Current Genetics

Current Genetics (1982) 5:39-46

© Springer-Veriag 1982

Isolation of the TRP2 and the TRP3 Genes of Saccharomyces cerevisiae by Functional Complementation in Yeast Markus Aebi, Peter Niederberger, and Ralf HOtter Microbiologisches Institut, Eidgen6ssische Technische Hochschule, Universit~itsstr. 2, ETH-Zentrum, CH-8092 Ztirich, Switzerland

Summary. This paper describes the isolation of the TRP2 and the TRP3 genes of Saecharomyces cerevisiae. Two pools of plasmids consisting of BamHI and SalGI yeast DNA inserts into the bifunctional yeast - Escherichia coli vector pLC544 (IOngsman et al. 1979) were constructed in E. eoli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast. The TRP2 gene was isolated on a 6.2 kb BamHI and a 5.8 kb SalGI yeast DNA fragment which shared an identical 4.5 kb BamHI-SalGI fragment. The TRP3 gene was located on a 5.2 kb BamHI fragment. By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes. Key words: Saccharomyces cerevisiae - TRP2 gene TRP3 gene - Cloning in yeast

Introduction

The enzymes catalyzing the five steps of tryptophan biosynthesis in Saeeharomyces cerevisiae are encoded for by five genes which are located on four different chromosomes (Mortimer and Schild 1980). The expression of these genes is controlled by the general control of amino acid biosynthesis (Schttrch et al. 1974; Miozzari et al. 1978a; Niederberger et al. 1981), a regulatory system also found in other fungi ( Carsiotis et al. 1974; Piotrowska 1980). The cloning of these genes should allow for an analysis of this regulatory system at the molecular level. Offprint requests to: M. Aebi

Two tryptophan biosynthetic genes, TRP1 and TRP5 of S. cerevisiae have been isolated by complementation of equivalent mutations in Escheriehia coli (Walz et al. 1978; Struhl et al. 1979). Particular characteristics of the TRP2 and the TRP3 gene products makes it necessary to isolate the corresponding genes by complementation in yeast. These two enzymes form a protein complex in vivo, even though they are transcribed from two separate genes (Doy and Cooper 1965; Schiirch 1972). Whereas the TRP2 gene product anthranilate synthase is active only in the aggregated form, the TRP3 gene product indole-3-glycerol-phosphate synthase is active irrespective of its aggregation with the anthranllate synthase. Accordingly there exist mutations in the TRP3 gene influencing only the indole-3-glycerol-phosphate synthase activity (trp3B), only the anthranilate synthase activity (trp3C) or both activities together (trp3A) (Schttrch 1972). In this communication we describe the isolation of yeast DNA fragments carrying the TRP2 or the TRP3 gene respectively. As a vector we have used the autonomously replicating hybrid plasmid pLC544 (Kinsman et al. 1979). We show that the cloned DNA fragments contain the complete coding sequences, including the regulatory sites for the general control system.

Materials and Methods Strains and Plasmidz Mutant strains of S. cerevisiae used in this study are listed in Table 1. They were all derived from the two haploid wild type strains X2180-1A (mating type a) or X2180-1B (~). E. coli strain JA196 (trpC1117 leuB6 thi hSrk) was obtained from A. Hinnen. The bifunctional yeast E. coli vector pLC544 (Kingsman et al, 1979) was obtained from J. Carbon. The plasmid pJDB219 (Beggs 1978) was from A, Hinnen with the premission of J, Beggs.

0172-8083/82/0005/0039/$ 01.60

M. Aebi et al.: Cloning of the TRP2 and TRP3 Genes of Yeast

40 E-

\

/

Fig. 1. Hybrid yeast-E, colt vectors used in this study, pLC544 is composed of pBR313 ( - - ) and a 1.45 kb chromosomal yeast DNA fragment (r:v~) carrying the TRP1 and ARS1 genes of yeast (Kingsmanet al 1979). pJDB219 consists of pMB9 ( - - ) , yeast 2 ~umDNA (l----l) and the chromosomal LEU2 gene region of yeast (t77~) (Beggs 1978)

\

Media. YEPD complete medium and minimal-MV medium for yeast have been previously described (Miozzari et al. 1978a). E. colt media were prepared according to Vogel and Bonnet (1956). Media were solidified with 2% (w/v) agar.

ing), E.C. 4.1.1.48] were performed according to Miozzari et al. (1978a).

DNA Preparation. Plasmid DNA from 17. colt was isolated follow-

Results

ing the method of Humphreys et al. (1975). Yeast plasmids were prepared according to Livingston and Klein (1977). For theisolation of total yeast DNA the method of Cryer et al. (1975) was used.

1. Construction o f Plasmid Pools

Endonuclease Digestion and Gel EIeetrophoresis. All restriction enzymes were from Boehringer, Mannheim (FRG). Digestions were performed in TA-buffer (33 mM Tris-acetate, 60 mM Kacetate, 10 mM Mg-acetate, 5 mM dithiothreitol, 1 mg/ml BSA, pH 7,5) according to O'Farretl et al. (1980) Horizontal electrophoresis of DNA samples was performed in 0.8% or 1% agarose (Sigma type II) gels in TBE-buffer (89 mM Tris; 89 mM boric acid; 2.5 mM EDTA, pH 8.2) at 2 V/cm for 16 h. DNA bands were visualized after staining in a 1.5 ~zg/mlethidium bromide solution and photographed under ultraviolet illumination.

Ligation. T4 ligase from Boehringer was used as recommended by the supplier.

Isolation of Integrated Plasmids in Yeast. 0.5 t~g of total yeast DNA was digested either with HindIII or BgllI. Ligitation of DNA (10 #gjml) was carried out at 14 °C for 16 h. This mixture was used to transform E. colt and ampicillin resistant clones were selected.

Transformation of E. colt was done according to Tabak et al. (1979).

Yeast Transformation was performed as described by Hsiao and Carbon (1979).

The plasmid pLC544 (Kingsman et al. 1979) used a cloning vector is composed of the E. colt plasmid pBR313 (Rodriguez et al. 1976) and a 1.45 kb EcoRI yeast DNA fragment, carrying the TRP1 gene of yeast (Fig. 1). This gene is able to complement a trpCmutation in E. coli. In addition the closely linked gene ARS1 enables the plasmid to replicate autonomously in yeast at a l o w c o p y number (Struhl et al. 1979, Kingsman et al. 1979). Two plasmid pools were constructed in E. colt: Pool A contained BamHI generated yeast DNA fragments, for pool B SalGI digested yeast DNA was used. 10/~g of yeast DNA cut with BamHI (SalGI respectively) were ligated with T4-DNA ligase to 5 ~g BamHI (SalGI respectively) cut plC544 DNA at a concentration of 25 /~g DNA/ml. The ligation mixtures were used to transform E. colt strain JA196 to ampicillin resistance. At least 1 x 104 transformants per pool or 5% of all transformants were tetracycline sensitive indicating inserts into the tetracycline resistance gene of the plasmid pLC544. Enrichment for tetracycline sensitive cells with the cyloserine method (Rodriguez et al. 1976) let to a population of 95% Amp r Tet s Trp + cells from which two plasmid pools were isolated.

Genetic Techniques. Tetrad analysis was done as described by Sherman et al. (1970).

Enzyme Assays. Yeast cells were grown overnight to a density of 3 x 107 cells per ml. They were permeabilized by the Triton X100 method (Miozzari et al. 1978b) and enzyme assays for the TRP2 gene product anthranilate synthase [chorismate pyruvatelyase (amino-accepting), E.C. 4,1.3.27] and the TRP3 gene product indole-3-glycerol-phosphate synthase [1-(2'-carboxyphenylamino)- 1-deoxyribulose-5-phosphate carboxy-lyase(cycliz-

2. Isolation o f the TRP2 Gene 2.1. Isolation o f trpl trp2 Complementing Plasmids. Several trpl trp2 double mutant strains ofS. cerevis&e were tested for good transformability. Strain RH963 turned out to be the best; it yielded 5 x 104 transformants per

M. Aebi et al.: Cloning of the TRP2 and TRP3 Genes of Yeast ~F

41

~

TRPI

pME500

~ p M E 5 0 1 ~.pME500-int 1B { ~ pME500-intlH BamHI, SaIGI '} Sgt11

Hindln

f

ct~,I

P~I EcoRI

Pvull

~

{

t

t

f

f

I

f t I

1 kb

i

X

i

Ampr

"F//A

© N coE ~3

Bg[I]~ pME500-intlB

~HindIlI pME500-intlH

Fig. 3. Reisolation of the integrated plasmid pMES00-intl. Integration of plasmid pME500 into the yeast chromosome by homologous recombination led to a duplication of the BamHI fragment. Digestion of total DNA from the stable transformant RH963 • pME500-intl with BglII or HindIII, circularisation of linear fragments and transformation of E. coli selecting for Ampr led to the plasmid pME500-intlB or pME500-intlH respectively which carry additional DNA not yet present in pME500. - - p B R 3 1 3 sequences, r///~ 1.45 kb yeast EcoRI fragment of pLC544 carrying TRP1 and ARS1, . yeast chromosomal sequences of the trp3 locus

/lg pLC544-DNA and 1.2 x 104 transformants per ~g of DNA from the hybrid plasmid pools when selection was made solely for TRP1 expression by growth on MV-agar supplemented with antrhanilic acid. Selection of transformants on unsupplemented MV-medium, requiring expression of both the TRP1 and the TRP2 gene for

Fig. 2. Restriction map of pME500, pME501, pMES00-intlB and pME500-intlH. The plasmids are drawn in a way that overlapping inserts are aligned and only parts of the vector pLC544 are shown. Restriction sites are shown for the inserts only. Boxed sections represent vector sequences, pBR313 sequences are hatched

growth, yielded 15 transformants per/Jg DNA from the BamHI-pool A and 4 transformants per ~g DNA from the SalGI-pool B. 10 transformants from each pool were tested for loss of the Trp+-phenotype when grown on YEPD-agar. All of them lost the Trpl- as well as the Trp 2-phenotype with high frequency indicating the plasmid location of the trpl and trp2 complementing genes. Plasmid DNA was isolated from such single unstable transformants form each hybrid plasmid pool and used to transform E. coli strain JA196 to ampicillin resistance and tryptophan prototrophy. From pool A plasmid pME500 with a 6.2 kb BamHI insert and from pool B plasmid pM501 with a 5.8 kb SalGI insert into plC544 could be isolated. Both plasmids transformed S. cerevisiae strain RH963 (trpl-21g trp2-315) with a frequency of 104 prototrophs per/Jg DNA. They shared a common BamHI-SalGI yeast DNA fragment incorporated into plC544 in the same orientation (Fig. 2).

2.2. PIasmid pME500 Integrates into the Yeast Chromosome V by Homologous Recombination. Hybrid plasraids derived from pLC544 are unstable in S. cerevisiae but can stabilize by integration into chromosomal DNA (Kingsman et al. 1979), most likely by homologous recombination (Hinnen et al. 1978). As shown in Fig. 2, both plasmids pME500 and pME501 carry additional yeast DNA adjacent to the common 4.5 kb BamHI-SalGI fragment. These sequences were used to verify that integration of plasmid pME500 into the yeast genome can occur by homologous recombination within the cloned BamHI fragment (Fig. 3). Yeast strain RH963 (trp1-218 trp2-315) was transformed with pME500 plasmid DNA to prototrophy and stable transformants were selected as described by Nasmyth and Reed (1980). From one of the stable transformants, strain RH063 • pME500-intl, total DNA was isolated, treated with HindIII or BglII respectively, ligated with T4-DNA ligase and used to transform E. coli strain JA196 to ampicillin resistance. Two transformants from HindIII digested DNA and four transformants from BglII digests were obtained. Subsequently plasmid DNA from one transformant of each type was isolated and the

42

M. Aebi et al.: Cloning of the TRP2 and TRP3 Genes of Yeast

Table 1. Mutant strains of S. cerevisiae used Strain

Genotype

RH962 RH963 RH974 RH978 RH986 RH989 RH991

trp l-218 trp3B-23 a trp l-218 trp2-315 trp3A-56 leu2 a trp3C-lO0 leu2 cdrl.1 a trp1-218 ural ade2 trp3B-23 leu2 ade 2 met8 trpl-218 ilvl ade2 a

Table 2. Marker segregation of a cross between strain RH963 (trp1-218 trp2-315 c~) • pME500-intl and strain RH991 (trpl218 ilvl ade 2 a) Markersregarded

Parental ditype asci

Nonparental ditype asci

Tetratype asci

i l v l . trpl il vl . ade2 ade2 - trp l

25

0

1

4 4

3 4

19 18

plasmids were designated pME500-intlH and pME500intlB respectively. Comparative restriction analysis with the two new plasmids revealed a close relationship with the original plasrnids pME500 and pME501. The two plasraids pMES00-intlH and pME500-intlB contained the whole 6.2 kb BamHI fragment present in the parent plasmid pME500. But in addition they carried DNA sequences only present in plasmid pMES01 (Fig. 2 and 4). It can therefore be concluded that integration of pME500 had occurred by recombination within the chromosomal sequence homologous to its 6.2 kb BamHI yeast DNA fragment. Transformation of the yeast strain RH963 (trpl-218 trp2-315) with plasmid pME500-intlB and selection for TRP1 expression on MV-agar supplemented with anthranilic acid yielded 12 transformants per gg DNA, indicating the expected loss of the ARS1 function due to the isolation procedure (Tschumper and Carbon 1980). None of these transformants was capable to grow on minimal medium. Possibly pME500-intlB carried the mutated trp2-315 allele present in strain RH963. Accordingly, transformation of strain RH963 with plaSmid pME500-intlH yielded no transformants neither on MVmedium nor on MV-medium supplemented with anthranilic acid since the TRP1 gene was destroyed due to the isolation procedure. The integration of plasmid pME500 into the yeast genome by homologous recombination within the 6.2 kb BamHI fragment led to the prediction that this integration had to occur at the trp2 locus of chromosome V. To verify this hypothesis, strain RH063 • pME500-intl was crossed with strain RH991 (trpl-218 i l v l ade2) and a tetrad analysis was performed. The results are shown in Table 2. Since both parental strains carried the same trpl mutant allele, the TRP1 allele on the vector marked the site of integration. It was found that in strain RH963 • pME500-intl the TRP1 gene was tightly linked to i l v l , a locus close to trp2 (Mortimer and Schild 1980). Both physical and genetic data thus supported the hypothesis that the plasmids pME500 and pME501 carry the TRP2 locus of yeast.

3. Isolation o f the TRP3 Gene 3.1. Isolation o f a trp l trp3 Complementing Plasmid. The isolation of a trpl trp3 complementing plasmid was per-

Fig. 4. Comparative restriction analysis of the plasmids plC544, pME500, pME501 and pME500-intH. 1: EcoRI/HindIl double digest of ~.-DNA (marker); 2, 6: pLC544; 3, 7: pME500; 4, 8: pME501; 5, 9: pME500-intlH; 2-5: ClaI digests; 6-9 ClaI/ HindIII double digests. Number on the left side indicate size of x-fragments in kb. Arrows indicate the fragments which show the correlation of pME500-intlH with pMES00 or pMES01. Electrophoresis was done in a 1% agarose gel in TBE-buffer at 2 V/cm for 16 h

formed by a procedure analogues to that described above (see 2.1). The yeast strain RH962 (trpl-218 trp3B-23) was transformed to prototrophy with the hybrid plasmid pool A carrying the BamHI yeast DNA inserts. From these experiments plasmid pME502 carrying a 5.2 kb yeast DNA fragment was obtained (Fig. 5). pME502 plasmid DNA was able to transform the yeast strain RH962 to prototrophy with a frequency of 104 transfor-

M. Aebi et al.: Cloning of the TRP2 and TRP3 Genes of Yeast ClaI

TRP 1

~

43

SaI.G1 i

~lkb

Fig. 5. Restriction map of the trp3-complementing plasmid pME502. The boxed sections represent pLC544 sequences, pBR313 sequences are hatched. Restriction sites are shown for the BamHI-insert only. The BamHI fragment contains no restriction site for EcoRI, HindIII, PstI, AvaI, PvuII, BglII or KpnI

Table 3. Marker segregation of a cross between strain RH962 (trpl-218 trp3B-23 a) • pME502-intl and strain RH986 (trp12 1 8 ural ade 2 ~) Markersregarded

trp l - ura l trp l - ad e 2 ade2 - ura] T R P 3 - trp3

Parental ditype asci

23 7 6 25

Nonparental ditype asci

Tetratype asci

0 3 4 0

2 15 15

analysis and crossed to strain RH982 (trp1-218 ade 2 ural). Marker segregation in the dissected tetrads showed a new linkage of the TRP1 marker, introduced by pME502, to ural, which is itself closely linked to trp3 (Table 3), thus indicating an integration of plasmid pME502 at or near the trp3 locus. The appearance of one tetrad with 3 : 1 segregation of the trp3 marker (see last line in Table 3) but a normal 2: 2 segregation of the plasmid marker TRP1 demands that both the wild type and the mutant allele of the trp3 locus are present in the stably transformed strain RH962 • pMES02-int 1.

4. Expression of the Cloned TRP2 and TRP3 Genes in Saccharomyces cerevisiae

mants per pg DNA. No transformants were obtained from the SalGI generated plasmid pool B.

3.2. Plasmid pME502 Integrates into the Yeast Chromosome X by Homologous Recombination. In analogy to the situation described for the TRP2 gene, stable prototrophic transformants of the yeast strain RH962 (trpl218 trp3B-23) were isolated using pME502 plasmid DNA. Strain RH962 • pME502-int 1 was chosen for further

4.1. Expression in Stably Transformed Strains. In order to demonstrate that the cloned yeast DNA fragment of pME500 harbours the whole TRP2 gene sequence, the enzymatic activity of its product anthranilate synthase was determined in the stably transformed strain RH963 • pME500-intl. Since anthranilate synthase is only active in complex with indole-3-glycerol-phosphate synthase, this enzyme was measured as a control. As shown in Table 4, anthranilate synthase activity in strain RH963 • pME500-intl was only at half the wild type level. Yet the same derepression factor was obtained under tryptophan limitation induced by 5-methyltryptophan. Analogously the stably transformed strain RH962 • pME502-intl was cultivated in MV-medium with or without 5-methyltryptophan. Normal levels of both anthranilate synthase and of indole-3-glycerol-phosphate synthase as well as normal regulation of both activities were found (Table 4).

Table 4. Expression of the TRP2 and the TRP3 gene in stably transformed strains Strain

Genotype

Growth conditions

Anthranilate synthase

Indole-3glycerolphosphate synthase

X2180-1A

wild type

MV-medium MV-medium + 5MT (5 x 10 - 4 M)

1 1.9

1 2.1

RH963. pME500-intl

trp-218 trp2-315 pME500-int

MV-medium MV-medium + 5MT (5 x 10 - 4 M)

0.5 1.1

0.9 2.1

trp1-218 trp3B-23 pME502-int

MV-medium MV-medium + 5MT (5 x 10 - 4 M)

0.9 1.7

0.9 1.9

RH962 - pME502-intl

Relative specific activities compared to the wild type levels (1.2 nmoles per mg of protein per rain for anthranilate synthase, 1.5 nmoles per mg of protein per rain for indole-3-glycerol-phosphate synthase) on MV-medium are given. They are derived from two independent measurements. 5MT = DL-5-methyltryptophan

44

M. Aebi et al.: Cloning of the TRP2 and TRP3 Genes of Yeast

Table 5. Tetrad analysis of the two crosses RH989 • pME504 x RH974 and RH989 - pMES04 x RH978 Strain

Genotype

Anthranilate synthase

Indole-3-glycerolphosphate synthase

X2180-1A RH974 RH978 RH989

wild type trp3A-56 leu2 trp3C-l O0 leu2 cdrl-1 trp3B-23 leu2 met8 ade2

1 0 0 0.6

1 0 3.2 0

0.9 1.1 1.1 1.0

22.5 23.6 25.4 23.2

0.9 1.2 2.5 2.3

25.1 29.7 106.0 85.4

Segregants of a tetrad from the cross RH989 - pME504 x RH794 A B C D

trp3 leu2 met8 ade2 trp3 leu2 trp3 leu2 met8 ade2 trp3 leu2

pME504 pME504 pME504 pME504

Segregants of a tetrad from the cross RH989 • pME504 x RH978 a b e d

trp3 leu2 met8 trp3 leu2 met8 ade2 trp3 leu2 ade2 cdrl-1 trp3 leu2 cdrl-1

pME504 pME504 pME504 pME504

Strains were grown in MV-medium supplemented with casamino acids (0.5% w/v) and adenine (30/~g/ml). For strains RH974, RH978 and RH989 this medium was supplemented with trytophan (20 gg/ml). Relative specific activities compared to the wild type levels are given (see legend to Table 4)

4.2. Expression o f the TRP3 Gene Located on a Multicopy Plasrnicl. In contrast to the TRP2 gene product an excess of the TRP3 gene product can be determined in a strain since indole-3-glycerol-phosphate synthase is active irrespective of its aggregation with anthranilate synthase. This offered the possibility to study the regulation of the TRP3 gene when located on the stable multicopy plasmid pJDB219 (Beggs 1978, and Fig. 1). This plasmid appears to replace the endogenous 2 #m-DNA in transformed strain (Erhart and Hollenberg 1981)and thereby establishes stably at a high copy number. Plasmid DNA of pME502 and pJDB219 were both digested with BamHI and ligated with T4-DNA ligase thereafter. The ligation mixture was used to transform E. coli strain JA 196 to leucine prototrophy and the transformants were screened for Amp s Tet s Trp- phenotypes. One of the clones was analyzed further and the plasmid it carried was named pME504. Restriction analysis revealed that pME504 was composed of the whole pJDB219 plasmid and the BamHI fragment originally cloned on plasmid pME502, The orientation of the BamHI fragment was the same with respect to the pMB9 sequences of pLC544 and pJDB219 (data not shown). pME504 plasmid DNA was used to transform the yeast strain RH989 (trp3B-23 leu2 ade2 met8) to both leucine and tryptophan prototrophy. One of the transformants, strain RH989 - pME504, was crossed with strains RH974 (trp3A-56 leu2) and RH978 (trp3C-lO0

leu2 cdrl-1) respectively. Mutation cdrl leads to a constitutive derepression of genes under the general control of amino acid biosynthesis, as e.g. the TRP3 gene (Miozzari et al. 1978a). In tetrads of the two crosses the markers TRP3 and LEU2 both segregated in the non-Mendelian fashion 4 : 0, confirming their location on the plasmid. The activities of anthranilate synthase and indole-3-glycerol-phosphate synthase were determined in selected tetrads. In the first tetrad (Table 5, A to D) from the cross with strain RH974 an excess of indole-3-glycerol-phosphate synthase was synthesized from the plasmid coded TRP3 gene. In the second tetrad resulting from the cross with strain RH968 (Table 5, a to d) two segregant strains contained the cdrl mutant allele and exhibited approximately three fold higher levels of both enzyme activities measured. It was thus confirmed that the 5.2 kb BamHI fragment cloned into the multicopy vector pJDB219 contains the whole TRP3 structural gene of yeast since it complements all three kinds of trp3 mutations. Apparently the cloned TRP3 fragment carries also a postulated regulatory region involved in the general control.

Discussion With the help of the hybrid vector pLC544 (Kingsman et al. 1979) we have isolated yeast DNA fragments capable

M. Aebi et al.: Cloning of the TRP2 and TRP3 Genes of Yeast of complementing yeast trp2 and trp3 mutations. In principle the procedure described by Nasmyth and Reed (1980) and by Clarke and Carbon (1980) was followed. In contrast to these authors our plasmid pools did not contain random or quasirandom yeast DNA fragments e.g. resulting from partial Sau3A digestion of yeast DNA, but BamHI or SalGI fragments. The TRP2 gene was isolated on a BamHI as well as on a SalGI yeast DNA fragment, sharing a common region of 4.5 kb (Fig. 2). The TRP3 gene was isolated on a BamHI fragment only. As the TRP3 gene does not contain a SalGI site (data not shown) the failure to isolate a trp3 complementing SalGI fragment may originate from the pool construction and the selection procedure in which e.g. large DNA fragments are underrepresented. As pointed out by Nasmyth and Reed (1980) complementation of a mutation in a specific gene by a cloned sequence is not sufficient proof that the cloned sequence corresponds to the gene being sought. The characteristics of the vector pLC544 allow for a more stringent test of identity of the cloned TRP2 and TRP3 gene respectively. The vector pLC544 is known to be very unstable in yeast (Kingsman et al. 1979) but the plasmid can stabilize by integration into the chromosome by homologous recombination. For plasmid pME500 carrying presumably the TRP2 gene sequence we showed that integration could occur within a DNA sequence corresponding to the cloned BamHI fragment. This was demonstrated by the reisolation of the stably integrated plasmid of strain RH963 • pME5OO-intl after excisions with HindIII or BglII and religation. The resulting new plasmids pME500-intlH and pME500-intlB carried the missense trp2-315 allele of strain RH963 suggesting integration of pME500 in or near the trp2 site. Direct proof for this configuration was obtained by tetrad analysis of a cross between strain RH963 - pMES00-intl and strain RH991 (trpl-218 ilvl ade2). The TRP1 gene carried by pME500-intl signalled a point of integration close to the ilvl locus which is itself closely linked to the trp2 site on chromosome V (see Table 2). In contrast to the trp2 complementing plasmid pME500, the integration of the trp3 complementing plasmid pME502 within the chromosomal sequence corresponding to the cloned BamHI fragment was not rigorously proven. Yet, the stable integration of the plasmid in strain RH962 • pME502-intl led to a new linkage of the plasmid marker TRP1 to the ural locus, which is linked to the trp3 locus on chromosome X. The simuhaneous presence of the closely linked wild type TRP3 gene and the mutant trp3B-23 allele in strain RH962. pME502intl was shown by the 3:1 segregation of the Trp3 +phenotype in one tetrad of the cross between strain

45 RH962 • pME502-intl and strain RH986 (trpl-218 urn1 ade2) (Table 3). The expression of the cloned TRP2 gene was studied in the stably transformed strain RH963 • pME500-intl and found to be reduced to half the wild type level (Table 3). As indicated above this strain carried both the wild type TRP2 allele together with a trp2 missense mutation. The missense allele used does not influence the complexing ability of the corresponding inactive anthranilate synthase with the TRP3 gene product indole-3glycerol-phosphate synthase (Landolt 1974). The observation that strain RH963 •pME500-intl exhibited only half the anthranilate synthase activity thus indicated that the TRP3 gene product was not present in excess but in approximately equimolar amount of the TRP2 gene product. The presence of two complexing anthranilate synthase products from the TRP2 and trp2-315 alleles would resuh in about half active and half inactive complex with the indole-3-glycerol-phosphate synthase. The expression of the TRP3 gene located on the multicopy plasmid pME504 was studied. This TRP3 gene was able to express indole-3-glycerol-phosphate synthase as well as to restore normal anthranilate synthase activity in trp3A, trp3B and trp3C mutant strains, confirming that the cloned 5.2 kb fragment contained the whole TRP3 gene. In the wild type CDR1 background a 20 to 30 fold higher indole-3-glycerol-phosphate synthase activity was found in comparison to the wild type strain carrying only one copy of the gene. This increase was most likely due to the increased copy number as described for the URA3 gene (Loison et al. 1981) or the ARG3 gene (Crabeel et al. 1981)ofS. cerevisiae. In the mutant cdrl background a further increase by a factor of 2 to 3 occurred (Table 5), indicating that also the multicopy plasmid coded TRP3 gene was subject to the general control system and that the trans-acting CDR1 gene product can cope even with a 2 0 - 3 0 fold increased of a single gene. The isolation of the TRP2 and the TRP3 gene will contribute to the understanding of the regulation elicited by the general control system. Furthermore, strains transformed with multicopy plasmids carrying the two genes might be helpful in the isolation of the anthranilate synthase/indole-3-glycerol-phosphate synthase complex.

Acknowledgements. The authors want to thank Dr. John Watson for giving substantial advise in the DNA technology and his helpful discussion. This work was supported by the grant no. 3.002-0.81 of the Swiss National Science Foundation.

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Communicated by C. P. Hollenberg Received February 16, 1982