Gene, 107 (1991) 149-154 0 1991 Elsevier Science Publishers B.V. Al1 rights reserved. 0378-l 119/91/SO3.50

149

GENE 0608 1

Cloning and transcriptional (Recombinant biosynthesis)

S&a

analysis of the ADE6 gene of Saccharomyces cerevisiae

DNA; yeast; pulsed-field gel electrophoresis;

phosphoribosylfo~yl

glycinamidine

synthetase;

purine

Giani *, Marco Manoni * and Diego Breviario

Istituto Biosintesi Vegetaii C.N.R., 20133 Milan (Italyj Received by J.-P. Lecocq: 22 March 1991 Revised/Accepted: 13 June/ 14 June 1991 Received at publishers: 2 August 1991

SUMMARY

The Sacc~uro~yce~ cerevisiae gene, ADE6, encoding 5’-phosphoribosylfo~yl glycin~idine synthetase (EC 6.3.5.3) has been cloned by complementation of an ade6 auxotroph. Transformation of ade6 mutants with ADE6-carrying centromeric plasmids restored normal, adenine-independent growth behavior in the recipients. Strains containing a disrupted ade6 allele were constructed and behaved as stable adenine auxotrophs. Southern transfer and genetic analyses of strains carrying a disrupted aded allele demonstrated that the cloned gene was ADE6 and not a suppressor. The cloned ADE6 DNA was mapped on the ~D2-pro~mal fragment of chromosome VII by hyb~dization on yeast chromosomes separated by pulsed-field gel electrophoresis. Northern-blot hybridization experiments show that the ADE6 region produces two different mRNA species of approx. 5 and 2 kb. Disappearance of the larger, but not the smaller, transcript is associated with ade6 mutations. A threefold repression in the amount of the 5-kb ADE6 mRNA is observed when growth medium is supplemented with exogenous adenine.

INTRODUCTION

The purine de novo synthetic pathway consists of ten enzymatic activities leading to the production of IMP and

Correspondenceto: Dr. D. Breviario, Istituto Biosintesi Vegetah, Via Bassini 15, 20133 Milan (Italy) Tel. (39-2)70600170; Fax (39-2)2362946. * Current address: (S.G. and MM.) Istituto Tecnologie Biomediche Avanzate, Via Ampere 56,20131 Milan (Italy) Tel. (39-2)70630741 Abbreviations: aa, amino acid(s); ade, mutation in the biosynthesis pathway of adenine; ADE6, gene encoding FGAMSase; bp, base pair(s); EtdBr, ethidium bromide; FGAMSase, 5’-phosphoribosyifo~yl giycinamidine synthetase; HIS3, gene encoding imidazoleglyceroi phosphodehydratase; kb, kilobase or 1000 bp; LB, Luria-Bertani (medium); LEU2, gene encoding j%opropylmalate dehydrogenase; nt, nucleotide(s); p, plasmid; PFGE, pulsed-field gel electrophoresis; R, resistance/resistant; RAD2 P, see Fig, 3 legend; S., Saccharomyces; wt, wild type.

other purine nucleotides. These enzymatic activities are well conserved across the species from yeast to mammals (Jones and Fink, 1982). Nevertheless, many of these functions are encoded by multifunction~ genes that differ between organisms (Henikoff, 1986). The cloning of four human multifunctional de novo purine biosynthetic genes by functional complementation of corresponding yeast mutations has been recently reported (Schild et al., 1990; Minet and Lacroute, 1990). Enzymes encoded by these and other genes of the purine synthetic pathway, have relevant medical significance since they are potential targets for chemotherapeutic agents. In S. cerevisiae, mutations defining genes that encode the enzymes for purine nucleotide synthesis have been mapped and several of these genes have been cloned (Mantsala and Zalkin, 1984; Mortimer and Schild, 1985; White et al., 1985; Henikoff, 1986; Staben et al., 1986; Stotz and Linder, 1990). More specifically, all the genes involved in the first steps of the pathway up to the closure of the

150 imidazole ring have been cloned and studied with the exception of the ADE6 gene encoding FGAMSase. FGAMSase of glutamine

is a glutamine amidotransferase amide transfer and aminator

characterization and a preliminary scriptional regulation.

study on ADE6

tran-

composed functional

domains. In Escherichia coli and S. typhimurium domains are fused into a single protein chain (French

both et al.,

1963 ; Smith and Daum, 1987). In Bacillus subtilis two genes pure andpurL encode the glutamine amide transfer and the aminator subunit respectively (Ebbole and Zalkin, 1987). pure and purL belong to a cluster of overlapping genes

EXPERIMENTAL

AND DISCUSSION

encoding the enzymes for de novo purine nucleotide synthesis. Transcriptional expression of these genes is regulated by adenine and guanine nucleotides. Similarly, in S. cerevisiae it has been shown that the presence of adenine in the culture medium negatively modulates mRNA level of

(a) Cloning and subcloning of ade6-complementing fragments The plasmid pool from which the ade6-complementing fragments described here were isolated was a partial Sau3A digest of genomic DNA from strain S288C cloned into the BamHI site of YCPSO, a centromeric plasmid that contains yeast URA3 as a marker (original source is M. Rose). Clones presumably carrying the ADE6 gene were identified by their ability to complement an ade6 ura3 strain (JC501;

the ADE4, ADEI and ADE2 genes (Mantsala and Zalkin, 1984; Alenin et al., 1987). Here we report the isolation of a genomic fragment able to complement that ade6 mutation, its physical and genetic

Table I). Two Ade’ transformants were isolated and showed a clearly associated loss of phenotypes Ade + and Ura+ after several generations at 28°C in nonselective medium. Both transformants contained pER017 that

TABLE

I

Vectors and strains Vector or strain=

Description b

Source ’

Yeast centromeric plasmid containing CEN4 and URA3 sequences Yeast artificial chromosome (YAC) plasmid containing HZS3, UZU3, TRPI, CEN4 and SUP4 sequences Yeast episomal plasmid containing the 2%kb BglII fragment of LEU2 and 2 pm plasmid sequences Yeast integrating plasmid containing yeast LEU2 and pBR322 sequences

CSH M. Olson

MATa aded ura3 met3 his4 srab-15 ras2 : :LEU2 leu2

MATa ma1 gal2

J. Cannon L. Panzeri L. Panzeri K. Tatchell J. Cannon CSH CSH K. Tatchell d K. Tatchelld This work CSH

pro _ thi- thr- lacy1 strR r- m- recA

Sambrook et al. (1989)

Plasmids YCP50

YAC3 YEP213 pHLES

CSH This work

Yeasts

JCSOl 6154/5D 6017/l lb DB112 JC302WT DB328 GRFl8 LRA3Ei LRA2 DB6a S288C

MATa aded cyh2 met13 MATa ade5/7 t@-101 cyh2 MATa ade2 leu2-112 ura3-1 can1 his3 MATa his4 ura3 lys2 leu2 MATa Ieu2-3 leu2-112 his4-480 hisj stell MA Ta leu2 his3

canR

MATa ura3-52 lys2 ade2 adel his7 trpl MATa ura3-52 lys2 ade2 adel his7 trpl MATa ade6: :LEU2 leu2 (~2 ura3-52 his4

E. coli

HBlOl DHl

F-

recA thi- r- m+

a Cells were grown aerobically at either 30°C (yeast) or 37°C (Z?.coli). The composition of the rich (YEPD) and synthetic media, variousomission media

and sporulation medium for yeast (Sherman et al., 1986) and LB medium for E. coli (Sambrook et al., 1989) were as described. Synthetic complete media lacking uracil (- ura) and/or lacking adenine ( - ade) were used for selecting and screening transformants. When added, the concentration of adenine was 20 fig/ml. Solid media contained 2% agar. E. coli HBlOl and DHl strains were used for preparation of plasmid DNAs. b See Jones and Fink (1982) for references to gene symbols in yeast. c CSH, Cold Spring Harbor Laboratory collection. These vectors and strains were given at the Yeast Genetic Course, Cold Spring Harbor 1985. iiChromosome VII of this strain has been split into two fragments at the RAD2 locus. d Original source is P. Hieter.

151 the ade6 lesion, have failed (Fig. 1). These results indicate that over 6 kb of the Hind111 insert are required to successfully complement the ade6 mutation. This suggests that either the coding region of the ADE6 gene is rather long or its genomic organization is complex (i.e., with exons separated by noncoding intervening sequences). (b) Identification

of the cloned DNA as ADE6

by gene

disruption Plasmid pDER065 has a single BamHI site that is located within the Hind111 insert and splits it into two

He”

H I 0

BfEx

VE

XH 1 I 6

pDER065

+

Ba

Ii

H

Ba E

H

I

I X I

X I

1 kb

Fig.

I. Restriction

ade6 mutation.

map of DNA Thick

yeast DNA. Thin lines indicate contains

fragments

(B) More

detailed

restriction

or inability

are the cloned

isolated

from Ade6’

pER017

transformants.

map of the 8-kb Hind111 fragment

Symbols

to complement

of complementing

segments

the YCPSO vector. (A) Plasmid

the 23-kb insert originally

cloned in pDER065.

capable

lines and double-lined

+ and - refer, respectively,

ade6 mutations.

Ba, BarnHI;

Bg, BglII;

EcoRI; H, HindIII; Hp, HpaI; K, KpnI; S, SalI; Sau, Sau3AI; V, EcoRV; X, XbaI. The boxed BamHI used for gene-disruption between BamHI

the Sau3AI

partially

site indicates Ba/Sau

digested

yeast

transformed

by Beggs (1978) by the CaCl,

of yeast plasmid

E,

Sp, SphI;

the integration

site

refer to the sites of ligation genomic

DNA

site ofthe YCPSO vector. Yeast cells were transformed

as described isolation

experiments.

sub-

to the ability

and the

essentially

and Ito et al. (1983). E. coli cells were

method

(see Sambrook

DNA was done according

et al., 1989). Rapid to Nasmyth

and

Reed (1980).

carries a DNA insert of 23 kb (Fig. 1) and transforms the JC50 1 strain to the Ura + and Ura + Ade + phenotypes with the same efficiency (1-5 x lo3 pg DNA). S. cerevisiue strain 6154/5D, originally isolated as an ade6 mutant (a gift from L. Panzeri), was also transformed by the purified pER0 17 to the Ade + phenotype with a similar efficiency. Restriction mapping of the cloned 23-kb DNA segment showed the presence of three Hind111 restriction sites located within the insert (Fig. 1). These sites were used to generate four different subclones (pDER025, 33, 40 and 65) by cutting and religating the mixture of fragments generated by a complete Hind111 digestion of pER017 (data not shown). Of the four, only pDER065 was able to transform adeb-deficient recipient strains to adenine prototrophy (Fig. 1). Further attempts to identify smaller regions of the S-kb Hind111 insert contained in pDER065, able to complement

fragments of 5 and 3 kb, respectively. Subcloning of these fragments did not yield plasmids that could complement the ade6 mutation, suggesting that the BamHI site is critical for maintaining gene function (Fig. 1). Therefore, we developed a strategy to disrupt ADE6 by inserting at the BamHI site either a 1.8-kb BamHI fragment containing the yeast HIS3 gene (isolated from plasmid YAC3) or the 2.8-kb BglII fragment carrying the sequence of the yeast LEU2 gene (isolated from plasmid YEP213). Insertion of both these markers at the BamHI site of pDER065 abolished its ability to complement the ade6 mutation (data not shown). We then cloned the Hind111 fragment containing the LEU2 gene inserted at the BamHI site in a pBR322-based vector generating pHLES2 and pHLES3 in which the LEU2 fragment was inserted in both orientations with respect to the insert. These plasmids were digested with HindIII, and the Hind111 LEU2-containing fragments were used to direct site-specific integration in two different leu2 ADE6 strains (JC302WT, DB328; for reference on onestep gene disruption see Rothstein, 1983). Integrative transformation of these strains would be expected to yield, by homologous recombination at the ADE6 locus, a high proportion of constructs in which the Hind111 fragment carrying LEU2 has replaced the corresponding wt ADE6 sequences. Performing these experiments we found for both strains that a large majority of Leu+ transformants has also become Ade -. The Leu + Ade- phenotype was stable over several generations of growth under nonselective conditions. Strains containing the ade6::LEU2 disruption were named DB6a. We used two different approaches to show that the expected ADE6 gene replacement had indeed taken place. First, a Southern transfer analysis was performed. Genomit DNA from strain JC302WT (leu2, ADE6) and from three strains in which ADE6 disruption had presumably occurred was exhaustively digested with HindIII, separated electrophoretically on a 0.8% agarose gel, transferred to nitrocellulose and probed with the 4-kb X&I fragment from the ADE6 region (Fig. 1). The results shown in Fig. 2 were consistent with the predictions. In fact, with the wt DNA

152

kb

1234

TABLE

II

Complementation

analysis

of Ade

auxotrophs

Cross o

-Adeb growth

DB 112 x JC302WT ade2 ADE6

10.8 -

ADE2 ADE6

DB 112

x

8-

DB6a2,

DB6a3

ADE2 ade6: :LEV2

ade2 ADE6

6017/11b x DB6a2, ade5/7 ADE6

DB6a3

ADE5/7

ade6 : :LEV2 JC501 x JC302WT ade6

ADE6

JC501 x DB6a2, 6134/5D

x JC302WT

ade6 6134/5D

ADE6 x DB6a2,

ade6 ’ Strains DB6a2,

Fig. 2. Southern was performed digested

transfer

by the procedure

strain JC302WT

[a-32P]dCTP

national,

was carried

“P-

are listed

trans-

a nick-translation

out as described

chromosomal were labelled

kit (Amersham

Inter-

of Rigby et al. (1977). Filter by Sambrook

in Table I. Only

represent

two strains

and the LEV2 marker

to the ADE6 insert. Genetic

complementation yeast genetics

tests were carried (Sherman

b + or - indicate, without

DB6a3

::LEV2 relevant

genotypes

are

given.

in which ADE6 gene replacement is present crosses,

in both orientations random

spore plating

out by the standard

procedures

with and for

et al., 1986).

respectively,

the ability or not to grow on medium

adenine.

has taken

filter (0.45 PM) in 10 x SSC

. citrate pH 7). DNA fragments

Bucks, U.K.) based on the methods

hybridization

was

with 0.25 M HCI for 30 min at and neutralization

to a nitrocellulose

by using

DNA

DNA from

DNA from three different

gels were treated

DNA was transferred

XbaI

Lanes: 1,genomic

of wt ADE6 gene by replacement

After denaturation

(1.5 M NaCl/O. 15 M Na,

et al. (1983). DNA

with the nick-translated

(ADE6); 2-4, genomic

place. EtdBr-stained

of yeast chromosomal

of Winston

isolated from pDER065.

in which disruption

room temperature.

with

Isolation

with Hind111 and probed

labelled fragment formants

analysis.

respect

ade6

DB6a3

has occurred

DB6a3

adeb: :LEV2

ade6

et al. (1989).

the expected 8-kb fragment hybridized to the probe whereas with the DNA that presumably contained a disrupted ADE6 gene, the size of the hybridizing fragment increased to the predicted 10.8 kb as no Hind111 sites are present in the LEU2 fragment. The results also suggest that the wt genome contains only one ADE6 gene. Second, two types of genetic crosses were performed. In the first one, two strains presumed to contain the ADE6 disruption-replacement (DB6a2, DB6a3) were crossed to a leu2 canR strain (GRF18). Random spores analysis of the canR haploid progeny (30 spores) showed that the adenine deficiency and the LEU’ phenotype of the ade6 strains cosegregated. In the second one, DB6a strains were crossed to both original ade6 mutants. No complementation of the ade6 mutation was observed in the diploid cells suggesting that mutations were allelic. On the other hand, complementation of the Ade- defect was observed when the DB6a

strains were crossed to strains carrying ade2 or ade5/7 mutations (Table II). Altogether these results demonstrate that gene disruption-replacement had occurred at the ADE6 locus and that the cloned gene must therefore be ADE6, as opposed to some suppressor. (c) Chromosomal mapping of the ADE6 locus Genetic mapping places ade6 on the right arm of chromosome VII between alg7 and supll2 and closely linked (5 CM) to sru6 (Mortimer and Schild, 1985; Cannon et al., 1986; Breviario et al., 1986). We mapped the cloned ADE6 fragment by hybridization on yeast chromosomes electrophoretically separated by PFGE and blotted onto nitrocellulose filters. To this purpose we used a yeast strain (LRA3) in which all the 16 chromosomal DNAs can be separated by a single PFGE run. This is possible because chromosome VII that normally corn&rates with chromosome XV has been split at the RAD2 locus by the chromosome fragmentation method developed by Vollrath et al. (1988). The ADE6 cloned sequence is expected to hybridize to the RAD2 proximal fragment (RAD2 P) of the chromosome VII just below the band corresponding to chromosome XV (approx. 1200 kb). The RADZ-distal fragment runs before the smallest yeast chromosome. As shown in Fig. 3A the labelled 8-kb

153

VII-XV

-

VII (RADSP)

+

kb

LRA2

LRA3

LRA2

LRA3

ADE6 + RASl

ADE6 Fig. 3. Southern-blot

analysis

PFGE.

VII of the LRA3

fragments

at the RAD2 locus. IUD2

hybridized

with the radiolabelled

B) The same nitrocellulose bound,

of yeast

chromosomes strain

separated

P refers

was rehybridized

gene. Agarose

to the RADI-proximal

a Pulsaphor

LKB Biotechnology

fragment

yeast chromosomes

with an hexagonal AB, Bromma,

array

Sweden)

gel, 150 V, a running

After electrophoresis, photographed. 48 h. Blotting

of ADE6.

were (Panel

containing

were prepared

(model

still

the RASI

by Carle and Olson (1985). PFGE was performed

system

tions: 1 y0 agarose

fragment

filter of panel A with the ADE6 probe with a DNA

plugs containing

tially as described

Hind111

essenusing

2015; Pharmacia

under the following

condi-

time of48 h and 100 s pulse time.

the gel was stained

Separated yeast chromosomes and hybridization conditions

2

by

has been split into two

LRA2 is the control strain. (Panel A) Yeast chromosomes

fragment.

*

-

5

Chromosome

’

in 0.5 pg EtdBr/ml

and then

were blotted for as long as were as described in the

legend of Fig. 2.

Fig. 4. Northern

(d) Transcription of the ADE6 gene Fig. 4 shows the results of a Northern-blot hybridization experiment. Probing with the 4-kb X&I fragment of the ADE6 region (Fig. l), two species, approx. 5 and 2 kb in length, were detected in the total RNA isolated from wt ADE6 cells. Of these two transcripts only the 5-kb mRNA was detected when an internal 1.5-kb HpaI-BamHI subfragment (Fig. 1) was used as a probe (data not shown).

analysis.

Total RNA extracted

with the ADE6 XbaI 32P-labelled

1, JC501 (ade6); 2, JCSOI transformed (ADE6)

cells grown

culture

medium.

without

Transcript

the presence

of 2.2 M formaldehyde.

nitrocellulose

filter (0.2 pm) and hybridized

of hybridization graphs

RNA

(Carlson

and Botstein,

and electrophoresed

II (CAMAG,

in

were blotted

with [32P]DNA

to a

probes

of the relative

between RNA samples was done by scanning

with a TLC Scanner

in the

using a BRL-Gibco

species

et al. (1989). Quantitation

Lanes:

3-4, JC302WT

(lane 3) or with (lane 4) adenine sizes were established

1982). Each RNA sample (IO pg) was denatured

by Sambrook

from log phase fragment.

with pDER065;

RNA ladder. Total RNA was isolated as described

described

Hind111 fragment correctly hybridized with either the fragmented RAD2 P (LRA3) or the intact form (LRA2) of chromosome VII. As a control, we rehybridized the same filter with a labelled DNA fragment containing the RASI gene that maps on chromosome XV. A band corresponding to this chromosome and clearly separated from the RAD2 P fragment of chromosome VII can be observed (Fig. 3B). We conclude that the cloned ADE6 DNA mapped on chromosome VII.

transfer

cells were hybridized

as

levels

autoradio-

Berlin, F.R.G.).

Furthermore, only the 5-kb mRNA was selectively lost in the ade6 mutants we analyzed and its presence restored by transformation with pDER065 (lanes 1 and 2, Fig. 4). This suggests that only the larger transcript encodes the ADE6 gene product. Alternatively, the 2-kb transcript could code for just one of the two biochemical domains of the FGAMSase, left unaltered by the ade6 mutation but unable by itself to give functional complementation. This would be a situation similar to the one observed in B. subtilis (Ebbole and Zalkin, 1987). The reason why ade6 original mutations reduce 5-kb mRNA to almost undetectable level is not known, but it could be that these are regulatory mutations that severely affect gene transcription. The unusually large size of the ADE6 transcript suggests that the encoded protein might be equally large. This would be consistent with the fact that FGAMSases from

154 S. typhimurium, E. coli and B. subtilis are all proteins M,s over 100000 (Ebbole and Zalkin, 1987).

with

Transcriptional regulation by adenine has already been reported for the ADE4, ADE2 and ADEI genes of S. cerevisiue (Mantsala and Zalkin, 1984; Alenin et al., 1987) and for the entire gene cluster encoding enzymes for de novo purine biosynthesis of B. subtilis (Ebbole and Zalkin, 1987). In all cases, addition of adenine decreases the steady-state level of the corresponding mRNA. We performed similar experiments and measured the amount

of the 5-kb mRNA

in cells grown in presence

or

absence of adenine. As shown in Fig. 4 (lanes 3 and 4) the level of accumulation of the 5-kb transcript was reduced by the presence of adenine in the culture medium. A threefold decrease in the amount of the 5-kb mRNA was consistently observed. In addition, consistent with the fact that the 2-kb mRNA is likely unrelated to the ADE6 gene, the amount of this transcript remains constant in both culture conditions. These data indicate that adenine can modulate ADE6 transcription and suggest the possibility that in S. cerevisiae common regulatory sequences may be shared by genes involved in the pathway of de novo purine nucleotide synthesis.

Carlson, M. and Botstein, D.: Two differentially different 5’ ends encode secreted invertase. Cell 28 (1982) 145-154. Ebbole,

D.J. and Zalkin,

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mRNAs forms

and characterization

with

of yeast

of a 12-gene

nine enzymes

for de novo

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T.C., Dawid,

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sulfhydryl

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(e) Conclusions (I) We have cloned the ADE6 gene from the yeast S. cerevisiue. (2) This gene was mapped on the RAD2proximal fragment of chromosome VII. (3) Insertional mutations in the ADE6 gene caused adenine auxotrophy. (4) ADE6 gene encoded a 5-kb mRNA the amount of which is negatively modulated by adenine.

H.: Cloning

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ACKNOWLEDGEMENTS

Cold Spring

We wish to thank Maurizio Baroni for helpful discussions and critical reading of the manuscript. This work was partially supported by the C.N.R. target project on Biotechnology and Bioinstrumentation.

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