arginase gene in Saccharomyces cerevisiae. A study of ... - Europe PMC

The EMBO Joumal Vol.1 No.9 pp.1125-1131, 1982

Molecular cloning, DNA structure, and RNA analysis of the arginase gene in Saccharomyces cerevisiae. A study of cis-dominant regulatory mutations Jean-Claude Jauniaux*, Evelyne Dubois', Stephan Vissers, Marjolaine Crabeel2, and Jean-Mane Wiame Laboratoire de Microbiologie, Universite Libre de Bnxelles, Institut de Recherches du CERIA, and 2Laboratorium voor Microbiologie, Vrije Universiteit BIussd, 1, av. E.Gryson, B-1070 Bruxeles, Belgium Communicated by J.-M. Wiame Received on 16 August 1982

The Saccharomyces cerevisiae gene cargA + or CAR], encoding arginase has been cloned by recovering function in transformed yeast cells. It was used to analyse RNA and chromosomal DNA from six strains bearing cis-dominant regulatory mutations leading to constitutive arginase synthesis. The DNA from the four cargA + 0- strains in which constitutive arginase synthesis was independent of the mating-type functions showed no detectable differences with the wild-typye. The cargA + 0- mutations were, therefore, small alterations, possibly single base substitutions. On the other hand, the cargA + '-I and cargA + O-2 mutations, leading to a constitutive and mating-type dependent arginase synthesis, were idendfied as insertions. Their size and restriction pattern strongly suggested that they were induced by the Tyl yeast transposable element. This was confirmed by cloning and analysis of the cargA + Oh-I mutant gene. The concentration of arginase RNA was significantly increased in the mutants, indicating that the regulation of arginase synthesis was exerted, at least in part, at the level of RNA synthesis or stability. In the cargA O+'-2 strain the Tyl element was located at a distance of -600 base pairs from the insertion present in the cargA + Oh-l strain. This result suggests either a surprisingly large arginase regulatory region or an indirect influence of the Tyl element on gene expression over long distances. Key words: arginase/Northern analysis/regulatory mutation/ transposable element (Ty)/yeast

Introduction The synthesis of arginase (EC 3.5.3.1) in Saccharomyces cerevisiae is regulated by at least four distinct circuits (Wiame and Dubois, 1976). The best known regulations are specific induction and nitrogen catabolite repression. Nitrogen catabolite repression is observed when cells are grown on the best nitrogen sources, namely ammonia, glutamine, or asparagine. It is a general regulation acting on other amino acid catabolic enzymes as well as on permeases (Grenson and Hou, 1972; Dubois et al., 1973). The signal of this regulation is not the same for all the enzymes (Dubois et al., 1977). For arginase as well as for several permeases the signal appears to be ammonia and needs the NADPdependent (anabolic) glutamate dehydrogenase for its transmission (Grenson and Hou, 1972; Dubois et al., 1974). Specific induction of arginase occurs upon addition of arginine to the growth medium. If arginine is the sole nitrogen source, catabolite derepression and specific induc*To whom reprint requests should be sent. © IRL Press Limited, Oxford, England. 0261-4189/82/0109-1125$2.00/0.

tion both operate. The analysis of the process of specific induction led to the first indications of the existence in eucaryotes of a regulatory mechanism that behaved according to the operator/repressor model of Jacob and Monod (Wiame, 1971). This proposition was based on the isolation of mutants bearing: (i) recessive mutations, cargR -, interpretable as affecting a repressor acting pleiotropically on arginase (cargA + or CARl) and omithine transaminase (cargB+ or CAR2) genes; (ii) cis-dominant mutations, cargA + 0- and cargB+ 0-, strongly linked to their respective structural genes. These mutations led to a constitutive expression of their adjacent structural genes but they did not affect the nitrogen catabolite repression controlling arginase synthesis (Dubois et al., 1978). Another type of cis-dominant mutations linked to the arginase structural gene was isolated. Curiously, the expression of these mutations is dependent on the mating-type functions. The first to be identified had a higher constitutivity than the 0- type and was designated cargA + (-lI. The mating-type effect consisted of a reduction of arginase constitutive synthesis in diploids heterozygous for the mating-type locus, i.e., in cargA+(j, MAT a/cargA + O&, MA Toe diploid strains, as compared with the arginase level expressed either in haploid mutant strains or in diploid mutant strains that were homozygous for either allele at the MA T locus (Wiame and Dubois, 1976; Dubois et al., 1978). The constitutive arginase synthesis was also diminished in haploid oh strains containing the ste 7 mutation, which prevents conjugation (Errede et al., 1980). This type of behaviour was also found for urea-amidolyase (dur&O mutations, Lemoine et al., 1978), omithine transaminase (cargB + O mutations, Deschamps and Wiame, 1979), and for iso-2-cytochrome c (CYC7-H2 mutation, Rothstein and Sherman, 1980). In the latter case the mating-type effect resulted from an insertion of a transposable and reiterated Tyl element (Cameron et al., 1979) in the 5' non-coding region of the CYC7 structural locus (Errede etal., 1980). The similarity of oh and CYC-H2 mutations led us to designate these mutations ROAM mutations (Regulatory Overproducing Alleles responding to Mating-type). It was proposed that they all consisted of an inserted Tyl element (Errede et al., 1980). To investigate the mechanism(s) of regulation of arginase expression, we have cloned and characterized the cargA + gene. We have used it as a probe to analyse the nature of the cis-dominant regulatory mutations and their effects on the size and concentration of arginase RNA in the mutants. A preliminary report of part of this work has been published as an abstract (Jauniaux et al., 1981).

Results Molecular cloning of the cargA +DNA The gene coding for arginase (cargA + or CARI) was cloned into arginase-deficient yeast recipient cells. The pool of donor DNA was constructed by inserting yeast DNA fragments restricted with BamHI into the BamHI site of the multi-copy pFL1 vector. The pFLI vector was a chimeric 1125

J.-C.Jauniaux et al.

bacterial-yeast plasmid containing Escherichia coli plasmid pBR322, part of the yeast 2g plasmid and the yeast URA3 + gene (Chevalier et al., 1980). The recipient yeast strain 01417d was deficient in arginase and uracil biosynthesis (cargA-, ura3-). Transfonnants were recovered as cells which were simultaneously uracil prototrophs and able to grow on arginine as sole nitrogen source. The study of the transformant progeny showed that the CargA+ phenotype derived from a plasmid-associated cargA + gene. Indeed, diploids derived from crosses between transformed cells and the mating-type allele cells, 01420 c, of the initial recipient strain gave tetrads mostly simultaneously 4 + /O - for both CargA + and Ura3 + phenotypes. A second useful characteristic of yeast cells transformed with the 24 DNA-containing plasmid is the loss of these plasmids during mitotic growth under non-selective conditions (Beggs, 1978). Independently transformed clones were grown for about eight generations in rich liquid medium, plated onto rich medium, and tested by replica plating onto M.am medium (see Materials and methods) to detect their Ura3 - phenotype and onto M.arg medium (see Materials and methods) supplemented with uracil to detect their CargA - phenotype. About 20% of the cells simultaneously lost their CargA + and Ura3 + phenotypes. Hence the CargA + phenotype cosegregates with the Ura3 + phenotype and is mitotically unstable, indicating that the CargA+ phenotype is associated with the plasmid. Nevertheless, a large number of cells lost their CargA+ phenotype but retained their Ura3 + phenotype suggesting that many original transformants had incorporated several types of hybrid molecules. This cotransformation by multiple plasmids was confirmed in transformation experiments with DNA extracted from the original clones. DNA was extracted from five transformants and used to transform E. coli to ampicillin resistance. Plasmid DNA was then isolated from different ampicillinresistant bacteria and used to transform the original (cargA -, ura3 -) yeast strain to the Ura3 + phenotype, checking the transformants afterwards for their CargA + phenotype. Only a fraction of the purified plasmids co-transformed the recipient simultaneously to uracil prototrophy and arginine utilisation. These plasmids were submitted to a restriction enzyme analysis. Digestion with BamHI and HindIII revealed that all consist of the same BamHI fragment of 5.6 kb inserted into the pFLI vector, the insertion being in both of the two possible orientations. Plasmid pJCJ1 bears the insertion in one orientation, pJCJ2 in the other. This 5.6-kb BamHI DNA fragment was part of the Saccharomyces genome and contained an unaltered arginase structural gene (Figure 2). Moreover, the arginase expressed from the plasmids in the transformants had the properties of S. cerevisiae arginase, in-

cluding its very specific capacity to bind omithine transcarbamylase and to inhibit its activity (Messenguy and Wiame, 1969); data not shown. Expression and regulation ofplasmid-coded arginase in yeast To determine whether the regulatory regions of the

arginase gene had been cloned and to analyse how they functioned on a plasmid, we measured the arginine-specific activi-

ty in cells transformed with purified plasmids growing in various media. Under all conditions tested, cultures of transformants 01417d (cargA-, ura3-, YEpURA3+, cargA+) synthesized 10 times more enzyme than the wild-type strain (Table I). However, the level of arginase is clearly dependent on the presence of arginine and ammonia in the medium. Since the number of plasmids per cell seemed to be constant (as indicated by the constant level of procaryotic ,B-lactamase expressed from the plasmids in the transformants on the various media; data not shown) arginase synthesis appeared to be regulated by arginine and ammonia as in the wild-type strain. The expression and the regulatory properties of the cloned cargA + (7-l mutation were also analysed. The plasmid pJCJ3 bears the cargA + (7-I gene in one orientation, pJCJ4 bears it in the other. They were selected in E. coli by colony hybridization without selection for arginase expression (see below). We used each plasmid to transform cells of the haploid yeast strain 01417d (ura3-, cargA -) and those of the corresponding diploid [01417d x 01420c] to uracil prototrophy. We found that the transformants were also able to utilize arginine as sole nitrogen source. Measurement of arginase specific activity in these transformants (Table I) showed that the cargA + O"-I mutation preserves its regulatory properties when it is located on a plasmid; in particular its constitutive synthesis of arginase is reduced in diploids heterozygous for the mating type as compared with haploids. The orientation of the cloned BamHI DNA fragment containing the cargA + or cargA + Oh-] gene in the pFLI vector did not influence arginase expression. Expression ofplasmid-coded arginase in E. coli Arginase activity was not detected in E. coli cells transformed and selected to ampicillin resistance no matter which YEpURA3+, cargA + plasmid was used. Location of the cargA + gene on the cloned 5.6-kb BamHI fragment and identification of the arginase RNA A restriction map of the 5.6-kb cloned BamHI fragment is shown in Figure 1. The position of the restriction sites was determined by gel electrophoretic analysis of the products of single and double digestions of the pJCJl and pJCJ2 plasmid DNAs. Various fragments generated by restriction endonuclease cleavage were purified and labelled by nick transla-

Table I. Arginase-specific activitya in wild-type and plasmid-harbouring strains

Strain

E1278b 01417d 01417d 01417dx01420c

Recipient genotype

wild-type cargA -ura3cargA -ura3-

cargA-ura3-MATa pti cargA-eura3 MA

aExpressed asu~mol urea formed/h/mg protein. 1126

Plasmid

-

YEpURA3+cargA+ YEpURA3+ cargA + 0'-J YEpURA3+cargA + -I

Nitrogen nutrient am

am + arg

arg

6 59 1450

20 226

250 1990 3000

800

700

DNA and RNA analysis of yeast

tion. They were used as probes to detect homologous RNAs in order to both identify RNA homologous to the cargA + gene and to map the cargA + gene on the cloned DNA fragment. A single band showed intensity variations related to arginase regulations. Hence it corresponds to the arginase RNA. The results are shown only for the e-g BglII DNA fragment (Figure 4). The e-f, f-g, g-h, and h-i fragments gave a positive signal for the band corresponding to arginase RNA, the signal from fragment a - e was very weak, fragment i-j did not react (data not shown). Hence the cargA + gene begins close to the e BglHI site and extends up into the h - i PstI fragment (Figure 1). DNA restriction fragment analysis in arginase constitutive strains The nature of the cargA + (^-L, cargA + oh-2 ROAM mutations and that of the cargA+0--i, cargA+O--2, cargA + 0 --3, and cargA + 0 --4 mutations were examined at the molecular level. The cargA+ (-2 mutation was previously believed to be of the cargA + 0- type. Its matingtype sensitivity (see below) was not realised. Total DNA was prepared from the mutants and their wild-type parent, cleaved by various restriction endonucleases, and the fragments were separated by electrophoresis on agarose gels. After transfer of the separated restriction fragments to a nitrocellulose filter by the Southern (1979) blot procedure, the DNA on the filter was hybridized to a radioactively labelled DNA probe. This probe consisted of the total 5.6-kb BamHI DNA fragment carrying the arginase wild-type gene. The restriction fragments showing homology with DNA from the probe were visualized by autoradiography. The results show that the cargA+O-4-, cargA+O--2, cargA+O--3, cargA+O--4, and the wild-type strains have the same restriction pattern (Figure 2). In contrast, the cargA regions in the cargA + f-I and cargA + oh-2 mutants have different restriction patterns. The wild-type cargA + gene is present on a single 5.6-kb BamHI DNA fragment corresponding to the cloned fragment, while in the cargA + OY-] and cargA + Oh-2 mutants, the cargA gene is present on a 12-kb BamHI fragment (Figure 2a,b). As a single BamHI fragment was found in the cargA + (Y-I and cargA + O-2 strains, these results suggested that the cargA + (-I as well as the cargA + oh-2 gene contained an insertion of - 5.6 kb devoid of a BamHI restriction site. A similar analysis performed for the HindlII restriction fragments (Figure 2d) confirmed that both mutants had an insertion of 5.6 kb which was located on the DNA fragment corresponding to the b -f HindIII segment of Figure 1. From the analysis of the fragments generated by restriction with BgIII, BamHI + BglII, BamHI + XhoI, and BamHI + HaeII (Figure 2c,g,e,f) it appeared that both insertions had similar BgII, XhoI, and HaeII restriction sites, indicating that both insertions were alike and with the same polarity. However, the insertions had a different location as indicated by the systematic 600-700 bp shifting between the corresponding bands of the two mutants. The insertion of strain cargA + hl-] was located very close to the e BglII restriction site whereas the insertion of strain cargA + O(-2 was very close to the d HaeII restriction site (Figure 1), the two insertions being on the same d -e DNA segment. Cloning and characterization of the cargA + G'-I gene The cargA + Olhi mutation was cloned to identify precisely its location and to study its relationship with members of the Tyl family. The entire cargA + (/-I mutated gene was pre-

arginase regulatory mutations

sent on a BamHI DNA fragment (Figure 1). Total DNA, extracted from strain 7094a, was cut with BamHI and ligated with pFLl DNA. The ligation mixture was used tor transform E. coli cells to ampicillin resistance. E. coli colonies containing the cargA + (Y-I gene were identified by colony hybridization using the nick-translated e-g BglII DNA fragment (Figure 1) as a probe. The E. coli transformants obtained yielded plasmid DNA with a size and restriction sites consistent with the results of the Southern analysis. The size as well as the distribution of the restriction sites of the inserted element were very suggestive of Tyl. The XhoI sites, near the insertion boundaries, are characteristic of 6 sequences that are 300-bp direct repeats and usually border Tyl elements (Gafner and Philippsen, 1980). The insertion of strain cargA + (/-l is located 100 bp in front of the e BgIII site (Figure 1) if, as is probably the case, the XhoI site in the cargA + C/-I insertion has the same position as the XhoI site in the 6 repeat sequenced by Gafner and Philippsen (1980). To test further the hypothesis that the cargA + (Y-I insertion is a Tyl element, total DNA prepared from the wild-type strain and from the cargA + (/-l mutant was digested with BamHI. The resulting restriction fragments from each strain were fractionated by gel electrophoresis, transferred to nitrocellulose sheets, and probed with the labelled 5.6-kb cargA + fragment, the I 1.2-kb cargA + C/-I fragment, and the 5.6-kb EcoRI Tyl fragment isolated from plasmid S13 (Cameron et al., 1979). As shown in Figure 3, the cargA + probe hybridized to a single 5.6-kb fragment from the wild-type DNA and to a fragment of the cargA + -lI DNA of - 12 kb as expected. In contrast, the cargA + Oh-] probe hybridized with a large number of fragments from genomic DNA of the wildtype and of the cargA + (Y-I strains. The pattern of reiterated sequences obtained for the cargA + O(-i probe was very similar to the pattern observed with the Tyl probe (Figure 3). To determine the homology between the insertion element in the cargA + O-] mutant and the Tyl element, cargA + OC-l and Tyl plasmid DNAs were digested with various restriction -

cargA+Oh-2

X

Bg

Ha

Bg

insertion

-

Ba HiHi X

carg A+ b c

a

H4

-

-

-

Ha X

--

Bg Hi Bg P P

d

f

g

Ba

h

cargA+Oh1 insertion

i

X

Bg

i

S Ha

EE

i

Bg I =

P

I

Bg P

Ii

Ha X

1000 base pairs.

Fig. 1. Restriction maps of the arginase region of the wild-type and the cargA '+ &-I and cargA + (t-2 mutants. Location of the DNA region homologous to the arginase RNA. Cleavage sites in the doned BanHI seg-

ment containing the cargA + gene were determined by a series of single and double restriction digests of either the intact plasmid or pertinent fragment purified from a preparative agarose gel. The following restriction endonucleases were used: Ba, BamHI; Hi, HindIII; X, XhoI; Ha, HaeII; Bg, BgIII; P, PstI; S, Sall, and E, EcoRI. The fragment sizes used to determine the maps and insertion positions of the cargA + C-l and cargA + &-2 mutations were approximated from the genomic blots in Figure 2. The arrows indicate the cargA +-DNA regions where the cargA +&-I and cargA +CO-2 mutations are located. For the cargA + Ct-I DNA region the data were confirmed and completed by restriction analysis of the doned 11.6-kb DNA region containing the cargA + Ct-I mutation. The cross-hatched area approximates the DNA region homologous to the arginase RNA.

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Fig. 2. Autoradiograms of Southern analysis of DNA from the wild-type strain and six mutants producing arginase constitutively. DNA was purified from the six analysed mutants and the wild-type strain. DNA was restricted, electrophoresed on a 0.7%70 agarose gel (a,b,c) or 0.8% agarose gel (d,e,f,g), transferred to nitrocellulose, and hybridized to the 32P-labelled 5.6-kb BamHI fragment carrying the arginase wild-type gene. Yeast DNA was cleaved with BamHI (a,b), BgIIl (c), BamHI + HindIII (d), BamHI + XhoI (e), BwnHI + HaeII (f), and BamHI + BglIl (2). Size markers were XDNA cut with HindIII and with HindII + EcoRL. Each lane was loaded with 3 -5 jig DNA. +: E1278b (wild-type), 0--l: 7204b (cargA+0-4), 0--2: DH4314 (cargA+0--2), 0--3: CD33 (cargA 0--3), 0-4: CD62 (cargA +0-4), Ct-i: 7094a(cargA O'-I), ok-2: DH4312 (cargA + -2).

enzymes and analysed by Southern hybridization using cargA + c-lI and Tyl probes. Only the restriction fragments of the cargA + (9-I insertion hybridized with the Tyl probe. There was no cross hybridization between Tyl and cargA + DNAs (data not shown). These results indicated a strong homology between Tyl element and the cargA + (9-I insertion element over the whole length of both elements. Regulatory properties of the cargA + O&-2 mutation Southern hybridization analysis indicated the presence of a similar insertion for both the cargA + (9-L and the cargA + 0b-2 mutants. The data presented in the preceding section demonstrated that the cargA + CX-I mutation results from a Tyl element insertion. Hence the cargA + 06-2 mutation is most probably also a Tyl insertion, despite the fact that the cargA + C-2 mutation possesses the same level of constitutivity of arginase synthesis as the cargA + 0- mutants and was previously classified in the 0- group of mutations (Dubois et al., 1978). This led us to investigate in detail the regulatory properties of the cargA + Oh-2 mutation to determine whether it had the characteristics of oh or 0- mutations. The segregant 80G0a (Table II), obtained from mutant DH4312 crossed with wild-type 3962c, bore the cargA + OY-2 mutation alone. The cargA + 0-2 mutation disturbed arginase synthesis in the same way and to a similar extent as the cargA + 0 mutation. Indeed, arginase synthesis was

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constitutive for induction but was still subject to nitrogen catabolite repression (Table II). However, in a diploid homozygous for the cargA + 0h-2 mutation but heterozygous for the mating-type locus, the arginase activity was strongly reduced. This behaviour classified the cargA + oh-2 mutation in the ROAM group of mutations (Errede et al., 1980) to which the cargA + Oh-] mutation belongs. Therefore, we named this mutation oh-2. Arginase RNA analysis in the cis-dominant regulatory mutants To determine the level of action of the mutations leading to constitutive arginase synthesis, we extracted total RNA from the cargA + 0-i-, cargA + 0-]J, cargA + oh-2, and wild-type

strains growing exponentially in M.am medium. The RNAs were denatured in glyoxal, fractionated on agarose gel, and transferred from the gel to diazotized paper. The RNAs were then probed with the labelled e -g DNA fragment carrying most of the cargA gene. The RNA homologous to the cargA + DNA was visualized by autoradiography. As a control, the same RNAs were hybridized independently to a 2.2-kb EcoRI restriction fragment that contained the CYC7 structural gene and an unidentified gene that hybridized to an RNA of 1450 nucleotides. This RNA was abundant and its level was not influenced by the arginase regulations. This RNA was therefore chosen as an internal control. Its signal

DNA and RNA analysis of yeast arginase regulatory mutations

4

+

Fig. 4. Autoradiogram of a Northern blot assay of arginase RNA from wild-type, cargA `0-. cargA d' 4, cargA +0"-2 strains grown on M.am. Total RNA was extracted from cells growing exponentially, denatured in glyoxal, and fractionated on 1.407o agarose gel. After transfer to DBM paper, the RNA was hybridized with 32P-labelled probe consisting of the arginase-specific DNA fragment e-g (Figure 1). Each lane was loaded with 100 Ag total RNA. +: E1278b (wild-type), 0--1: 7204b (cargA + 0 -1). 0i-i: 7094a (cargA + o-i), oh-2: 80G0a (cargA +0h-2).

Fig. 3. Restriction fragments of cargA + and cargA + o(t-i genomic DNA complementary to cargA +, cargA + 0-l, and Tyl probes. Genomic DNA samples from E 1278b (wild-type) and 7094a (cargA + /-li) strains were deaved with BamHI, co-electrophoresed in 0.77o agarose gel, transferred to nitrocellulose sheets, and hybridized to either the 32p -labelled 5.6-kb cargA + BamHI fragment or to the 11.2-kb cargA + (j-h BamHI fragment or to the 5.6-kb Tyl EcoRI fragment from plasmid S13 (Cameron et al., 1978). The figure shows the autoradiograms.

Table H. Arginase-specific activitya in wild-type, cargA + 0-, and cargA + mutants

Strain E 1278b 7204a 7094a

80GOa DH4312x80GOa

Genotype wild-type rgA+ 0-1

cargA + O4-i cargA + -2

Nitrogen nutrient am

am + arg

arg

6 138 230 100

20 137 240 110

250 350 350 480

MATa cargA +Oh-2 cargA + &-2 MA Tai

50

aExpressed as Amol urea formed/h/mg protein.

was constant in the different lanes corresponding to the experiment shown in Figure 4 (data not shown). The cisdominant regulatory mutations caused a significant increase in the arginase RNA concentration (Figure 4). This increase was slightly larger in the cargA + O(Y-J strain, as was the arginase specific activity (Table II). Within the limits of precision allowed by the Northern technique, the size of the arginase RNA in the cargA + 0--1, cargA + (j-J, and cargA + O&-2 strains were the same as in the wild-type strain (Figure 4). Discussion A 5600-bp DNA fragment which contains the arginase gene of S. cerevisiae has been cloned. This fragment contains a 1300-bp DNA region that hybridizes to an RNA species, the concentration of which varies in the same direction as the arginase activity. Hence this 1300-bp DNA segment most probably includes the arginase coding region. This segment

has a length adequate to encode the 39 000-dalton arginase subunit (Penninckx et al., 1974). The expression of the cargA + gene on a multi-copy plasmid is normally regulated both by specific induction and by nitrogen catabolite repression, while the amount of arginase is increased. This indicates that the regulatory regions have also been cloned and that the regulatory elements (e.g., the repressor molecules) are not limiting. Although the cloned DNA fragment seems to contain all the structural and regulatory regions required for normal expression of the gene in S. cerevisiae, this is not sufficient to allow its expression in E. coli. To investigate the mechanism of arginase specific induction we have analysed mutants that express arginase constitutively due to cis-dominant mutations adjacent to the structural gene (Dubois et al., 1978). According to their behaviour towards signals depending on the mating-type two classes of mutants are distinguished. Mutants of the first type, cargA + 0-, have a constitutive level of arginase independent of the matingtype constitution. In Southern experiments, they show hybridization patterns identical to that of the wild-type. Hence, their mutations are small alterations, possibly single base substitutions. In the second class of mutants, cargA + Oh, constitutive arginase synthesis is sensitive to the mating-type constitution. Southern analysis and molecular cloning have shown that they result from insertion of elements of the Tyl family. This result is in agreement with the suggestion that all ROAM mutations might be due to Tyl insertions (Errede et al., 1980). However, all the Tyl insertions do not cause constitutive expression of the adjacent gene since Tyl insertions in the 5' non-coding sequence of the HIS4 locus inactivate expression of the gene (Roeder et al., 1980). The analysis of the restriction patterns of the oft strains indicated, in addition, that the cargA + Oh-) element is inserted close to the putative cargA + coding region whereas the cargA + o-2 insertion is located 600 bp farther away. Further indications concerning the regulatory mechanism of arginase induction are provided by the analysis of the arginase RNA by gel-transfer hybridization. The oh and 0mutations provoke an important increase in arginase RNA concentration, the largest increase is observed for the cargA + (-I mutant which also has the highest arginase 1129 -


specific activity (Figure 4 and Table II). These variations in arginase RNA concentration are compatible with a control at the level of transcription. If a post-transcriptional control is considered, it must be noted that the dominant cis-acting regulatory mutations might produce their effect only as far as the arginase RNA itself contains a modification. On the other hand, no significant difference in size was

detected among the arginase RNAs from the cargA+O-, cargA + O(-l, and cargA + Oh-2 strains (as determined within the limits of precision allowed by our experimental conditions: 100 bp). This result is particularly striking for the cargA +Oh-2 strain since its insertion is located >600 bp away from the arginase structural gene. The fact that no long RNA was detected indicates that the whole region between the insertion and the structural gene is not transcribed (assuming rapid processing does not occur). Regarding the mechanism by which the Tyl insertions can provoke constitutive synthesis of arginase, it is clear that the operator, defined as the receptor of the negative signal neutralized by arginine, is either inactivated or put out of service in oh as well as in 0- mutants, but not necessarily by the same mechanism. Since -600 bp separate the two insertion sites of the Tyl elements it seems difficult to imagine that they are inserted in the supposed operator region of this gene, which would then be unusually long. There is more likely to be a long-distance effect of the cargA + O/-2 Tyl element inserted outside the arginase regulatory region which might influence the DNA conformation (e.g., induce negative supercoiling) favouring separation of the DNA strands at the arginase promoter. Materials and methods -

Strains and culture conditions The AGI (cargA -, MA To) mutant obtained from M.Grenson and the FL 100 (ura3-, MA Ta) mutant obtained from F.Lacroute were crossed to produce the 01417d (MATai) and 01420c (MATa) cargA-ura3- recombinants. Yeast mutants 7204b (cargA + o--), DH4314 (cargA + O- -2), 7094a (MATa, cargA+oC-1), DH4312 (MA Ta, cargA+O&-2), 8KOGa (MAT., cargA +Ol-2), CD33 (cargA +O--3), and CD62 (cargA4+0-4) were described in Dubois et al. (1978); only the pertinent mutation is quoted in parenthesis. The E. coli C600 r- m- deletion (pro-lac-argF) arg I (Crabeel et al., 1979) was used as recipient. The basic minimal medium (M. medium) containing 3% glucose, vitamins, and mineral traces was described previously (Ramos and Wiame, 1979). M.am medium is M. medium supplemented with 0.02 M (NHJ2SO4 as a nitrogen nutrient. Arginine (0.1 % w/v) was added when indicated either to the M. medium or to the M.am medium. Uracil, 50 jig/ml, was added when indicated. DNA preparations Large-scale yeast DNA was prepared by the method of Cryer et al. (1975) with minor modifications. Small-scale yeast DNA was prepared by the method of Davis et al. (1980). Large-scale E. coli plasmid DNA was recovered from E. coli as described by Crabeel et al. (1979). Small scaleE. coli plasmid DNA was prepared by the method of Bimboim and Doly 1979).

Transformation procedures Yeast cells were transformed according to Hinnen et al. (1978) with the following modification. Glucuronidase/arylsulfatase (from Boehringer Mannheim) was used instead of glusulase to generate protoplasts. Media for yeast protoplast regeneration were the usual Wiame's laboratory media containing 1.2 M sorbitol, 3% (w/v) glucose and 3% (w/v) agar. The yeast plasmid pool containing the cargA + gene was constructed by F.Lacroute. E. coli cells were transformed according to Petes et al. (1978), except that after the cells were diluted 10-fold with LB broth, they were grown for 90 min at 37°C before being plated directly on selective medium, without suspension

in soft agar. Cloning of the cargA + O&-I mutation Total DNA extracted from strain 7094a (cargA +0b-J) was restricted with BamHI. The fragments were ligated into BamHI-cut pFLI plasmid that had been treated with alkaline phosphatase. After ligation the DNA was used to

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transform our E. coli C600 recipient to ampicillin resistance. Ampicillinresistant E. coli colonies were screened by the method of Grunstein and Hogness (1975) with minor modifications, using the labelled e-g arginase specific DNA fragment as probe.

Restriction mapping Restriction sites

were

located by determining the lengths of the

fragments

generated by single and double digests with a variety of restriction enzymes. DNA restricted with HindIII or HindIII + EcoRI was used as high mol. wt.

marker. pBR322 restricted with AluI or HaeIII was used as low mol. wt. marker. Southern analysis DNA restriction fragments were fractionated by electrophoresis through 0.7 or 0.8%1o horizontal agarose gels; 3-5 jig of yeast DNA were loaded per slot. Transfers to nitrocellulose filters were performed by the Southern (1979) blotting procedure. After denaturation of the probe by incubation at 100°C for 10 min, hybridization was carried out in 4 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5 x Denhardt (0.1% polyvinylpyrrolidone, 0.1%o bovine serum albumin, 0.10% Ficoll), 0.1%o SDS, 100 jig/ml denatured sonicated herring sperm DNA and 10%o dextran sulfate in heat-sealed plastic bags at 65°C for - 16 h. Filters were washed twice in 4 x SSC/0. 1 % SDS at room temperature and then in 0.2 x SSC/0.1Io SDS at 65°C, until the background detected with a monitor was very low. The filters were dried and autoradiographed at - 80°C using Siemens special intensifying screens and Kodak X-Omat S1 X-ray film. Northern analysis Total RNA was prepared from exponentially growing yeast cells (0.5-1 x 106 cells/ml) by a method described previously (Waldron and Lacroute, 1975) except that the organic mix is replaced by a 0.2 M Tris buffer pH 7.4, 0.5 M NaCl, and 0.01 M sodium EDTA. After two phenol extractions and three precipitations by ethanol, the RNA was resuspended in 30 mM phosphate buffer pH 7.0 at a concentration of 10 mg/ml. Horizontal 1.4% agarose electrophoresis of glyoxal-treated RNA was performed as described by McMaster and Carmichael (1977). The procedure for transferring RNA molecules from agarose gels to diazobenzyloxymethyl cellulose (DBM) paper was performed as described by Alwine et al. (1979). Treatment and hybridization of diazotized paper sheets were described by Alwine et al. (1979) but SDS was replaced by 10%o dextran sulfate. Hybridizations with appropriate 32P-labelled probes were performed for 2 days. The papers were washed three times at room temperature with 2 x SSC/0.Io SDS followed by two washes with 0.2 x SSC at 50°C. The papers were exposed at - 80°C for 2 days under Kodak XR-5 ray film with two Kodak X-Omatic intensifying screens. Probes and labelling Appropriate radioactive fragments were made by nick translation according to the procedure of Rigby et al. (1977). The DNA fragments were purified on a low melting agarose gel after restriction of the appropriate plasmids as specified by Bethesda Research Laboratories Inc., who furnished the special agarose. Protein assays Arginase was measured as described by Messenguy et al. (1971). Protein concentration was determined by the Folin method (Lowry et al., 1951). Enzymes and chemicals

Agarose was purchased from Bethesda Research Laboratories. Restriction

endonucdeases, DNA polymerase I, deoxyribonuciease, and T4 DNA ligase were Boehringer or Bethesda Research Laboratories products. [32P]dATP was

purchased from the Radiochemical Centre, Amersham.

Acknowledgements We wish to thank Dr.M.Grenson for reading the manuscript and providing helpful suggestions. We are very grateful to M.De Baerdemaeker and E.Joris for their efficient and skilful technical contribution. We are grateful to Dr.F.Lacroute for the yeast plasmid pool, to Dr.R.W.Davis for the S13 plasmid containing the Tyl DNA, and to Dr.Cardillo for the pAB25 plasmid containing the CYC7 gene. This work was supported by an 'Action de Recherche Concertee' no. 80/85-15 between the Belgian Government, the UniversiteLibre de Bruxelles, and J.-M.Wiame, and by grant 1-5-300-82F from the Fonds National de la Recherche Scientifique to E.D.

References Alwine,J.C., Kemp,D.J., Parker,B.A., Reiser,J., Renart,J., Stark,G.R., and Wahl,G.M. (1979) in Wu,R. (ed.), Recombinant DNA, Methods in Enzymo/ogy, 68, Academic Press, NY, pp. 220-242. Beggs,J.D. (1978) Nature, 275, 104-109.

DNA and RNA analysis of yeast arginase regulatory mutations

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Dubois,E., Vissers,S., Grenson,M., and Wiame,J.M. (1977) Biochem. Biophys. Res. Commun., 75, 233-239. Dubois,E., Hiernaux,D., Grenson,M., and Wiame,J.M. (1978) J. Mol. Biol., 122, 383406. Errede,B., Cardillo,T.S., and Sherman,F. (1980) Cell,2 2, 427436. Errede,B., Cardillo,T.S., Sherman,F., Dubois,E., Deschamps,J., and Wiame,J.-M. (1980) Cell, 22, 427436. Gafner,J., and Philippsen,P. (1980) Nature, 286, 414-418. Grenson,M., and Hou,C. (1972) Biochem. Biophys. Res. Commun., 48, 749756. Grunstein,M., and Hogness,D.S. (1975) Proc. Natl. Acad. Sci. USA, 72, 3961-3965. Hinnen,A., Hicks,J.B., and Fink,G.R. (1978) Proc. Natl. Acad. Sci. USA, 75, 1929-1933. Jauniaux,J.-C., Dubois,E., Crabeel,M., and Wiame,J.M. (1981) Arch. Int. Physiol. Biochim., 89, Blll-112. Lemoine,Y., Dubois,E., and Wiame,J.M. (1978) Mol. Gen. Genet., 166, 251258. Lowry,O.H., Rosebrough,N.J., Farr,A.L., and Randall,R.J. (1951) J. Biol. Chem., 193, 265-275. Mc Master,G.K., and Carmichael,G.G. (1977) Proc. Natl. Acad. Sci. USA, 74, 4835-4838. Messenguy,F., and Wiame,J.-M. (1969) FEBSLett., 3, 47-49. Messenguy,F., Penninckx,M., and Wiame,J.-M. (1971) Eur. J. Biochem., 22, 277-286. Penninckx,M., Simon,J.P., and Wiame,J.-M. (1974) Eur. J. Biochem., 49, 429-442. Petes,T.D., Broach,J., Wensink,P., Hereford,L., Fink,G.R. and Botstein,D. (1978) Gene, 4, 37-49. Ramos,F., and Wiame,J.-M. (1979) Eur. J. Biochem., 94, 409-417. Rigby,P.W.J., Dieckmann,M., Rhodes,C., and Berg,P. (1977) J. Mol. Biol., 113, 237-251. Roeder,G.S., Farabaugh,P.-J., Chaleff,D.T., and Fink,G.R. (1980) Science (Wash.), 209, 1375-1380. Rothstein,R.J., and Sherman,F. (1980) Genetics, 94, 891-898. Southern,E.M. (1979) in Wu,R. (ed.), Recombinant DNA, Methods in Enzymology, 68, Academic Press, NY, pp. 152-176. Waldron,C., and Lacroute,F. (1975) J. Bactefiol., 122, 855-865. Wiame,J.-M. (1971) in Horecker,B.L., and Stadtman,E.W. (eds.), Current Topics in Cellular Regulation, 4, Academic Press, NY, pp. 1-38. Wiame,J.-M., and Dubois,E. (1976) in McDonald,K.D. (ed.) 2nd Int. Symp. Genetics of Industrial Microorganisms, Academic Press, NY, pp. 391-406.

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