Gene, 119 (1992) 49-56 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

49

0378-I I19~92/%05.00

06628

A multisite integrative

cassette for the yeast Saccharomyces

cerevisiae

homologous recombination; genomic integration; multiple targets; sigma elements)

(Transformation;

Bernard Kudla* and Alain Nieolas Institut de GCnetique et Microbiologic, UniversitC:Paris-Sud, Orsay, France Received

by .J. Marmur:

3 February

1992; Revised/Accepted:

30 March/

31 March

1992; Received

at publishers:

25 May 1992

SUMMARY

We have developed a cassette for the integration of cloned DNA sequences at multiple sites in the Sacc~aromyces cerevisiae genome, taking advantage of the naturally repeated sigma sequences. This cassette contains one engineered sigma element which allows the targeting of an embedded gene at different genomic sig?na elements by gene replacement. Two yeast genes, ARG4 and URA3, were thus integrated in the absence of any bacterial sequences, individually or sequentially on twelve chromosomes. Consequently, these studies led to the genetical tagging of individual members of the sigma family.

INTRODUCTION

During tr~sformation, the inte~ation of exogeneous DNA can occur by recombination with an homologous or an unrelated sequence. The ratio between homologous vs. nonhomologous integration events varies among organisms and experimental systems. In most cases, nonhomologous integration is more common than homologous integration (Fincham, 1989). In contrast, in S. cerevisiae, integrative transformation almost always occurs by homologous recombination (Hinnen et al., 1978). Illegitimate integration can occur but is rare (Schiestl and Petes, 1991). Therefore, the difficulty in S. cerevisiae is to integrate, and preferably

Correspondence to: Dr. A. Nicolas, ogie, Bat. 400, Universite Tel. (33-l) * Present Rostand,

Institute

de Genetique

Paris-Sud,

91405 Orsay

69 41 62 08; Fax (33-l)

69 41 66 78.

address:

Eurolysine,

91893 Orsay,

Abbreviations:

France.

Pam

Club Universite

Tel. (33-l)

ARC4 , gene encoding

et Microbiol-

Cedex, France. Bat. E, 1 rue J.

69 41 24 00.

argininosuccinate

lyase; bp, base

pair(s); chr, chromosome; E., Escherichiu; EtdBr, ethidium bromide; kb, kilobasefs) or 1000 bp; MCS, multiple cloning site; nt, nucleotide(s); oligo, oligod~xy~bonucl~tide; PCR, polymerase chain Sacc~~ro~~e~; URA3, gene encoding oroti~ne-5’-phosphate

reaction; S., decarboxyl-

ase; sigma, long terminal repeat of the yeast retrotransposon junction (insertion or fusion).

Ty3; ::, novel

target, a desired construction in various locations of the genome. One possibility is to use the naturally repeated sequences of the host genome as multiple target sites. Indeed, sequences of the yeast retrotransposon Ty2 were probably responsible for the integration of a LEU2-carrying plasmid in novel locations of the genome (type II integrations in Hinnen et al., 1978). More recently, a 1%kb Ty-Cl yeast fragment was used to obtain the integration of a circular plasmid at different locations in S. cerevisiae (Sakai et al., 1990). Insertion of genetic markers at several telomeric Y’ elements by gene replacement was also obtained (Louis and Haber, 1990). Here, we report the use of another naturally repeated sequence of the yeast S. cerevisiae for multisite gene replacement. This sequence is the highly conserved 340-341bp sigma element that corresponds to the long terminal repeat of the retrotransposon Ty3 (Clark et al., 1988), and also occurs as isolated sigma element repeated about 30 times on different chromosomes of the haploid genome (Sandmeyer et al., 1988). The most striking feature of the sigma elements is their specific association with the 5’ end of tRNA genes, located 16 or 19 bp upstream from the 5’ transcriptional startpoint. This close association apparently does not affect tRNA expression and the solo sigma elements are considered to be nonessential. Thus, we reasoned that they might be suitable for dispersed multisite

50

gene targeting and allow gene expression. We show here that these predictions are fulfilled.

RESULTS AND DISCUSSION

(a) Isolation and characterization of the sigma VZZZ.Z element We have isolated and cloned a sigma element located on the yeast chr. VIII distal to the ARG4 gene, hereafter named s&ma VIII.1. The presence of a sigma in this region was detected by hyb~d~ation of a set of 1 phages containing overlapping DNA segments of chr. VIII with a sigma probe (Dr. M. Olson, personal communication). Indeed, subcloning the region from plasmid pSPO13-2 (Wang et al., 1987) and sequencing revealed the presence of a sigma element of 341 nt located 16 bp 5’ from a tRNAA’” gene. The sigma VlII.1 and surrounding region sequences showed complete identity with the nt sequence of the 1 clone 7 previously reported (Sandmeyer et al., 1988). This identity strongly suggests that clone 7 belongs to chr. VIII. The sigma VIII.1 sequence was found to have a m~imum of nine differences out of 341 bp (97.5 % identity) when compared with the sigma elements previously sequenced (Sandmeyer et al., 1988). The gene order in this region of the right arm of chr. VIII is shown in Fig.1. (b) ~onstructiou of the signra integrative cassette The construction of the sigma integrative cassette required several steps creating the plasmid pBK614 (Fig. 2). In pBK614, the size of the engineered sigma WI.1 is 367 bp instead of 341 bp. This results from the substitution of the central .5’-TGGAAGCGCGGA sequence by a 38-bp MCS sequence containing the unique Bali, PstI, EcoRI and EcoRV restriction sites that facilitates gene cloning. It is located 156 bp from the left side and 173 bp from the right side of the sigma VIII.1 element (Fig. 2B). The engineered sigma element is flanked on both sides by several restriction sites (Fig. 2A).

BEBg

SLOVUI-I

NdX

EE$EP

stgma tt?NAAla DE082 VIII. 1

Hp EV

DE081

AR04

CIBgHp

CEN VIII

Fig. 1. Map of the chromosomal .s&ua WI.1 region. B, BnmHI; Bg, BgfiI; Cl, ClaI; E, EcoRI; Ev, EcoRV; Hp, HpaI; Nd, NdeI; P, PsrI; X, XhoI. Gene order is from Nicolas et al. (1989) and present work. Symbols for loci are: lightly shaded box, SLUVIII-I; darkly shaded box, sigmaUII.1 element; open box, tRNA*‘“; blackened boxes, DED81 and DED82; hatched box, ARG4; circle, CEN VIII centromere. Arrows and arrowhead indicate the direction of transcription.

(c) Yeast transformation with the sigma integrative cassette To assess the possibility of targeting the integration of a sigma VIII.1 disrupted sequence in the yeast genome, the ARG4 or URA3 selectable genes were cloned into the central polyl~ker sites of the engineered sigma VW.1 of pBK614 to generate plasmids pBK615 and pBK617, respectively (Fig. 3). The haploid strain MGD131-102A (MATa, his 3-1, ade2, arg4-A2060, ura3-52, trpl-289) which is deleted for the ARG4 gene was electrotransformed with 100 ng of the 2.4-kb Hind111 sigmaVHI. 1::ARG4 fragment obtained from pBK615 (Fig, 3). In several independent experiments, 150 to 1200 Arg+ transformants were obtained per pg of exogenous DNA. To test if transformants could be obtained for another marker and in strains carrying a resident mutant copy of the marker, we attempted to transform yeast cells with the 1.37-kb CfaI-CZaI sigmaVIII-I::URA3 fragment purified from plasmid pBK617 (Fig.3). This fragment contains 1 and 7 bp of MCS on the sides of the sigma halves. Two strains were used: MGD 13l-102A (genotype above) and OIL1 (data, his3-(11,15), ieu2-(3,112), ura3(257,373), gal2). Both strains are auxotrophic for uracil because they contain a ura3 mutant allele on chr. V. In several independent experiments, we obtained 150-350 Ura+ transformants in strain MGD131-102A and 300600 Ura+ transform~ts in strain OLl per yg of s~gmuVr~I.l::~~3 fragment. (d) Characterisation of yeast transformants A sample of randomly chosen Arg’or Uraf transformants obtained in strain MGD 131-102A were genetically analyzed by mating with the strain MGDl3 l-21: (MAT a, Ieu2-(3112), cyh”, arg4-d2060, ura3-52, trpl-289). The resulting diploids were sporulated and tetrads analyzed. In all cases, the Arg’ and the Ura+ markers were found to segregate as stable chromosomal Mendelian markers (2 + :2-). The vegetative growth of the transfo~~ts was in~stinguishable from the parental strain cultivated on minimal supplemented media, and no altered phenotypes were ever observed. Randomly chosen Arg* and Ura+ transformants were analyzed by chr. DNA pulsed-field gel electrophoresis, DNA transfer and hyb~dization with ARG4 and URA3 specific probes, respectively (Fig. 4). All the At-g+ tr_ansformants, but not the parental strain, present one hybridization band on different chromosomes. This demonstrates the dispersed integration of the transforming ARG4 gene. All the Ura+ transform~ts and the parental strain OLl present a common hybridization band corresponding to the resident ura3 mutant alleles (chr. V). In addition, six out of nine transformants exhibit a novel hybridization signal on different chromosomes. This demonstrates the dispersed

51 Ev

Up SI E

P S XbS

P S Sm SI E

/I I \

I I

\,__________________________________,I pBK505:

7.3 kb Ea P EEV

pBK217: 4.4 kb

pBK611: 5.4 kb

Fig. 3. Structure

of the plasmids

(2060 bp HpaI fragment,

pBK615

The ARG4 gene

and pBK617.

Fig. 1) was subcloned

in the SmaI

pUCl8 polylinker and then isolated as a 2.1 kb EcoRI-PstI UR43 gene was isolated as a l.l-kb EcoRI-NsiI restriction plasmid pHS 113 (gift from H. Sun, Harvard introduced pSK614:

3.3

pBK615

Lb

between the EcoRI-PsrI and pBK617,

University).

sites of pBK614

respectively.

Symbols

site of the

fragment. The fragment from Both genes were

to generate

plasmids

are as in Figs. 1 and 2

(B)

integration of an additional URA3 gene in the genome (Fig. 4B). The absence of a second detectable band in the last three transformants could be explained either by an integration event on chr.V (gene conversion of the resident ECORV t ura3 allele or integration of an additional URA3 gene) or BfL TGCAGwaiGCTCC TCTCGGATCTMACTAATTGTTCAGGCATTTATA on chr. VIII that is not electrophoretically separated from EcoRl CTTTTGGGTAGTTCAGCTAGGGAAGGACGGACGGCTTTTGTCTCATGTTGTTCGTTTTGTTATA chr. V in the OLl strain. Thirty independent Ura+ transformants obtained in strain MGD131-102A in which && AGGTTGTTTCATATGTGTTTTATGAACGTTTTAGGATGACGTATTGTCATACTGACGTATC chromosomes V and VIII are electrophoretically separable were analyzed by chromosomal blot. All Ura+ transformants exhibited two hybridization bands with a URA3 Fig. 2. Construction of pBK614 containing the sigmaVIII.1 integrative probe. One band corresponds to the resident uru3 mutant cassette. (A) The 2.1-kb BglII-EcoRI DNA fragment (Fig. 1) was used as allele (chr. V) and the second is on different chromosomes. template for a PCR amplification to add flanking restriction sites to the sigma VIII. The DNA was amplified (30 cycles) with a DNA Thermal This demonstrates the dispersed integration of an addi-

7

Cycler using the AmplitaqTM

polymerase

(Perkin

Elmer Cetus) and two

oligos (A and B). The oligo A: 5’-ACTAGTCGGCCGATCGATGTTGTATTACGGGCTCGAGTAATACCGGAG,

includes the recognition

sites for the SpeI, EagI and CIaI enzymes

to the sigma VIII.1 end containing

nt homologous B:

and presents

31

the XhoI site. The oligo

5’-GTCTAGAGTCGACAAGCTTGTTGTATCTCAAAATGA-

GAT, includes opposite

sites for the XbaI, Sail and Hind111 en-

the recognition

zymes (underlined)

and presents

end. Newly introduced

bp PCR product

obtained

were

21 nt homologous

two mutagenic

oligo (Rev)

in the 377

used to subclone the sigma VIII. I between vector to give pBK605.

Cloning

in the middle

of the sigma VIII.2 sequence First, the reverse sequencing

(Sequenase

and the mutagenic

TGGCCACTTAGT,

VIII.1

PCR amplifications.

sites were then introduced through

to the sigma

SpeI and Sal1 sites included

the XbaI and Sal1 sites of a Ml3mpl8

OH.)

(underlined)

Version

2.0 kit, US Biochemical,

Cleveland,

were used to amplify the left half of sigma VIII.1

(PCR product 1). Secondly, the Universal sequencing oligo (Un) (Sequenase Version 2.0 kit, US Biochemical, Cleveland, OH) and the mutagenic

modifications

introduced

by oligo C and D created

flanking

striction

restriction

were 2). Sequence

Bali and SruI restric-

tion sites (underlined in the oligo C and D sequences) in both PCR products at the same central location. The PCR products 1 and 2 were then successively cloned in pBluescriptKS+ taking advantage of the

sites and of the newly introduced

StuI re-

1 was cleaved with SmaI+StuI,

and then

site. The PCR product

cloned into the SmaI site of pBluescriptKS+ product

2 was cleaved by SalI+StuI

HindIII,

filled-in with the Klenow

I (Boehringer) bp CluI-ClaI

restriction

sigmaVIII.Z element glassbeads

fragment

fragment

of pBK610

was isolated

The PCR

into pBK608 cut with

of E. coli DNA polymerase plasmid

pBK610.

containing

on a 0.8%

which had the SmuI-EcoRV

agarose

MCS deleted, to generate

of the PstI-EcoRI-EcoRV

The 383

the reconstructed gel, purified

and cloned into the CluI site of a pBluescriptKS+

to the introduction between

to give pBK608.

and introduced

and cleaved by Sal1 generating

on

derivative,

pBK614.

MCS sequence

This led

(open box)

the two halves of the sigma VIII.1. In all steps, the DNA liga-

tions were performed

oligo C: 5’-ATCCGAGATAGGCCTCTT-

oligo D: 5’-ACTAAGTGGCCAAAGAGGCCTATCTCGGAT, ~ used to amplify the right half of sigmn VIII.1 (PCR product

Ml3mpl8

with T4 DNA ligase (Boehringer),

cells were used for transformations,

and amplification

E. coli DH5 of plasmids

LY

was

performed using standard procedures (Ausubel et al., 1987). Oligos were synthesized on an Applied 381A DNA synthesizer. Restriction sites are as in Fig. 1 and additionally

Ap, ApuI; Bx, Ξ

Ea, EaeI; H, HindIII,

Kp, KpnI; Nt, NotI; SI, SacI; SII, SacII; S, SalI; Sm, SmnI; Xb, XbuI. (B) Nucleotide sequence of the engineered sigma VIII.. cassette (389-bp HindIII-ClaI

fragment

of pBK614).

ternal sigma VIII.1 cloning upward

arrowheads.

region

Other symbols

The 38 bp corresponding are indicated

to the in-

in italics between

are as in Fig. 1.

two

52

(A)

1

2

ARG4 probe

1

12

3 4

5 6 7

8 9

1

3

4

in the OLI

Chromosomal respectively.

5

pattern

6

7

of Arg+

8

5

6

7

8

9 10 11

URA3 probe

recipient

hybridization

(Panel A) Chromosomal

in agarose

gel stained

of various

Yeast cells (10’) resuspended

with 100 ng of purified fragment

transformants

in 0.8 ml of transformation with a BioRad

were extracted

strains

with an ARG4 probe. (Panel B) Chromosomal Ura+

according

Genepulser

ORT786.1

from MGD131-102A. buffer (sucrose (450 volts/cm,

patterns

of various

with EtdBr (chromosomes

to transformant

with URA3. OLI is in lane 1 and transformants

patterns

or uracil). Yeast chromosomes

transformants.

separation

strain, lanes Z-11 correspond

to the same gel hyb~diz~

strain probed

123456789

9 10

and Ura’

Panel 1 shows the chromosomal

Lane 1 is the untransformed

tively. Panel 2 corresponds

formed

2

hybridization

in strain MGD131-102A.

obtained

4

w URA3 probe

merals).

3

10 11

w

Fig. 4. Chromosomal

2

ORT720-ORT726

hybridization to 0RT786.9

and ORT728-ORT730,

patterns

nu-

respec-

of various Ura” transfo~ants

ORT731

to 0RT739

10 mM pH S/MgCI,

250 pF, 200 a), and spread

to Chu et al. (1986) and separated

obtained

with roman

are in lanes 2-10, respectively.

Transformants

270 mM/Tris.Cl

Arg + transformants are numbered

0.1 mM) were electrotrans-

on selective medium

by pulsed field electrophoresis

(Panel C)

are in lane 1-9,

(BioRad

(without CHEF

arginine

DRH,

200

volts for 19 h with 60 s switch and for 16 h with 90 s switch) at 11’C through a 1% agarose gel in 0.5 x TBE buffer (89 mM Tris-base/89 mM boric acidi2.5 mM EDTA pH 8.3). For molecular analysis, the chromosomes were hybridized with the ARG4 DNA purified as the 2.1-kb EcoRI-PstI fragment of plasmid

pBK615,

and the URA3 specific DNA purified as the I.l-kb

beled using the ECL Gene detection the supplier

(Amersham).

system (Amersham);

Yeast experimental

procedures

Hind111 fragment

(Bach et al., 1979). These fragments

probe labeling,

hybridization

procedures

and signal detection

were performed

as described

by Ausubel

et al. (1987).

tional URM gene into the genome. Interestingly, the absence of an integration event on chr.V suggests that the + 150-bp segments of sigma sequence flanking the URA3 gene are sufhcient to prevent gene conversion with the endogenous uru3 mutant allele despite the l.l-kb internal

were chemically

were performed

as indicated

laby

sequence homology. Fig. 4C shows the hyb~dization pattern of a random sample of nine transformants. In order to correlate the chromosomal pattern of integration events and the distribution of the sigma elements on the chromosomes, we examined the sigma content of strains

53 ARG4 (Fig. 6). Total DNA was cleaved byXhoI+NdeI 2

1 sigma VIII. 1 hromosomal lybridization

4

3

Number

sigma

Chromosome

copy

cut at both ends

XII

2

5

IV XV+VII

1 4

2”1

XVI XIII

3

sequence and with BgiII enzyme that cuts once in ARG4 (see Figs. 1 and 3 for restriction maps). The DNA of all transformants analyzed exhibited a single 2.35kb XhoINdeI band corresponding to the length of the XhoI-NdeI fragment of the sigmaVZZZ.l::ARGI cassette (Fig. 6, panel 1). This suggested the occurrence of an integration event that preserves most if not all the structure of the transforming molecule. The BamHI digests generate a unique band of variable size whereas the BglII digests lead to two

0”

1 2

xiv

4 :

XI

1

1

1

135

IX

2

5

III VI I

1

VT11

Fig. 5. Sigma content gration

events.

tograph

assignment not shown).

per chromosome

5

of strain MGD13 l-102A

Columns:

MGD13 l- 102A, probed mosomal

1 0

1

and distribution sigma

1, chromosomal

content

of inteof strain

with the specific sigma VIII. I element. 2, chro-

based on the EtdBr-stained 3, quantitative

estimation

based on the analysis

1 y0 agarose

gel (pho-

of the sigma copy number

of the autoradiogram

by densit-

ometry tracing with a Photometric

Image Analyzer

(Bio Comm 200). The

dash symbol reflects the absence

of hybridization

signal. 4, summary

the distribution Ura’ ) obtained

per chromosome in the MGD13

of 68 integration l-102A

sigma VIII. 1 specific DNA was isolated

345 bp ClaI-Hind111

restriction

strain.

out overnight

of

(Arg’

and

For hybridization,

the

and purified by glass beads as the

fragment

DNA (200 ng) was 32P labeled by random ization was carried

events

from pBK605.

This purified

priming (Boehringer).

that

and by the &I

enzyme that cuts once in the ARGl gene. The chr. DNA of three transformants was further characterized by cleavage with BamHI that has no recognition site in the ARG4

of Integration events

number

of the sigma element

Hybrid-

at 65 “C.

MGD131-102A (Fig. 5) and OLl (data not shown) with a sigma probe. Hybridization signals on all chromosomes but VI, X and XIII were observed. Altogether, 68 Arg+ and Ura+ transformants of this strain have been analyzed, and the distribution per chromosome of the integration events is summarized in Fig. 5. Integration on 12 out of the 16 chromosomes of S. cerevisiae have been obtained. As expected, no integration event was found on chromosomes which do not display sigma specific signal. The variations in targeting efficiency on the different chromosomes probably reflects two factors: (I) the sigma copy number per chromosome, (2) the fact that all genomic sigma sequences are not identical, with deletions and microheterogenity (Sandmeyer et al. 1988). These heterologies may reduce targeting potentiality for homologous recombination. To determine the nature of the integration events, five Arg+ transformants were further characterized by Southern blot analysis of restriction digested DNA, probed with

bands of variable size (Fig. 6, panel 2). These patterns are consistent with the integration of a single ARG4 gene at various positions in the genome. The sizes of the BglII, C/n1 and BamHI restriction fragments for 0RT724 (chr. VIII) are consistent with an integration event taking place in the resident sigma VZZZ.1 element (Fig. 6, panels 1 and 2). In this case, the CZuI pattern (one 12-kb band) is explained by a doublet of junction bands in accordance with the known restriction map. The nt sequence of the junction between the integrated DNA and the flanking region was established for the transformants ORT722, 0RT723 and ORT724 by the inverse PCR method (Ochman et al., 1988). The PCR product obtained from the Bg111 genomic restriction digest of ORT724 corresponded to a 2.3-kb band (Fig. 7) as expected from the restriction map of the chromosomal sigma VZZZ.1 region (Fig. 1A). This was confirmed by partial sequencing of this fragment which was found identical to the original sigma VIII.1 and flanking region. This demonstrates that the integration of the sigma VIII. I ::AR G4 molecule occurred by homologous recombination similar to a classical targeted gene disruption event (Rothstein, 1983). With the same methodology, transformants 0RT722 and ORT723 (ARG4 on chr.11) were characterized and found to correspond to an integration event in the same sigma target, hereafter termed sigma II.1 (Fig. 7). Sequencing of the flanking region of sigma II.1 site of integration reveals 16 bp of a spacing sequence to a putative tRNA different from published ones (Sandmeyer et al., 1988). This genetical tagging of sigma II.1 followed by the inverse PCR method illustrates a powerful approach to isolate flanking regions of other sigma sequences. The examination of the genomic sequences of these transformants reveals important features of the integration process. First, that the heterologous nucleotides ATCGA, which correspond to the CZaI site present at the end of the transforming molecule, were not incorporated in 0RT722, 0RT723 and 0RT724. Secondly, whereas ORT723 and 0RT724 present the exact sigma VZZZ.I sequence, ORT722

54

h

III

Fig. 6. Southe~-blot were prepared

bp HpaI ARC4 probe. XhoI+NdeI fragment

analysis

by standard

of Arg’

methods

transformants (Ausubel

(Panel 2) transformants

(XN) and C/a1 (C) digestions sites: panel 1, 21226-1375

obtained

were repeated

0RT722

by XhoI+NdeI and 0RT724

using independent

bp; panel 2, 21226-831

probed with ARG4. (Panel 1) Total DNA from five transformants

from MGD131-102A

et al., 1987), cleaved ORT720,

CRT724 OUT722 ORT720 B Bg C XN B Bg C XN B Bg C XN

bp. Experimental

enzymes

(XN) and C.&z1enzyme

were further

DNA preparations. methods

characterized i III, DNA

are as described

(C), and hybridized

by BumHI

with the 2060

(B) and BglII (Bg) digestions.

size marker

(Boehringer);

top to bottom

in Fig. 4.

Nd

SgIll restriction of ganomic DNA dilution and llgatlon

(4 T~ns~~~

Sequence of the PCR

PCR ampllflcation with ollgos C and E

product wtth ollgo C

5

mokule I -20

10

-10

20

30

40

50

60

80

70

I’ oRl724

. ~~~TATT~TT~T~~A~~~T~TMZCC~~TCTT~~TM~

OR7723

I ~~c~~~~~~~r---------------_--~-----~~~~______~~~~~~~______________-________________________~

ORT722

ccAcA&M

&ACAGTCT&GAXT

l&M?aGTTGTW

I Te~~~T~T~~~----------------------------~~~~~~~~~~~~~____~~~_~_____-_______________________--

I

I

1

I

1RNA ’ 16bpspaclng

4

sigms sequence

W

Fig. 7. Sequencing characterization of transformants. (A) Genomic DNA of transformants ORT722,ORT723 and ORT724 was obtained by inverse PCR. 2 pg of total DNA was cleaved with BglII enzyme which cut once in ARC4 , ligated at 11’ C, phenol extracted and ethanol precipitated, and resuspended in 20 ~1 of TE buffer (10 mM Tris.Ci/l mM EDTA). (Fig. 2) and E (5’-~GGTGAGTGTGTCG~ACT)

Then 25 to 30 amplification cycles were performed using oligos C specific to the sigma sequence specific to ARG4. T’he PCR products were separated on agarose gel, sized as a single band of 2.3

kb for 0RT724 and of 1.8 kb for ORT722 and ORT723, and sequenced using the oligo C. (B) Genomic sequences of tr~sfo~~ts and 0RT724 compared with those of the transfo~ing molecule (see Fig. 2). Sequence identity is indicated by dashes. Symbols

ORT722,ORT723 are as in Fig. 1.

55

W

(A)

URA3

probe ARG4

B

B

3 *w

8 b

B

probe B

XII

I Fig. 8. Chromosomal from 0RT723

patterns

of Arg’ Ura’

(panel B). Separated

sequential

chromosomes

transformants.

were blotted

Transformants

and successively

obtained

hybridized

with sigmaVIII-Z:.tIRA3

from 0RT720

(panel A) and

with an URA3 and an ARG4 probe. Experimental

methods

are as in Fig. 4.

exhibits two nt differences (Fig. 7) that are common in the sigma family (Sandmeyer et al., 1988). It probably reveals the nt sequence of the resident sigma II.1 used as a target in transformants 0RT722 and ORT723 but that was retained only in the first one during the recombination process. In conclusion, the DNA analysis of various transformants demonstrates that the sigma integrative cassette allows the insertion of the cloned gene at multiple sites of the yeast genome by homologous recombination with resident members of the sigma family. (e) A multiple target application: sequential transformations with the sigmaVIII..I::URA3 and the sigmaVZZZ.1:: ARC4 fragments To test if the multiplicity of target sites could be used to introduce several genes in the same cell, the primary transformants 0RT720 and 0RT723 carrying the sigma::ARGI locus on chr. IX and II respectively, were re-electroporated with the ClaI sigma VIII.l::URA3 fragment. Ura+ transformants were selected and characterized. All of them were found to have retained their Arg’ phenotype indicating that transformation did not occur by substitution of the resident sigma::ARG4 locus but more likely by integration of the transforming cassette into another sigma target. Indeed, the chromosomal blot analysis of five transformants, 0RT720.6, 0RT720.8 (obtained from 0RT720) and 0RT723.8, 0RT723.9 (obtained from ORT723) demonstrate the insertion of URA3 at a novel location distinct

from chr. V and distinct from chr. IX or chr. II carrying the sigma::ARG4 genes (Fig. 8). (I) Conclusions (I) We have constructed a novel vector for the yeast S. cerevisiae that allows the integration of a cloned DNA sequence at multiple sites in the genome. This feature is provided by the inclusion in the vector of a repeated yeast sigma sequence present in approx. 20-30 copies per genome on the different chromosomes. This vector named pBK614 was engineered as a cassette containing a fulllength 341-bp sigma element disrupted by a MCS to allow the introduction of any cloned DNA sequence. In addition, it was flanked by convenient restriction sites to permit the isolation of the sigma disrupted cassette without external sequences. These features enable the purification of a DNA fragment with sigma extremities which confers a very efficient targeting to yeast resident sigma sequences. (2) The molecular analysis of numerous transformants demonstrates the integration of the sigma embedded genes at novel locations on 12 out of the 16 chromosomes of S. cerevisiae. These integrations occurred by homologous recombination with a resident sigma sequence which is then genetically and physically marked. This should facilitate the identification of tRNA genes associated with sigma elements. (3) The natural repetition of the target allows the use of the same vector several times for sequential transformations because a successful integration event does not elim-

56 inate the overall targeting potential. The integration of a defined number of gene copies should be useful for the overexpression of proteins in S. cerevisiue. The effectiveness of this vector was demonstrated by the genomic dispersion of two different genes in the same cell without integration of bacterial or vector sequences as desired for industrial applications. (4) Practically, the advantages of this methodology using the sigma elements are: First, its targeting feature which is not the case in random integration procedures (Schiestl and Petes, 1991); secondly, the predictable position of the insertions in a reasonably low number of locations; thirdly, the well defined sequence context allows a better control of gene expression than in random integration; fourthly, it allows the disruption of these sequences without apparent side effects. (5) The methods described here should be also applicable to other yeast repeated target sequences and to other organisms that contain repeated sequences and are proficient for homologous recombination.

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