Chromatin structure in the nuclei of the ciliate Stylonychia mytilus

BIOCHEMICAL

Vol. 74, No. 1, 1977

CHROMATIN

STRUCTURE

AND BIOPHYSICAL

IN THE NUCLEI

STYLONYCHIA H.J. MRC Laboratory Received

October

OF THE CILIATE

MYTILUS and N.R.

Lipps'

RESEARCH COMMUNICATIONS

of Molecular

2

Morris

Biology,

Cambridge,

U.K.

13,1976

: Nucleosome repeat EFEF of the unicellular transcriptionally inactive of 202 base macronucleus found. Three

pairs, in a repeat

different

types

trichous

ciliate

nucleus,

the

contains

polytene

macronucleus

of nuclei

Stylonychia macronuclear

which

macronucleus

nucleus

is

transcriptionally

little

The presence that

its

this

paper

peat Material

micro-

DNA - rich

fragmented

which vegetative

chromosomes

transcriptionally

active, and the

the

macronuclear

(1).

microanla-

or no RNA (1).

DNA is

in Stylonychia

organized

we present of the

length

hypo-

macronucleus)

and the

inactive

the

The diploid

(developing

contains is

from

2):

(1,

chromosomes

of histones

organization

mytilus

probably

the

can be isolated

anlage

giant

Whereas

ge makes

length was studied in three different organism Stylonychia mytilus. The micronucleus has a nucleosome repeat the macronuclear anlagen and the vegetative length of 217 resp. 220 base pairs was

between

as in

evidence chromatin the

other

nuclei eucaryotic

for

a regular

which

differs

different

(2,

nuclear

3) suggests chromatin.

nucleosome in

nucleosome

In - like re-

types.

and Methods

Stylonychia mytilus (syngen I) was grown in neutral Prin sheim solution (1) Nuclei were isolated as described earlier 7 1, 2). Rat liver nuilei were prepared according to the method of Hewish and Burgoyne (4). Digestion of nuclei with micrococcal nuclease Present

Copyright All rights

addresses:

H.J. Lipps, Institut fiir Biologie III, Abt. Zellbiologie, Auf der Morgenstelle 28 74 Tiibingen, Federal Republic of Germany 2) N.R. Morris, CMDNJ - Rutgers Medical School Pitscataway, N.J. 08856, U.S.A. 1)

0 1977 by Academic of reproduction in any

Press, Inc form reserved.

230

BIOCHEMICAL

Vol. 74, No. 1, 1977

Figure coccal

1. Electrophoresis nuclease digest

micrococcal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on 1.7 96 agarose

gel

of

a short

micro-

nuclease.

was performed as described previously (5). Digested nuclei were deproteinized with SDS and chloroform - isoamylalcohol until the 280 : 260 ratio was 0.54. Electrophoretic analysis and calibration of digestion products on agarose and polyacrylamide gels fragments was done as described by with &X 174 - RF restriction Morris (5). Results

and Discussion

Brief

digestion

liver

nuclei

approximate

with generates multiples

micrococcal

nuclease

a series of the

of chromatin

of DNA fragments

nucleosome 231

repeat

which length

from are of

200

rat

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2. Electrophoresis on 6 % acrylamide gel of a long micrococcal nuclease digest of a) rat liver nuclei, b) St lon chia macronuclei, c) Stylonychia micronuclei, d) rat liver *nucle . 0.2 ml of StylQnychia nuclei at 25 CD 15 min. at 37 "C with 225 units/ml of260~ia~o~~~aa~i~~~~~~s~~r

base

pairs

fragment is

Digestion

(6).

pattern,

organized

differed

of Stylonychia

indicating

that

into

nucleosomes.

according

to nuclear

had a repeat

of

pairs

gestion

from

the

macro-

pairs

and that

from

the

macro-

liver

chromatin

core

8) which

gave

fragments

therefore nuclei

a quasi

of Stylonychia

conditions

must

DNA between

a similar

- limit

from

nucleosome

with

fragment

the

nucleosome

represents

- and micronuclei

fragment

in

The micronucleus

digestion

(Fig.

macronucleus

difference result

of rat

macro

from

the

length

repeat

digestion

(7,

DNA repeat

the

was 217 2 4 base

gives

the

202 2 3 base pairs;

220 2 3 base pairs.

base

However,

of Stylonychia

1).

nucleus

nuclease

chromatin

a similar

(Fig.

anlage

extensive

the

gave

type

nuclear

More

nuclei

the

2).

of about core.

a difference cores.

232

in

Di-

comparable

The size

of the

length the

140

under

and micronucleus DNA repeat

micrococcal

length

DNA

was identical; between

these

of the

linker

Vol. 74, No. 1, 1977

Correlative linker

BIOCHEMICAL

evidence

suggests

may be specified basic

HI's

have

short

more

basic

HI's

have

long

amino

acid been

composition

than

that

histone

riants

calf

thymus

the

three

length

HI.

In

general

(5,

9,

IO)

12).

the

different

known to

HI

It

(13).

from

of nuclei

only

the

very

active.

as in

the

(11)

and in

change

in

of us (NRM)

(e.g.

According must

repeat.

signals

would

this

criptional

active

alter

associated

In

(liver

that with

inactive

more

be suggested more

basic

va-

to macronuclear

in

chicken

the

in

urchin

sperm

has led

the

have

nucleosome

signals

repeat

than inactivity

nucleosome anlage

233

repeat.

re-

(e.g. to

nucleosome

repeat to

recognition

the

repeat

active is

inactive

the

the inactive

macronucleus. not

The fact

has a repeat

trans-

than

Stylonychia the

pro-

proteins

and modulating

a longer In

one

of trans-

be accessible the

(12)

to a

control

in

is

Stylonychia

and sea urchin

and gastrula).

macronuclear

Thus

accessibility

transcriptional a long

macronucleus

of nucleosomes

and sperm)

has a shorter

We conclude

could

coarse

to

changes

their

(11).

(erythrocyte

micronucleus

related

recognition

relationship

altering

tissues

HI of the be somewhat

changes

nucleosomes Thus

the

activity

tissues

has not

relationship

for

hypothesis

between

thereby

This

by HI mediated to

lie

the

seems to be related

a mechanism

RNA polymerase).

length

highly

nucleosome

activity

moters)

activity

to propose

criptional peat.

erythrocyte

transcriptional

the

with

nuclei

vegetative

to be transcriptionally

in

with

Stylonychia

micronucleus

known

a change

the

macronucleus.

types

chicken

nuclei

by successively

progression

DNA in

and those In

(11,

is

histone

of the

macronuclear

Oxytricha

to vegetative

Of the

the

HI may be replaced

during

anlagen

however

ciliate

basic

repeats

RESEARCH COMMUNICATIONS

the

repeats

of HI from

determined;

hypotrichous

that

by histone

less

yet

AND BIOPHYSICAL

invariably that very

the similar

Vol. 74, No. 1, 1977

to

that

of the

many factors

BIOCHEMICAL

active

must

nucleosome

repeat

criptional

activity.

macronucleus

be involved need

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

not

in result

demonstrates gene in

that,

regulation, a gross

since

a change change

in

in

trans-

Acknowledgement: H.J.L. thanks Dr. F.H.C. Crick for the opportunity to work in the MRC chromatin group in Cambridge. This work was supported by a short term EMBO fellowship awarded to H.J.L. and by a Senior fellowship of the Fogarty International Center of the U.S. National Institutes of Health to N.R.M. References 1)

2) 3)

4) 5) 6) 7)

Ammermann, D., Steinbriick, G., v.Berger, L., Hennig, W. (1974) Chromosoma (Berl.) 45, 401 401 -- 429. 429. Lipps, H.J., Sapra, G.R., Ammermann, D. (1974) Chromosoma (Berl.) 2, 273 273 - 280. 280. Chromosoma Lipps, H.J., Hantke, K.G. K.C. (1975) (1975) (Berl.) 49, 309 - 320. Hewish, D.R., Burgoyne, L.A. (1973) Biochem. Biophys. Res. Comm. 52, 504 - 510. Morris, N.R. (1976) Cell 2, 357 - 364. Nell, M. (1974) Nature 251, 249 - 251. Shaw, B.R., Herman, T.M., Kovacic, R.T., Beaudreau, G,S., Van Holde, K.E. (1976) Proc. Natl. Acad. Sci. US. 73, 505 - 509.

sumitted for publication M., Kornberg, R.D.: 9) Nell, M. (1976) Cell 8, 349 - 356. 10) Thomas, J.O., Furber, V. (1976) FEBS Letters 66, 274 - 280. 11) Morris, N.R.: Cell, in press, 12) Spadafora, C., Bellard, M., Chambon, P.: submitted for publication, 13) Caplan, E.B. (1975) Biochem. Biophys. Acta 407, 109 - 113. 8) Nell,

234