Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

Vol.

59,

No.

4, 1974

BIOCHEMICAL

AUTONOMOUS

rDNA

AND

BIOPHYSICAL

MOLECULES

RESEARCH

CONTAINING

COMMUNICATIONS

SINGLE

COPIES

OFTHERlBOSOMAl-RNAGENES IN Jan

Engberg

,

Eiochemical DK

-

2 100,

Received

or CsCl

one

of

Vagn

pyriformis

l-eick

Copenhagen,

Medical

Juliane

Microbiology,

Mariesvej

30,

University

of

.

containing

the in

of

the

are

of

electron

microscopy

fractionated

@29

20%

5. 65

the

this

method.

of

The DNA

the

rDNA

2 0. 6 pm

25s

=

11

W.

to

of 17s

purified the

bases

rRNA

as

in de-

co-sediments

x 106).

that

whole

rDNA

(40% and

rDNA

demonstrates corresponding

lysed In

DNA

to

r-

considerably

gently

purified

(M.

called

gradients from

homologous

phage

(commonly

DNA by

about

rRNA

density

when

hybridization.

of

for

sucrose

sequences

with

of

genes

DNA

centrifugation

DNA-RNA

a length 6 .

Institute

part

consists

gradients

11x10

*

nuclei

gradient

by

of

University

sediments main

sucrose

with

DNA

isolated

termined

B,

Tetrahymena

El,1974

the

strand)

and

, Denmark

Tetrahymena than

OF

Christiansen:

.

8000

: The of

by

-

July

SUMMAR’f

cells

Gunna

Denmark DK

slower

MACRONUCLEUS

Institute

Aarhus,

DNA)

THE

the

in

Examination

molecules

a molecular

by are

weight

linear of

INTRODUCTION We

have

recently

the

rRNA

genes

contains radioactive

DNA

selective

In

detail

and

the

gradients present

it

is

hybridization of cules

Copyright All rights

the

(1).

density

DNA.

and

each

in

containing

the

this

DNA

during It

was

paper

concluded

the

that

we on

found

and

macronucleus one

@ 1974 by Academic Press, Inc. of reproduction in any form reserved.

DNA be

basis

the

the of

electron

than

the

in

macronuclear (t-DNA)

in

more

DNA-RNA

observations

preribosomal

induced

sedimented

bulk

in --

with

an

analyses,

exists for

to

DNA

DNA

microscopic

1356

labelled due

nucleolar

nucleelar

Tetrahymena

Tetrahymena

selectively

sedimentation

gene

of

shift-up

slower

of structural

can

that

describe the

nucleolar

a nutritional

considerably

experiments rDNA

that

precursors

replication

sucrose

shown

that vivo RNA

as .

free

most mole-

Vol.

59,

No.

4, 1974

BIOCHEMICAL

METHODS Cultivation

of

(amicronucleate) midine

and

as

detail

well

as

were

made

trifuged man

in

10

sodium at

centrifuge.

lyzed

5 mM

concentrated

with

isopycnic

banding

fugations

(see

purification it could

phage

DNA

Fig.

the

r ‘H I described

been

pH

on

a cushion

18

on

,

,

50

The

thyin

yield

35s)

of

rDNA

mM

NaCl, and

were

Seckdia-

EDTA

further

and

purified

gradient

across

by

centri-

collected

during

cen-

pooled,

5 mM

DNA

index

15-3070

a L2-65

density of

hybridization final

of

was

ml gradients

sucrose

NaCl,

CsCl

amount

34

50

80%

to

mM

of

nuclei

The

rotor

rDNA

of

the

top

EDTA,

1 ml

(15s

8.5

isolated

(4).

a SW27

fractions

From

the

1 mM

of

in

or

Andoh

7.4

hrs

pH

DNA-RNA

cells

and

rounds

2).

the

whole

buffer

successive

to

{Szo

M.W.

samples

at the

each

gradients

preparation

were

was’

Davidson

ref.

generous

were

used

from

as

Prof.

DNA

was

and

Schuell,

as

P.

Borst,

denatured

al-

B-6)

,

previously(2).

using

procedure

in

type

described

microscopy

Kleinschmidt

T 7

14C-labelled

.

processed

electron

6)

gifts

(Schleicher

and

for the

106,

‘H-labelled 5) and

respectively

filters rRNA

of

rDNA,

ref.

experiments,

nitrate

prepared

of

x 106,

11.0x

hybridization

modification and

=

Madrid,

32P-labelled were

analyses = 26.4

species

M. Salas,

cellulose

with

formamide

w = 24; DNA

Dr.

on

incubatecl

M. W.

DNA-RNA

loaded

gradient ;

pha;e

and For

density w = 32.0

These

Amsterdam

Simon,

with

has

sulphate

(Calbiochem).

two

sucrose

DNA

markers.

DNA

of

for

buffer

that

(Sgo

phage

kali,

GL

. For

9229

15OC

containing

bme calculated 20%

strain

labelled t-RNA

Miyagishi

sulphate

rDNA

and

uniformly

dodecyl

Tris-HCI

in

COM:utiNlCATlONS

pyriformis,

P-labelled

by

Aquacide

step

about

sodium

at

legend

DNA

: Suspensions

Tris-HCI

rpm The

against

of 32

described

mM

000

Tetrahymena

of

dodecyl

18,

RESEARCH

.

0. 5% as

up

0.5%

protozoan

rDNA

with

gradients

were and

3)

of

lysed

sucrose

the

BIOPHYSICAL

MATERIALS

preparation

(2,

Isolation (2)

AND

preparation

previously

AhD

the

aqueous

described

by

and

Davis,

(7).

RESULTS In genes

for

order rRNA,

sedimentation of avoid

the

DNA through

gradient or

to

minimize

determine

the

from

whole

sucrose

was

tested any

shearing

size cells

distribution

of

or

nuclei

gradients for

its

before

capacity or

isolated

DNase

1357

to

the

in with

during

DNA was

DNA

hybridize

cleavage

the

containing

the

fractionated

the rRNA. handling

by

different

parts In

of

order the

to DNA,

Vol.

59,

whole

No.

cells

Fig.

or

that

than

marker.

BIOCHEMICAL

isolated

1 shows

slower

or

4, 1974

the

most main

\‘irtually

isolated

sity

in

the

middle

CsCl part

2 subsequent

were

lysed

of

rDNA

sediments

the of

identical

of

the

DNA

were

analyzed

gradients

than

initial

sucrose

the

density

gradient

RESEARCH

directly

the

slower

obtained

when

bulk

rDNA DNA

gradient

centrifugations

of

in

sucrose

T 7 DNA

from

whole

a higher

in

cells

buoyant

rDNA

1) could detailed

the

DNA

the

gradients.

considerably

than

(3),

as

the

band

has

(Fig.

COM!JtiNlCATiONS

top

a narrow

slightly

. Since the

on as

and

results was

density

BIOPHYSICAL

nuclei

part

macronuclei

AND

den-

contained

in

be

purified

by

the

legend

to Fig.

T7 1

FRACTION

Fig.

1 . Sedimentation

sions

of

late

were

lysed

tions

of

and equal

radioactivity.

phase

on

and

hybridized

tractions properties

as were

DNA CsCl 32

with for

which

fractionated

preparative

T

t-DNA were

in

the

were 7 phage

for

pooled (14),

rRNA made.

as The

DNA.

1358

and

assayed

fractions,

as loaded

Methods.

acid

on

was

membrane

earlier indicates

Frac-

precipitable

indicated,

described arrow

Suspenthymidine

with

in Material and

step-gradients P-labelled

gradients. 3 r HI

prelabelled

described

collected

background of

of

cells

volume The

fied

tion

properties

log

NUMBER

(2). the

purifilters No

sedimenta-

sub-

2.

Vol.

59,

BIOCHEMICAL

Nom. 4, 1974

AND

BIOPHYSICAL

FRACTION

Fig.

Purification

2 .

fugation.

(A)

shown

of

The

in Fig.

dients.

Each

gradient

about

50

65

at

fractions

of

tube

(polyallomer, and

in sity

The

DNA

ments.

In

hybridized

to rRNA

fractions

of

was

the

rRNA.

rDNA Assuming

(8,g)

has

24

using

purified

rDNA

of that

a ZO”,$

the higher

32

25 and

common molecular

in

from

1359

the

was

rpm

used

The

was

and

rebanded Buoyant

was

den-

(3).

depicted

subsequent

experi-

H-labelled

17s

rDNA

rRNA

found

RNA

molecule

than

the

on DNA

gradient

for 3

60

loaded

.

CsCl

for

centrifuge

refractometer

the

strand)

weight

(A)

gracon-

000

(6)

in

that

About

conditions

where

precursor

buffer

37

pooled,

gradient

25s

one

of

of

line).

Abbe

P-labelled

bases

bottom were

experiments of

the

to

ml

to density

centrifuge.

the

similar

a Zeiss

CsCl

at

(dotted

the

under

hybridization

(40%

from

8.0

Beckman

fractions

22

a mixture

and

centri-

similar in

performed

rRNA

fractions

as

saturation against

made

the

defined

to

banded

from

P-labelled

gradient

were

l-2-65

collected

21

density

in

the

gradient

gradient

was

9. 4 g CsCl

Selected 32

density

a sucrose

DNA

was

of

were

to

contained 26

40

volume

determination

in Fig.

20%

the CsCl

in

the of

Beckman).

in

25

CsCl

Centrifugation

rotor

hybridized

a second

to

consisted

the

NUMBER

isopycnic

and

DNA.

equal

filters contained

in

15 pooled

pg

20°C

by

fractions

1 were

taining hrs

rDNA

COMMUNICATIONS

RESEARCH

combined

(Fig.

to

be to

was 3)about

homologous 17s

and

masses

25s of

Vol.

59,

No.

4, 1974

BIOCHEMICAL

AND

rRNA

3 * Saturation

l=ig* brane

filters

rDNA

hybridization

containing

(specific

with

increasing

25s

r.RNA

19,000

amounts

of

(specific chloride, earlier

described

and

25s

than

half

of

density

rRNA the

(lo), rDNA

gradient

dimenting (cf.

@29

were

to

, containing

19 hrs

formamide of

at

37OC and

citrate

and two

of

17s

M sodium

average

pg

P-labelted

0.03

50% the

Mem-

0.05

for 32

of in

rDNA. about

incubated

cpm/pg)

is

purified with

mixture

point

and

linears

of

classify

the

occasionally

Of

hundred

some

Taking

1.92 pm

x

uniform

106 6)

with electron

of

,

processed

separate

found

very

experi-

to

a single

sharp

rDNA

was

isolated the 5.

in Fig.

molecules No

one

was to

1 pm

a molecular

J. Gall

1360

weight

the of

described on

the

linear

unusually

(ll),

11 x

co-se-

IO6

for

about

11

in as

made circular.

long

(21.0 length

x 106

Tetrahymena

well to

or

Moreover, in

the

examined

average

above. rDNA

peak

were

as

found

sucrose

appeared

efforts

molecules

more

Neutral

the

tangled

by

rRNA,

of

analyses study

slightly

weight

of

equivalent

sedimentation

microscopy

seen

inspected, as

showed

majority

as

for

that

a molecular

When

size

corresponds

4)

indicating

great

indicate

genes

(Fig.

Methods).

Daltons

the

results

structural

rDNA

DNA

molecules

(Fig.

agreement

of

the

microscope,

spread

hybridization

consists

phage

Materials

electron

recent

7.0

rKNA loaded

cpm/@

Each

the

analyses

with

rDNA

5.65

pl+

of

equimolar

COMMUNICATIONS

1

filters

350.000

(2).

RESEARCH

.

17s

the

and

an

activity

as

(pg/rnl

curve DNA

activity

0. 3 M sodium

ments

no

BlOPHYSlCAl.

pm). of

in

close in

a

Vol. 59, NC). 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL

FFI ACTION

Fig.

4 . Neutral

sample

of

sucrose

‘H-labelled

and

fractionated

and

Methods.

rDNA on

(Combusto-Cone,

was DNA

(Proc.

NaLAcad.

174

DNA

natured

t-DNA

turation

map

from

and

se’quence c:ircles

volume

DNA.

U.

S. A.,

in

internal

was

was

also

shows and by

carried

that that

single

.

the the

out t-DNA

linear

breaks

the

molecules at

random

1361

of

sample

holders Sample

T 7 phage

a mixture

of

the

molecular

were

made.

weight

of

12.

microscopy

of

partially

de-

of

The

partial

dena-

may

to

($29

be

study

Gall. have

could places

DNA

Materials

background

estimated

molecules

phage

Packard Tri-Carb 3 H-labelled

12),

Electron in

A

in into

for

rDNA

@29

rDNA.

of

subtractions

ref.

standard)

led described

gradient

press;

macronuclear as

in

sucrose No

purified

collected

position

a parallel

T 7 phage

as

were

The

of

14C-label

combusted

counted.

from

Sci.

with gradients

dried,

and

non-replicating

@X

cal

306)

determined

phage

the

equal

Packard) (model

analysis

mixed

sucrose of

NUMBER

gradient

was

1530%

Fractions

Oxidizer QNA

density

RESEARCH COMMUNICATIONS

not in

a similar have

the

been

molecule.

6 x

or

106(using

identi-

produced Although

Vol. 59, No. 4, 1974

Electron

Fig.

5 .

were

spread

The

of

micrograph for

inserted

we

have

yet

we

have

denatured sequences)

located

Gali

of

is

out

easily at

rDNA as

COM:~*:JNICATIO~&

I-inear

rDNA

molecules

in Materials

denaturation

rDNA

end

(A).

described

of

map

molecules

distinguishable

each

RESEARCH

and

Methods.

.

a complete that

BIOPHYSICAL

purified

1 pm

observed two

AtiD

microscopy

worked

have

of

electron

scalemark

not

rDNA

results

BIOCI-IEMICAL

which

are

denaturation the

molecule

in

on

our more

bubbtes close

preparation than

20%

(AT-rich

agreement

with

the

(12). DISCUSSION

In hymena 11 from

x

this

paper

macronuclei lo! whole

purification reasonable

we as

the and

scheme to

assume

shown

a unique

Considering ceils

have

does that

DNA

gentle

isolated

that species

lysis

most

select of

can with

procedure

macronuclei not

rDNA

the

one rDNA

1362

isolated

a molecular used

and for

be

to

considering

distinct exists

liberate the

vivo

Tetra-

weight

molecular in--

from

as

of

the

DNA

fact

that

size,

it

molecules

about

the is with

Vol.

59,

No.

4, 1974

BIOCHEMICAL

AND

1

Fig

. 6 St !iistogt-am

The

molecules

and

the

The

solid

tally

the

length

and

the

into

each is

amount

+ 25s

molecule

is

very

the

bian

oocytes

ning

free

to

hymena DNA.

‘Wnen

shown

in

Fig.

remains

the

length length

DNA 1 was

study

DNA-RNA to

of

be

of

established

in used

if

the

some

bottom for

part

Daltons

amount

is

gene

for

free

rDNA

both

is to

which 17s

called

the

be

a convenient

cal-

combined

means

the

that

25s

rRNA.

from

Tetra-

repeating

DNA

unit

from

system this

be

the

and

nucleolar

of

can transcribed

molecules

been

structure

it

close

x 106)

in

amphi-

for

repeating

obtaiunit

by

methods. whether

are

6

molecules

3),

genes

fine

B).

mechanism

rDNA

(2O%,Fig.

has

may the

the

what

hybridization

or

of

(2.0

the

9100

experimen-

cleavage

x 10

molecule

rRNA

to

2.2 This

Tetrahymena

autonomous the

the

to

specific

structural

the

pm).

weight

to

random. 9108)

(model

around

value

rRNA one

units and

are

25s

Hence,

repeating

65

a very

molecule.

alternating

(15).

enzymological

rDNA

at (model

Calculator

(5.

equivalent

that

similar

regularly

It

note

collected digitizer

molecular

DNA

one

molecules

distribution

unless

contains

interesting

within

and

9

rDNA

molecules

COMMUNICATIONS

)

hybridization

per

17s

46

Gaussian

estimated

of

rRNA

o’f one

hymena

the

DNA-RNA

an

rDNA

the

7 (,um

a Hewlett-Packard the

of

5

RESEARCH

a Hewlett-Packard

mentioned,

From

17s

masses

It

weight

that

of

with using

length

saturation

culated

length

represents

mean

molecular postulated.

the

traced

calculated

curve

found

is

of were

3 LENGTH

BIOPHYSICAL

all

rDNA

integrated of

hybridization

a sucrose experiments

1363

in

molecules high

in

molecular

gradient

similar

a saturation

Tetraweight to value

that of

‘401.59, No. 4, 1974

0,02%

is

found

fractionated

(unpublished DNA

when

micronuclear

dized

with

was

is the

from

integrated

from

ACKNOWLEDGMENTS: Council

for

Dr. ted

M. to

Salas

Gall

thank

Prof.

M.

Brieg

for

rDNA

free

I-DNA micronucleus

This

work

was authors

Klenow expert

technical

found hybriof

Te-

observations found

in

the

ma-

mechanism (12,

the

Danish to

q’-29

13).

rqesearch

Prof.

P.

DNA.

sorst

We

communicating continuous

that

strains

amplification

and

un-

was

these

indebted

for his

Tetrahymena

by

T 7 DNA

for

of

when

to

macronucleus

are

Gorovsky

identical

molecules

a gene the

is

DNA,

supported

of

M.

t+.

by

0. 3%

micronucleate

rDNA

to

The

Dr.

in weight

the

the

her

#. 02%

molecular

samples

and

We

the

that

providing

J.

Mrs.

high

Sciences.

for

Prof.

licationand

Natural

of

COMMUNlCATtCINS

with

strains

that

integrated of

value

RESEARCH

compared

micronucleate

into

conversion

BIOPHYSICAL

as

The

possibility

arose the

(2).

Assuming

interesting

cronucleus

during

used

(1 1).

AND

observations)

DNA

r%NA

trahymena raise

BIOCHEMICAL

are

data interest

and indep-

before in

pub-

this

work

assistance.

i?EFEPENCE.S 1.

Engberg,

J.,

Proc.

Nat.

Nilsson,

Acad.

J. P.,

Sci.

U.

2.

Engberg,

J.,

and

3.

Engberg,

J.,

Mowat,D,,

2=, 4.

Miyag~shi, Studier,

6.

Ortin,

7.

Davis,

J,, z,

Xes.

Leick,

V.

(lQ74)

E. (1972)

Pearlman,

Eur.

J. Biochem.z,

393-400.

F?. E.

(1972)

Biophys.

Biochim.

Acta

Andob,

T,

J,Mof. E.,

(1973)

3iol.

Biochim. u,

Salas,

Biophys.

Acta

7x,

507-515.

)Nature

New

373-390.

M.,

and

Vasquez,

C.

(19’71

Simon,

M., L.,

and and

N.

Davidson?

Moldave,

(1971)

Methods

K. (Academic

Press,

in

Enzymology

New

York),

413-428, V.

6L,

Perry, and

and

275-277. ,Z.,

XXI,

9.

P. E.,

894-8Q8.

9. and

(1965)

Vinuela,

Grossman,

Prescott,

Pearlman,

and

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Vol. Leick,

10.

A,* F.

eds.

8.

3,

312-320.

5.

Biol.

Pearlman,

S, A.

Eur. Bostock,

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C.,

221-228.

Gamow,

E,,

and

Lauth,

M.

(1971)

Exp.

Cell

124-128. B,

Tartof,

(1969) D.M.,

P.,

Cheng, K.

0,

(1970)

T*

, Proc,

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Greenberg, .Sci.

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A.

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