A. nidulans AREA protein. The erythroid-specific protein GATA-1 found in both murine and human cells is similar to chicken GATA-1 shown here. .

GATA-1 aa 168

sequence contains features found in DNA-binding regulatory proteins. The central region of the GLN3 protein contains a zinc finger motif homologous to an erythroid-specific transcription factor, GATA-1, found in mammalian and chicken cells and to two nitrogen regulatory transcription factors, one found in N. crassa and one from A. nidulans (14, 16, 25, 44, 55, 56). Overall, the GLN3 protein is basic, with a net charge of +17. However, like other transcriptional activators, including NIT2 (16), AREA (25), and the chicken erythroid-specific factor (14), GLN3 contains an acidic region with a net charge of -20 in the first 140 amino acids. This acidic region could be the transcriptional activation domain of the protein as described by Ptashne (45). Analysis of genes under GLN3 control, including GLNJ, GDH2, and DALM (35, 36, 46) has delineated upstream sequences involved in nitrogen regulation. Interestingly, part of the sequence important for this regulation, GATAA, is a subset of the consensus sequence (AIT)GAT(A/T)(AIG) defined for the binding of the GATA-1 factor that has zinc fingers similar to the GLN3 zinc finger (14). This suggests that the region of homology between GLN3 and GATA-1 is involved in DNA sequence recognition. Immunoprecipitation experiments with an antibody against GLN3 indicated that GLN3 binds the upstream region of GLNJ. Not only does it bind to the GLNJ 108-bp fragment defined as the nitrogen UAS, but it also binds to a fragment composed of a repeated 32-bp oligomer that contains the GATAA sequences within the GLNJ nitrogen UAS. Prelim1

2

3 4

FIG. 8. Western blot analysis of the GLN3 gene product. Total protein extracts were prepared from cells grown on raffinose and glutamate and induced for 2 h with galactose. The protein extracts were subjected to electrophoresis on a sodium dodecyl sulfate-7.5% polyacrylamide gel and electroblotted to nitrocellulose. Binding of the primary antiserum raised against a TrpE-GLN3 hybrid protein was visualized by using the Vectastain ABC immunoperoxidase system. In lane 1, immune serum was used against an extract from strain PM77 (gln3A5::LEU2 carrying the GAL1O-CYCI-GLN3 fusion). In lane 2, immune serum was used against an extract from strain PM76 (gln3A5::LEU2 carrying the GALIO-CYCI promoter vector). In lanes 3 and 4, preimmune serum was used against extracts from strains PM77 and PM76, respectively. Between lanes 2 and 3 are Bio-Rad size markers of 205, 116, 77, and 46.5 kDa.

inary competition experiments indicated that GLN3 binds the repeated oligomer more strongly than it binds the single UAS, presumably because the repeated oligomer contains more GLN3-binding sites. In synthetic complete media with either glutamate or glutamine as the nitrogen source, increasing the copy number of GLN3 increased the levels of both GS and NADGDH, further supporting the idea that GLN3 is an activator. In minimal medium, increasing GLN3 copy number was detrimental to cell growth, which is most easily explained in one of two ways. One explanation is that the high number of GLN3 protein molecules titrates important cellular factors, leaving them unavailable for other interactions. Since GLN3 has been shown to be a transcriptional activator (1, 7), presumably these other factors would be general transcription factors. The other possible explanation is that the increased copy number of GLN3 leads to high constitutive levels of the proteins it regulates and that this is detrimental to the cell. This explanation seems less likely since mutations in URE2 lead to high constitutive levels of GS, NAD-GDH, and the general amino acid permease without causing the drastic decrease in growth rate in minimal medium, as ure2 strains double in 4 to 5 h, while strains carrying increased copies of GLN3 require more than 10 h to double. Furthermore, when comparing the activities of NAD-GDH and GS in synthetic complete media, these activities are slightly higher in a ure2 background than in a high-copy GLN3 background. Therefore, if the increased level of a GLN3-regulated protein is responsible for the poor growth of cells carrying GLN3 in high copy, this protein must be affected only by GLN3 and not by URE2. Previous genetic analysis has shown that gln3 mutations are epistatic to ure2 mutations which lead to high levels of GS, NAD-GDH, and the general amino acid permease when either glutamate or glutamine is the nitrogen source (11). Since gin3 ure2 double mutants have the phenotype of gin3 single mutants, that is, GS, NAD-GDH, and the general amino acid permease are low on both glutamate and glutamine, it is possible that the product of the URE2 gene prevents the ability of GLN3 to activate the transcription of these genes. However, it is also possible that GLN3 is simply an essential component for activation in response to the glutamate/glutamine ratio and that URE2 confers regulation through one or several other proteins which do not interact with GLN3 but are also essential for activation. The third possibility, that URE2 is a general repressor, seems less likely since URE2 specifically prevents activation by GLN3 in the presence of glutamine but does not prevent activation of GLNJ in response to general amino acid control or purine starvation in the presence of glutamine.

6226

MINEHART AND MAGASANIK

MOL. CELL. BIOL.

TABLE 6. Immunoprecipitation of GLN3-GLNI protein-DNA complexesa Expt

Radioactive

1

Nonspecific Multiple sites Multiple sites Multiple sites GLNI N-UAS GLNI N-UAS GLNI N-UAS Multiple sites Multiple sites gIn3l N-UAS GLNJ N-UAS GLNI N-UAS GLNI N-UAS

Expt

2 3 4

~~~~DNAb

Cold DNA' Extractd Serum' ClDNCEtatSeu

None None None None None None None None GLNI N-UAS (1:1) None Multiple sites (1:1) Multiple sites (1:5) Multiple sites (1:50)

GLN30P gln3 GLN30P GLN30P gln3 GLN30P GLN30P GLN3YP GLN30P GLN30P GLN30P GLN30P GLN30P

GLN3 GLN Preimmune GLN3 GLN3 Preimmune GLN3 GLN3 GLN3 GLN3 GLN3 GLN3 GLN3

% Counts

precipitatedf 0.2 ± 0.3 ± 0.3 9.5 + 0.01 ± 0.01 ± 12.0 ± 11.0 ± 12.0 ± 12.4 ± 1.3 ± 0.2 ± 0.1 ±

0.04 0.03 0.04 0.9 0.01 0.01 1.5 2.1 1.8 1.9 0.05 0.04 0.04

a Total cell lysates were incubated with radioactively labeled DNA to allow binding to occur. Antiserum was then added to the binding reaction mixtures, which were then immunoprecipitated with fixed S. aureus Cowan I. Radioactive counts in the pellet were determined. All experiments were repeated at least two times. b Three DNA probes were tested for the ability to bind DNA. The nonspecific DNA was a 222-bp Avall fragment from the Bluescript KS1+ plasmid. The multiple-sites DNA was a 224-bp fragment consisting of a seven-times-repeated 32-bp oligimer containing the GATAA motif thought to be the GLN3-binding site. The GLN1 N-UAS was a 108-bp Sal-AvaIl fragment defined as containing the GLNI nitrogen UAS. In the experiments shown here, the specific activity of the multiple-oligomer DNA was 500 cpm/ng, while the specific activity of the KS DNA and the GLNI N-UAS DNA was 5,000 cpm/ng. Approximately 10,000 cpm were added to each reaction. c Nonradioactive competitor DNA was added simultaneously with the radioactively labeled DNA in the gel binding reaction. The indicated ratio indicates the molar ratio of the radioactively labeled DNA fragment to the unlabeled fragment. d Total cell extracts were made from either PM76 (gln3), a gIn3l&5::LEU2 strain which carries pKP15, the GALIO-CYCI promoter vector, or PM77 (GLN30P), a gln3i&5::LEU2 strain which carries the GALJO-CYCI -GLN3 fusion. Cells were grown on raffinose and then shifted to galactose for 2 h. Protein (40 Fg) was added to each reaction. I Either preimmune serum or anti-GLN3 antiserum was added to the binding reactions. f Pellets were counted by Cerenkov counting in Eppendorf tubes without scintillation fluid.

Our results show that neither control of transcription nor initiation of translation of GLN3 is important for regulation of NAD-GDH and GS in response to the glutamate/glutamine ratio. If GLN3 is involved in conveying the regulatory signal responding to glutamine and glutamate levels, this signal must first be imparted to GLN3 by a posttranslational modification or interaction. URE2 remains a likely candidate for imparting this signal to GLN3 since its sequence analysis has shown that it is more likely to interact with other proteins than with DNA (8). Furthermore, it has been shown to be involved in GS inactivation (8, 28), presumably by another protein-protein interaction. Unless the cell contains a vast excess of URE2 over GLN3, which is unlikely as transcription of URE2 is quite weak (8), the presumed interaction between the two is not likely to be stoichiometric since slightly increasing the copy number of GLN3 by placing it on the low-copy vector YCp5O only slightly increases GS and NAD-GDH, but eliminating URE2 greatly increases GS and NAD-GDH. Furthermore, although increasing the copy number of GLN3 by placing it on the high-copy vector YEp24 does significantly increase the activities of GS and NAD-GDH, these activities are still lower than those in a ure2 deletion strain with only one copy of GLN3. Thus, our results suggest that the URE2 product catalytically inactivates GLN3 in response to an increase in the intracellular concentration of glutamine.

ACKNOWLEDGMENTS We thank members of the B. Magasanik's laboratory, especially Peter Coschigano, for helpful suggestions and discussions. We are grateful to Hilda Harris-Ransom for assistance in preparation of the

manuscript. This work was supported by Public Health Service research grant GM-07446 to B.M. P.M. was supported by a National Science

Foundation fellowship and a National Institutes of Health training grant. REFERENCES 1. Benjamin, P. M., J. Wu, A. P. Mitchell, and B. Magasanik. 1989. Three regulatory systems control expression of glutamine synthetase in Saccharomyces cerevisiae at the level of transcription. Mol. Gen. Genet. 217:370-377. 2. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heynecker, and H. W. Bover. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 3. Botstein, G., S. C. Falco, S. E. Stewart, M. Brennan, S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis. 1979. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17-24. 4. Carlson, M., and D. Botstein. 1982. Two differentially regulated mRNAs with different 5' ends encode secreted and intracellular forms of yeast invertase. Cell 28:145-154. 5. Clark-Adams, C. D., and F. Winston. 1987. The SPT6 gene is essential for growth and is required for 8mediated transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 7:679-686. 6. Cooper, T. G. 1982. Nitrogen metabolism in Saccharomyces cerevisiae, p. 39-99. In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), The molecular biology of the yeast Saccharomyces: metabolism and gene expression. Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. 7. Cooper, T. G., D. Ferguson, R. Rai, ahd N. BysanL. 1990. The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae. J. Bacteriol. 172:1014-1018. 8. Coschigano, P. W., and B. Magasanik. 1991. The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to nitrogen source and has homology to glutathione S-transferases. Mol. Cell. Biol. 11:822-832. 9. Coschigano, P. W., S. M. Mller, and B. Magasanik. 1991. Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomy-

VOL.

11, 1991

ces cerevisiae. Mol. Cell. Biol. 11:4455-4465. 10. Courchesne, W. E. 1985. Ph.D. dissertation. Massachusetts Institute of Technology, Cambridge. 11. Courchesne, W. E., and B. Magnik. 1988. Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes. J. Bacteriol. 170:708-713. 12. Dayhoff, M. O., R. M. Schwartz, and B. C. Orcutt. 1979. A model of evolutionary change in proteins, p. 345-352. In M. 0. Dayhoff (ed.), Atlas of protein sequence and structure, vol. 5, suppl. 3. National Biomedical Research Foundation, Washington, D.C. 13. Drillien, R., M. Aigle, and F. Lacroute. 1973. Yeast mutants pleiotropically impaired in the regulation of the two glutamate dehydrogenases. Biochem. Biophys. Res. Commun. 53:367372. 14. Evans, T., and G. Felsenfeld. 1989. The erythroid-specific transcription factor Eryfl: a new finger protein. Cell 58:877-885. 15. Forsburg, S. L., and L. Guarente. 1988. Mutational analysis of upstream activation sequence 2 of the CYCI gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site. Mol. Cell. Biol. 8:647-654. 16. Fu, Y.-H., and G. A. Marzluf. 1990. nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Mol. Cell. Biol. 10:1056-1065. 17. Fu, Y.-H., and G. A. Marzluf. 1990. nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a sequence specific DNA binding protein. Proc. Natl. Acad. Sci. USA 87:5331-5335. 18. Grenson, M., E. Dubois, M. Piotrowska, R. Drillien, and M. Aigle. 1974. Ammonia assimilation in Saccharomyces cerevisiae as mediated by the two glutamate dehydrogenases. Mol. Gen. Genet. 128:73-85. 19. Guarente, L., and T. Mason. 1983. Heme regulates transcription of the CYCI gene of S. cerevisiae via an upstream activation site. Cell 32:1279-1286. 20. Guarente, L., R. R. Yocum, and P. Gifford. 1982. A GALIOCYCI hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc. Natl. Acad. Sci. USA 79:74107414. 21. Hinnebusch, A. G. 1988. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol. Rev. 52:248-273. 22. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193197. 23. Ito, H., Y. Fukada, K. Murato, and A. Kimiru. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. 24. Koerner, T. J., J. E. Hil, A. M. Myers, and A. Tzagoloff. 1991. High-expression vectors with multiple cloning sites for construction of tprE fusion genes: pATH vectors. Methods Enzymol. 194:477-490. 25. Kudla, B., M. X. Caddick, T. Langdon, N. M. Martinez-Rossi, C. F. Bennett, S. Sibley, R. W. Davies, and H. N. Arst, Jr. 1990. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J. 9:1355-1364. 26. Kuo, C.-L., and J. L. Campbell. 1983. Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol. Cell. Biol. 3:1730-1737. 27. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)

227:6804685.

28. Legrain, C., S. Vissers, E. Dubois, M. Legrain, and J.-M. Wiame. 1982. Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis. Eur. J. Biochem. 123:611-616. 29. Lipman, D. J., and W. J. Pearson. 1985. Rapid and sensitive protein similarity searches. Science 227:1435-1441. 30. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

GLN3, A POSITIVE NITROGEN REGULATORY GENE

6227

cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 31. Martin, D. I. K., S.-F. Tsai, and S. H. Orkin. 1989. Increased gamma-globin expression in a non-deletion HPFH mediated by an erythroid-specific DNA binding factor. Nature (London) 338:435-438. 32. Mignotte, V., L. Wall, E. deBoer, F. Grosveld, and P.-H. Romeo. 1989. Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene. Nucleic Acids Res. 17:37-54. 33. Mifler, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 34. Miller, S. M., and B. Magasanik. 1990. Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae. J. Bacteriol. 172:4927-4935. 35. Miller, S. M., and B. Magasanik. 1991. Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:6229-6247. 36. Minehart, P. L. 1991. Ph.D. thesis. Massachusetts Institute of Technology, Cambridge. 37. Mitchell, A. P., and S. W. Ludmerer. 1984. Identification of a glutaminyl-tRNA synthetase mutation in Saccharomyces cerevisiae. J. Bacteriol. 158:530-534. 38. Mitchell, A. P., and B. Magasanik. 1983. Purification and properties of glutamine synthetase from Saccharomyces cerevisiae. J. Biol. Chem. 258:119-124. 39. Mitchell, A. P., and B. Magasanik. 1984. Biochemical and physiological aspects of glutamine synthetase inactivation in Saccharomyces cerevisiae. J. Biol. Chem. 259:12054-12062. 40. Mitchell, A. P., and B. Magasanik. 1984. Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2758-2767. 41. Mitchell, A. P., and B. Magasanik. 1984. Three regulatory systems control production of glutamine synthetase in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2767-2773. 42. Mortimer, R. K., and D. C. Hawthorne. 1969. Yeast genetics, p. 385-460. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 1. Academic Press, Inc., New York. 43. Orr-Weaver, T. L., J. W. Szostak, and R. J. Rothstein. 1981. Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 78:6354-6358. 44. Pevny, L., M. C. Simon, E. Robertson, W. H. Klein, S.-F. Tsai, V. D'Agati, S. H. Orkin, and F. Constanti. 1991. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature (London) 349:257-260. 45. Ptashne, M. 1988. How eukaryotic transcriptional activators work. Nature (London) 335:683-689. 46. Rai, R., F. S. Genbauffe, R. A. Sumrada, and T. G. Cooper. 1989. Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae. Mol. Cell. Biol. 9:602-608. 47. Rose, M., and D. Botstein. 1983. Construction and use of gene fusions to lacZ (3-galactosidase) that are expressed in yeast. Methods Enzymol. 101:167-180. 48. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202-210. 49. Sanger, G., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 50. Sherman, F., G. R. Fink, and C. W. Lawrence. 1978. Manual for a course: methods in yeast genetics, revised edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 51. Spindler, K. R., D. S. E. Rosser, and A. J. Berk. 1984. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J. Virol. 49:132-141. 52. Stanbrough, M. (Massachusetts Institute of Technology). 1991. Personal communication. 53. Struhl, K. D., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979. High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci.

6228

MINEHART AND MAGASANIK

USA 76:1035-1039.

54. Towbin, H., T. Staehelln, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:43504354. 55. Tsai, S.-F., D. I. K. Martin, L. I. Zon, A. D. D'Andrea, G. G. Wong, and S. H. Orkln. 1989. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature (London) 339:446 451. 56. Wall, L., E. deBoer, and F. Grosveld. 1988. The human 0-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein. Genes Dev. 2:1089-1100. 57. Wiame, J. M., M. Grenson, and H. N. Arst, Jr. 1985. Nitrogen

MOL. CELL. BIOL.

catabolite repression in yeasts and filamentous fungi. Adv. Microb. Physiol. 26:1-88. 58. WInston, F., F. Chumley, and G. R. Fink. 1983. Eviction and transplacement of mutant genes in yeast. Methods Enzymol. 101:211-228. 59. Yanofsky, C., T. Platt, I. P. Crawford, B. P. NIhols, G. E. Christie, H. Horowitz, M. VanCleemput, and A. M. Wu. 1989. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 9:6647-6668. 60. Youssoufian, H., L. Zon, S. H. Orldn, A. D. d'Andrea, and H. F. Lodsh. 1990. Structure and transcription of the mouse erythropoietin receptor gene. Mol. Cell. Biol. 10:3675-3682.