Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants

Gene,21(1983)233-231

233

Elsevier GENE

920

Short Communication Molecular cloning of the ADEl gene of Saccharomyces cerevisiae and stability of the transformants t (Recombinant DNA bank; yeast transformation; endonuclease maps)

yeast plasmid vectors; ,J Chat-on 30 phage; restriction

Kenneth Dimock, Allen P. James and Verner L. Seligy Molecular Genetics Section, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KlA OR6 (Canada) Tel. (613) 9926055 (Received

July 21st, 1983)

(Revision

received

and accepted

October

5th, 1983)

SUMMARY

Plasmid YEp(ADEl)la, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI-1 (Morris et al., 1981), results in high frequency, unstable transformation of adel yeast strains. A second plasmid, YRp(ADEI)2, containing adjacent 0.5kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from adel strains by hybridization analyis, and (3) a transformant carrying a multimeric form of YRp(ADEI)2. Cells transformed with either of the plasmids are free of the red pigment characteristic of adel mutants and indicate potential for direct colour-based selection of yeast transformants using ADEl plasmids.

INTRODUCTION

Protein-coding DNA sequences (Petes, 1980; Botstein and Davis, 1982), chromosomal replicators (ars; Stinchcomb et al., 1979; Struhl et al., 1979; Hsiao and Carbon, 198 1) and centromeric sequences (CEN; Clarke and Carbon, 1980; Hsiao and Carbon, 1981; Stinchcomb et al., 1982) have been isolated from several S. cerevisiae chromosomes. We chose

+ Dedicated Abbreviations:

to the memory

I. Bukhari.

ApR, ampicillin-resistant;

YPD, 1% yeast extract, yeast nitrogen

of Ahmad

2% peptone,

base, 2% dextrose

0378-l 119/84/$03.00

0

kb, kilobase

2% dextrose;

(Sherman

1984 Elsevier

et al., 1981).

Science

pairs;

SD, 0.67%

Publishers

to clone the ADEI gene of S. cerevisiae to gain access to other functional DNA sequences on yeast chromosome I, in particular FL01 (Miki et al., 1982a,b). The adel mutants characteristically accumulate a red intracellular pigment. Yeast cloning vectors which contain the ADEl gene are potentially useful for selection of transformants on the basis of colour. Transformation of adel yeast strains has been reported (Henikoff et al., 1981). Here we report the isolation of an ADEl gene using molecular cloning techniques and complementation of adel mutations. Some properties of the ADEI transformants and restriction maps of recombinant ADEI plasmids are presented.

directly

EXPERIMENTAL

reflecting

differences

in regeneration

after

sphaeroplasting. (a) Screening

of transformants (b) Stability

of transformants

Yeast strain C627-4B (a adel leu2 his3) was transformed

(Hsiao

and Carbon,

plasmid

bank of strain S646-8D (ADEI ; Miki et al.,

1982a,b) to take advantage quency

of transformation

Approx.

30000 Leu+

1979) with a pBTI-1 of the vector’s high fre(Morris

transformants

et al.,

were obtained

and screened for Ade + phenotype.

ined contained

and ana-

in Fig. 1. All transformants

two DNA

bands

exam-

which hybridized

the other, were identified by restriction endonuclease analysis. The monomer, YEp(ADEl)la transformed C627-4B with high frequency (0.6-2.0 x lo4 transformants/pg), consistent with frequencies for other plasmids containing the 2~ origin of replication. A diploid yeast S646-1B (Miki et al., 1982a,b) was

1

at a frequency

l/l00

2

4

Fig. 1. Hybridization

3

analysis

of DNA

that of C627-4B,

5

6

isolated

from

transformants.

DNA (10 pg) isolated

from overnight

four potential

ADEI

(lanes

transformants

3-6)

(lane 2) was electrophoresed

in a 1 y0 agarose

to nitrocellulose

1975). Nick-translated

(Southern,

used as a hybridization in lane 1 as a marker.

Ade’

cultures

of

and C627-4B

gel and transferred pBR322 was

probe. pBTI-1 DNA was electrophoresed

under

non-selective

during

was

conditions.

mitosis

was

concomitant

of growth

Loss of the Leu+ with loss of Ade’

Tetrad analyses were also carried out and results are summarized Mendelian

in Table I. ADEI fashion

segregated

as expected

somal marker. All segregants

strongly to pBR322. pBR322 did not hybridize to DNA from C627-4B (lane 2). DNA from one of the transformants (lane 5) was used to transform Escherichia coli HBlOl and ApR colonies were screened for plasmids. Two plasmids, one a dimeric form of

transformed

of YEp(ADEl)la

on selective plates after lo-12 generations phenotype

DNA was isolat-

ed from several Ade + Leu + transformants lyzed as described

1981).

Stability

tested. Roughly 40 y0 of cells lost their ability to grow

in a non-

for an extrachromo-

prototrophic

for aden-

ine were also prototrophic for leucine, therefore ADEl and LEU2 are tightly linked. Only white colonies were positive when hybridized with j2Plabelled pBR322 (Fig. 2). The Ade’ Leu+ phenotype therefore is perfectly correlated with the presence of YEp(ADEl)la. (c) Reconstruction

of ADEZ gene

To isolate the functional ADEI gene from the other yeast sequences in YEp(ADEl)la, the 0.5-kb BamHI fragment and an adjacent 3.0-kb BamHI fragment were isolated (Dretzen et al., 1981) and combined in pBR322, yielding YRp(ADEl)2 (Fig. 3). The large BamHI fragment was identified in a 2 Charon 30 bank of S646-8D DNA by hybridization with the EcoRI-E fragment ofYEp(ADEl)la. In the course of the ADEl reconstruction experiments we discovered that ADEl complements apurC mutation in E. coli (K.D. and V.L.S., manuscript in preparation). When inserted in pBR322, the small BamHI fragment did not transform C627-4B to Ade + ; however, the large BarnHI fragment results in low frequency (l-3 transformants/pg) integrative transformation (not shown). Colonies that appeared following YRp(ADEl)2 transformation were distinctly smaller than those from YEp(ADEl)la transformation and less than 1% grew when replated on selective media. Two classes of YRp(ADE1)2 transformants were identified: (1) transformants with an Ade + phenotype which was completely stable during 24 h of non-selective growth, and (2) transformants with an unstable Ade + phenotype (55-7 1 y0 of cells became Adee). Hybridization analyses of DNA isolated from the “unstable” transformants showed

235

TABLE

I

Tetrad

analyses

of ADEl

ADEI

transformants

micromanipulator. cytohelicase

transformants

of C627-4B

were mated

strains

of the opposite

mating

Transforming

C627-4B

: Ade-

Ade+

plasmid

were isolated

with a

were carried out following

distribution 1:3

0:4

1

2

11 a

0

15b

1

0’

4

5

2

19d

4:o

3:l

11

2

0 18

212

(a adel leu2 his3) YEp(ADEl)la

C636-25D

type and zygotes

and asci were picked 3 days later. Tetrad analyses

digestion.

Ade + diploid

C627-4B

with haploid

Ade + diploids were induced to sporulate

(a adel leu2 his3) (a adel leu2 his3) YRp(ADE1)2

C636-20C (a adel LEU2 HIS3) a All Ade’ segregants

segregants

were also Leu+

as expected

because

YEp(ADEl)la

carries

the yeast LEU2 gene as well as ADEl;

all Ade-

were also Leu-

’ LEU2 and HIS3 segregated ’ “Stable”

YRp(ADEl)Z

d “Unstable”

independently

transformants

YRp(ADEl)2

from ADEl

of C627-4B

transformants

and from each other.

as parent.

of C627-4B

as parent.

2

3

4

spore --a Fig. 2. Correlation tetrad

analysis

nitrocellulose

between

of diploid

colour Ade’

and hybridization yeast

filters were photographed

colonies

growing

colonies

are indicated

on nitrocellulose

transformants

patterns

and colony hybridization

following

tetrad

analysis

with the letter r. (B) Autoradiogram

1-7 specify asci selected

for tetrad

of Ade + and Ade - segregants.

were replica-plated

analysis

onto

was performed

(Sherman

of asci from S646-1B the individual

spores

on YEPD following

for 12 h on YEPD.

et al., 1981). (A) Photograph with YEp(ADEl)la.

hybridization

isolated

growing

and grown

transformed

of the same filter following

and a-d columns

Colonies

nitrocellulose

with 32P-labelled

from each ascus.

The

of segregant The red (dark) pBR322.

Lines

s -salI X -XhoI 3A - Sau3A Fig. 3. Restriction sequences; ADEl

maps

of YEp(ADEZ)la

and YRp(ADEI)2.

broad black line, yeast chromosomal

inserts

are represented

Thin lines represent

(LEU2) sequences

by a double line. The EcoRI fragments

on the inside of the YEp(ADEl)la

B

a

--130kb-78kb-

.

BK*

-4.36kb2

3

4

1

5

2

3

4

5

-1xokb -78kb -4.36kb

I_

G' Fig. 4. Nucleic acid hybridization YRp(ADEl)2

D

analysis

yeast transformants.

of DNA isolated from

DNA was extracted

man et al., 1981) from overnight

YNB (-adenine)

and from YEPD cultures

4B. 5-10 pg of PvuI-digested

or undigested

in each track

nitrocellulose

and

lane 1, C627-4B; mants;

probe,

lanes

2-5,

wavy lines, yeast 2~ plasmid

with capital

(Morris

et al., 1981). Yeast

letters A-E in order of decreasing

M,,

“stable”

of C627-

gel, transferred DNA

YRp(ADEZ)2 DNA

of

that extrachromosomal YRp(ADEI)2 was present in high copy (Fig. 4C,D) and indicate that the 3.5kb BamHI insert contains a functional chromosomal replication origin (ars) as defined by Stinchcomb et al. (1979), Struhl et al. (1979), and Hsiao and Carbon (198 1). With one exception (see below), the hybridization patterns of DNA from “stable” transformants were identical to that of C627-4B. This type of transformant may result from a double crossover event, via integration of YRp(ADEI)2 and excision of pBR322 and adel sequences (Hinnen et al., 1978; Hicks et al., 1979), or by some mechanism that repairs mismatched base pairs in DNA heteroduplexes. This type of transformant, however, can be distinguished from revertants only because of its frequency. Hybridization analysis of DNA from the atypical “stable” transformant identitied multiple copies of unit length YRp(ADEI)2 in PvuI digests (Fig. 4A and B, lane 5), however, no

DNA was electro-

(B) As for (A) except

transformants;

(D) Undigested

agarose

(A) PvuI-digested

(C) PvuI-digested

YRp(ADEI)2

[‘2P]pBR322. transformant

hybridized.

[32P]pBR322.

[3’P]YRp(ALJE1)2. “unstable”

of a 0.7%

(Sher-

cultures

C627-4B Ade + transformants phoresed

are designated

sequences;

lines, i, cos fragment

circle

A

1

pBR322

of pBTI-1; hatched

from:

to from:

transforprobe

was

lanes

l-4,

lane 5, C627-4B; probe,

DNA from: lane 1, the “stable”

shown in (A) and (B) lane 5; lanes 2-4, “unstable”

transformants

shown in (C) lanes 2-4; lane 5, C627-4B;

[3ZP]pBR322.

YRp(ADEI)2

the pBR322 phoresed

sequences.

were BarnHI-digested (ADEl)la

Marker

in neighbouring

YRp(ADE1)2

(approx.

(approx.13.0

YRp(ADEZ)2

(shown

contains

probe,

a unique PvuI site within

plasmid

DNAs

were electro-

lanes. For (A), (B) and (C) markers

pBR322

(approx.

7.8 kb)

and

4.36 kb), PvuI-digested HindIII-digested

kb) and for (D), undigested between

panels).

YEp-

pBR322 and

237

monomeric plasmid was detected in undigested DNA (Fig. 4D, lane 1). In addition, two PvuI fragments (approx. 14 kb and 11 kb), which contain pBR322 sequences, appear to be present as single copies and suggest that at least one copy of YRp(ADEI)2 integrated into chromosomal DNA. Two possible, as yet unresolved, explanations for these observations are: (1) multiple, tandemly-integrated copies (Orr-Weaver and Szostak, 1983) of YRp(ADE1)2 are present, or (2) an integrated copy (or copies) coexists with multimeric extrachromosomal plasmid (Stinchcomb et al., 1982).Tetrad analysis of spores produced from crosses between C63620C (a adel LEU2) and “stable” YRp(ADEI)2 transformants indicated that ADEI segregated in a Mendelian fashion (Table I). Spores obtained from crosses using the “unstable” transformants exhibited ADEl segregation which confirmed the extrachromosomal nature of YRp(ADEl)2.

ACKNOWLEDGEMENTS

We wish to thank Diana Zahab and Michael Dove for their expert technical assistance. NRCC22760.

REFERENCES Botstein, D. and Davis, R.W.: Principles and practice of recombinant DNA research with yeast, in Strathern, J.N., Jones, E.W. and Broach, J.R. (Eds.), The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 607-636. Clarke, L. and Carbon, J.: Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287 (1980) 504-509. Dretzen, G., Bellard, M., Sassone-Corsi, P. and Chambon, P.: A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem. 112 (1981) 295-298.

Henikoff, S., Tatchell, K., Hall, B.D. and Nasmyth, K.A.: Isolation of a gene from Drosophila by complementation in yeast. Nature 289 (1981) 33-37. Hicks, J.B., Hinnen, A. and Fink, G.R.: Properties of yeast transformation. Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 1305-1313. Hinnen, A., Hicks, J.B. and Fink, G.R.: Transformation of yeast. Proc. Natl. Acad. Sci. USA 75 (1978) 1929-1933. Hsiao, C.-L. and Carbon, J.: High frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. Proc. Natl. Acad. Sci. USA 76 (1979) 3829-3833. Hsiao, C.-L. and Carbon, J.: Characterization of a yeast replication origin (arr2) and construction of stable minichromosomes containing cloned yeast centromere DNA (CEN3). Gene 15 (1981) 157-166. Miki, B.L.A., Poon, N.H., James,A.P. and Seligy, V.L.: Possible mechanism for flocculation interaction governed by gene FL01 in Saccharomyces cerevisiae. J. Bacterial. 150 (1982a) 878-889. Miki, B.L.A., Poon, N.H. and Seligy, V.L.: Repression and induction of flocculation interactions in Saccharomyces cerevisiae. J. Bacterial. 150 (1982b) 890-899. Morris, D.W., Noti, J.D., Osborne, F.A. and Szalay, A.A.: Plasmid vectors capable of transferring large DNA fragments to yeast. DNA 1 (1981) 27-36. Orr-Weaver, T.L. and Szostak, J.W.: Multiple, tandem plasmid integration in Saccharomyces cerevisiae. Mol. Cell. Biol. 3 (1983) 747-749. Petes, T.D.: Molecular genetics of yeast. Annu. Rev. Biochem. 49 (1980) 845-876. Sherman, F., Fink, G.R. and Hicks, J.B.: Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 (1975) 503-517. Stinchcomb, D.T., Struhl, D. and Davis, R.W.: Isolation and characterization of a yeast chromosomal replicator. Nature 282 (1979) 39-43. Stinchcomb, D.T., Mann, C. and Davis, R.W.: Centromeric DNA from Saccharomyces cerevisiae. J. Mol. Biol. 158 (1982) 157-179. Struhl, K., Stinchcomb, D., Scherer, S. and Davis, R.W.: High frequency transformation ofyeast: autonomous replication of hybrid molecules. Proc. Natl. Acad. Sci. USA 76 (1979) 1035-1039. Communicated by AI. Bukhari.