rDNA replication in a synchronized culture of Saccharomyces cerevisiae

Vol. 46, No. 1, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

rDNA REPLICATION

IN A SYNCHRONIZED

CULTURE

OF -_I_--SACCHAROMYCES --I__ CERE.VISIAE

Gerlinde

M. Gimmler

Institut

fiir 87

and

Biochemie WErzburg,

Eckhart

Schweiser

der Universitat Germany

Received October 19, 1971

SUMMARY. From a synchronized culture of SaccharomJces cere?-visiae cells v\rere harvested at different intervals during S-phase. The purified DNA of each sample was challenged with ribosomal RNA by specific DNA/rRNA hybridization. From the constant rDNA/DNA ratio observed it is concluded that in yeast, the replication of the rRMA cistrons is not restricted to a distinct interval but proceeds at a constant rate throughout the entire S-period. INTRODUCTION A sequential well

established

bacterial tures tion in

for

cell

studies

the

the

eucaryotes.

mes present replicate

fore

methods

much less

However,

in

the

yeast,

replication

provide

only

marker

sequence

method

proved

especially

of gene

instance,

on the the

limited

in

0 1972

by Academic

can not

information

Press, Inc.

the cul-

determinauseful loci

to

be applied.

mode of DNA replication 16-18

synchronous

143 Copyright

mapping

different

cerevisiae --Saccharomyces simultaneously or sequentially. times

in

division-synchronized for

is known

is

present

of rRNA and tRNA gene

For

either

of gene

This

of replication

chromosome

Bacterial

position

usual

manner

single

be used

(5,6).

studying

the

(l-4).

may therefore

which

in

and polarized

yeast about

the

chromoso(i'),

may

The study will

there-

genetic

loca-

Vol. 46, No. 1, 1972

tion

BIOCHEMICAL

of a specific

however, this

like

study

those

yeast

to

during

of

genome.

genes,

or transfer

between In

140 different

the

redundant

ribosomal

was investigated

replication

case

for

on the

of the

genome

the

discriminate

arrangement

distribution

In

coding

may help

scattered the

gene.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RNA (8),

a clustered

the

present

paper,

rRNA cistrons

by following

DNA synthetic

the

period

or

on the

course

of the

of rDNA

cell

cycle

( = S-phase). MATERIALS Strains. -2,

The diploid

strain

his7-l/his7-2,

Esposito,

the

Z 65 (lysZ-l/lys2-2,

ade2/+, haploid

AND METHODS

Ural/+)

wild

tyrl-l/tyrl-

was obtained

type

strain

from

X 2180-IB

Dr.

from

R.E.

Dr.R.K.

Mortimer. Media. --

YEPD:

medium: (9)

1% yeast

YEPD t 0.1%

to

Cells of

were the

2% glucose; starvation

as described

isolated

culture.

"chase" medium

elsewhere.

by centrifugation The DNA was extracted

and subsequently

procedure

(13)

determined

as described

by

and MYERS (14). Z 65 cells

double-strength 8 cells/ml

grown

1 mC/ltr

were added.

After

the

was subject

culture

synchronization

procedure

SCOPES (3).

After

transferred

to unlabeled

3O'C.

70 minutes

Every

were

YEPD to which

(Schwarz/Mann)

tion

(8)

of BURTON's

Synchronization.

10

t 0.9% KH2P04;

OGUR and ROSEN (12)

by a modification

x

2% peptone,

medium

IO ml aliquots

according

GILES

K2HP04

and low phosphate

DNA Determination. I_-from

extract,

of

of

of 2.5

feeding-starva-

by WILLIAMSON cycles

YEPD medium

144

a density to the

described

an aliquot

1.5 liters

of 8-5H-adenine

reaching

4 feeding-starvation

in

the

and vigorously

cells

and were

shaken

250 ml was removed

for

at

Vol. 46, No. 1, 1972

BIOCHEMICAL

DNA isolation.

Other

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

aliquots

were

taken

for

cell

number

and

DNA determinations. 'H-DNA

Isolation.

ml samples collected -2O'C

were

be almost

instantaneously

a longer

to DUELL et

cation

ml/gr

to

SMITH

was achieved

gradient

cells.

containing cells

These

medium.

for

cell

mes were

isolated

them

32P-labeled

the

extraction

in

Final

DNA purifi-

equilibrium

density

(8).

For

obtained. from

X 2180

the

in unlabeled pressure

"chase"cell,

centrifugation

rRNA was isolated density

medium

Subsequently,

a French

by differential

and sucrose

DNA was isola-

500 ml of low phosphate

two generations rupture

with

the

rRNA was isolated in

to

according

previously

DNA were

at

happened

3 hours

CsCl

32 P-orthophosphate.

10 mC of

After

grown

kept

for

(11).

250

cells

Laboratories)

as described

were

were grown

Endo

32P-labeled

the

converted

spheroplasts

pg of pure

the

were

37'C

by two subsequent

each sample, 50-90 32 P-rRNA Isolation. -lB

at

and HALVORSON

centrifugations

period

cells

them

From the

on ice,

This the

wet cells;

al.(lO).

according

thawing

culture

and subsequently

time.

by treating

(0.3

the

poured

of

After

spheroplasts

from

at 0'2

period

1 year.

glusulase

ted

withdrawal

by centrifugation for

into

After

and from

by subsequent

gradient

riboso-

phenol

centrifugation

procedures. DBA/rRNA essentially

Hybridization. as described

Each nitrocellulose GmbH,

Gottingen,

45 u)

contained

"F-rRNA sequently

DNA-rRNA

10 ,ug of

in

paper

SM 11306,

was heat-treated cooled

by GILLESPIE

filter type

ice

hybridization

and SPIEGELMAN

(Sartorius

Before

5 min

and passed

145

pore

size

hybridization,

at 80°C through

(15).

Membranfilter

30 mm diameter,

3H-DNA. for

was performed

in

4 x SSC,

a nitrocellulose

the sub-

Vol. 46, No. 1, 1972

filter

BIOCHEMICAL

which

had been

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

prewashed

RESULTS By the

the

the

of period

amount

phase ( 16-18

procedure

yeast the

the

division with

a good

almost

(Fig.1).

underwent

one,

two distinct with

as well

as the

relative

earlier

reports

from

respect

length other

rounds to

of the

S-

laboratories

).

40

80 1M 160 TIME (km)

DNA replication and culture of S.cerevisiae. cultural conditions Methods. The arrows 250 ml-samples were According

cellular

to

Fig.2,

DNA remains

ways about synchronous

2.2

percent

the

(8). replicate

is

240

division in a synchronized For details of synchronization, and DNA assay, see Materials and indicate time intervals at which withdrawn for DNA isolation. proportion

of rDNA to total

throughout

The rDNA content

yeast

apparently

200

cellular

constant

of DNA replication.

yeast

number

of DNA replication

0

Fig.1.

applied,

was obtained

cell

culture

The timing

agrees

culture

studied,

of DNA in

of doubling. cellular

the

100 ml of 4 x SSC.

AND DISCUSSION

feeding-starvation

synchronization During

with

of

identical

Therefore,

the

to the

at a constant 146

the that

140

entire genome

period being

al-

in

non-

observed rRNA cistrons

rate

along

of with

the

Vol. 46, No. 1, 1972

BIOCHEMICAL

OL 1 20

other

rate

1.0 40

@I TIME(mln)

80

la,

120

rDNA/DNA ratios ( l ) determined at different intervals during synchronous replication of S.cerevisiae DNA (0). The diagram represents the same experiment as depicted in Fig.1. The hybridization values are averaged from almost identical values obtained at different three, 32-P-rRNA concentrations (5,lO and 15 pg/ml, respectiEach hybridization vial contained 10 ml of 4 x vely). 11 750 cpm/,ug) as indicated SSC, 32-P-rRNA (spec.act. 2 DNA filters and 2 controls without DNA. The above, specific activity of the T-H-DNA continuously decreased from 295 cpm/ug in the first sample to 205 cpm/ug in the last one. The filters were incubated in the hybridization mixture for 12 hours at 640C and subsequently treated as described earlier (8).

Fig.2.

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cellular

DNA. This

observation

The results

is

obtained

cistrons

are

either

combined

in

a large

requires

the

entire

rRNA cistrons

should

of

other

represents

in

may

the

cell

cycle.

indicate

that

in

yeast,

the

over

the

scattered the

be somewhat

larger

chromosomes unit

(8).

than Supposed

of replication,

or exclusively 147

the

rRNA

genome

replication

A DNA molecule

S-period.

explain

-Sacch.lactis -7

over

DNA segment

of mainly

that,

dosis

constant

randomly

an independent

some consisting

rRNA gene

of TAURO et a1.(19)

of rRNA synthesis

one of the

constant

or

of which containing the

140

average

each this

rDNA would

size

chromosome chromoreplicate

Vol. 46, No. 1,1972

BIOCHEMICAL

simultaneously this

along

DNA segment

zer

region

the

rRNA genes,

yeast lus

would

of higher

the

methods,

in

of higher

the

eucaryotes. these for

cells

(22)

the

it

Future

must

of

two the

systems

is

in

based

is

organi-

known

Although

replicate recently

to

the

late

in the

nucleo-

during

HeLa entire

correspondence

on a similar

carry

shown by BALAZS

synchronized

this

nucleolar

of a nucleolus

throughout

show if

Possibly,

By cytological

(20,21).

to

also

replicate

the

(20).

has been

that

region

existence

reported

28 S rRNA cistrons experiments

to the This

so far

is (20)

and SCHILDKRAUT

chromosomes.

organisms

equivocal,

division

other

be equivalent

evidence

has been

nuclear

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

genetic

cells

the

S-period. between

organization

rRNA cistrons. ACKNOWLEDGEMENT

This

work

was supported

by a grant

from

the

Deutsche

Forschungsgemeinschaft. REFERENCES

I. :: 4. 5. 6. 7.

8.

2:

J-Cairns. Cold Spring Harbor Symp. Quant.Bio1.28,43(1963). T.Nagata. Cold Spring Harbor Symp. Quant.Bio1.28,55(1963). F.Bonhoeffer and A.Gierer. J.Mol.Biol. '7, 534 (1963). M.Oishi, H.Yoshikawa and N.Sueoka. Nature a,1069 (1964). N.Sueoka. In "Cell Synchrony" (ed.by I.L.Cameron and G.M. Padilla) Academic Press, New-York 1966, pp.38-53. R.G.Cutler and J.E.Evans. J.Mol.Biol. 26, 91 (1967). R.K.Mortimer and D.C.Hawthorne. Genetics 53, 165 (1966). E.Schweizer, C.McKechnie and H.O.Halvorson. J.Mol.Biol. 40, 261 (1969). D.H.Williamson and A.W.Scopes. Nature 193, 256 (1962). A.E.Duell, S.Inoue and M.F.Utter. J.Bacteriol. 88, 1762

11. 11;:

(1964).

J.D.Smith and H.O.Halvorson. Methods Enzymol.12 538(1967). M.Ogur and G.Rosen. Arch.Biochem. 25, 262 (1950). K.Burton. Bi0chem.J. 62, 315 (1956T K.W.Giles and A.Myers. Nature 206, 93 (1965). D.Gillespie and S.Spiegelman. J,Mol.Biol. l2, 829 (1965). D.H.Williamson and A.W.Scopes. Exp.Cell Res.z,338 (1960). D.Smith, P.Tauro, E.Schweizer and H.O.Halvorson. Proc. Nat1.Acad.Sci.U.S.A. 60, 936 (1968).

148

Vol. 46, No. 1,1972

20. 21. 22.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

R.E.Esposito. Genetics 59, 191 (1968). P.Tauro, E.Schweizer, R.Epstein and H.O.Halvorson. In "The Cell Cycle" (ed.by G.M.Padilla, G.L.Whitson and I.L. Cameron) Academic Press, New York 1969. pp. 101-118. For review see H.Busch and K.Smetana "The Nucleolus". Academic Press, New York 1970. H-Moore and C.F.Robinow. In "The Yeasts" (ed.by P.Matile, A.H.Rose and J.S.Harrison) Academic Press, New York 1969. Vol. I, pp. 219-302. I.Balazs and C.L.Schildkraut. J.Mol.Biol. 57, 153 (1971).

149