BIOCHEMICAL

Vol. 88, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 27, 1979

Pages 1329-1336

THE MESOKARYOTE GYRODINIUM COHN11 LACKS NUCLEOSOMES Steven

Bodansky,

Lauri

B. Mints

and David

S. Holmes

Department of Biological Sciences State University of New York at Albany Albany, New York 12222

Received

May 3,1979

SUMMARY The dinoflagellate Gyrodinium cohnii has a distinct nuclear membrane but apparently lacks histones associated with its chromatin. Approximately 13% of the nuclear DNA is rapidly digested by micrococcal nuclease to acid soluble fragments and not to nucleosomal sized fragments as in the typical eukaryote. Moreover in the electron mbcroscope the chromatin of 5. cohnii appears as a thin filament of 40-60 A in width without regularly spaced nucleosomes. These observations support the view that the dinoflagellates exhibit characteristics of both prokaryotes and eukaryotes.

Gyrodinium is

cohnii --___

a unicellular

marine,

the common name for algae

in the plant

mesokaryotes organisms appears

(1).

There

are

microscopy fibrils

compared Moreover,

or morphological DNA per

cell

Abbreviations methylsulfonyl

have

Pyrophyta,

features

no normal

associated

to the

of both but

sections

(2). with reveal

prokaryotic

the nuclear remain

briefly

spindles

elements

seven

just

No histones the

never

the presence

division

is

and electron

of thin

(40-60

unicellular

with

differentiation

it

biochemical

the same amount sequences

used: EDTA, ethylenediaminetetraacetic fluoride; TCA, tricarboxylic acid.

acid;

(5).

All

rights

of

We

PMSF, pheny-

0006-291X/79/121 Copyright

1329

A)

eukaryotes

little

has approximately

DNA has reiterated

pro-

(3),

its

dis-

to mitosis

or histone-like

chromatin

nature

as

throughout

prior

and nuclear

of

and eukaryotic

condensed

of typical

as a human and this

divisions

envelope

100 i to 300 i fibrils

despite

is

have been classified

decondensing

mitotic

cohnii,

Dinoflagellate

one of the

The chromosomes

only

of nuclear

dinoflagellate.

has a nucleus

cycle,

can be detected

known as Crypthecodinium

Dinoflagellates

(1).

by microtubular

teins

(4).

they

mitosis cell

assisted

kingdom.

5. cohnii

during

also

heterotrophic

the division

in that

most of the (1).

(G. cohnii),

@ I979

329-08$01.00/O

by Academic

of reproduction

in any

Press,

Inc.

formreserved,

BIOCHEMICAL

Vol. 88, No. 4, 1979

have extended

this

nucleosome-type tron

survey

repeats

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and show that as judged

G. cohnii -___

by nuclease

has no nucleosomes

digestion

studies

or

and elec-

microscopy.

Materials

and Methods

5. cohnii strain Woods Hole was kindly provided by Dr. Beach. Cells were maiadmerile culture and nuclei were prepared essentially as described by Riszo and Nooden (3). Micrococcal nuclease digestion was carried out essentially by the method of No11 (14) as modified by Newman and Noon (15). 10 1.11 (150 units) of micrococcal nuclease (Worthington) were added to 90 ul of nuclei (130 A260 units per ml) in 10 mM Na borate-l mM CaC12, pH 7~6, and the suspensron incubated at 37'C for varying times. The reaction was quenched by placing on ice, and the nuclei sedimented at 2000 x g for 10 min at 4OC. The A260 of the supernatant was read directly or was made 7.5% tricarboxylic acid at 4OC, sedimented at 400 x g for 10 min and the A260 of the supernatant read. When nuclei were treated as described above without added en&me, 0.68% of the initial A260 absorbing material was liberated into the supernatant after 10 min of incubation at 37OC, indicating that endogenous nuclease digestion is not significant. Gel electrophoresis was carried out essentially by the method of Varshavsky Nuclei were digested for 10 min as described --et al. (17). above and the reaction quenched at 4O with the addition of an equal volume of 20 mM ethylenediaminetetraacetic acid (EDTA) and the nuclei sedimented at 2000 x g for 10 min. Nucleic acids were extracted from either the nuclei or the supernatant essentially by the method of Blin and Stafford (16) and concentrated by precipitation with ethanol. The nucleic acids were resuspended in 10 mM'Na borate - 5 mM EDTA, pH 9.2 - 20% glycerol and electrophoresed on a 5% polyacrylamide gel at 150 volts and visualized by staining with ethidium bromide (17). Chicken chromatin or chromatin isolated from G. cohnii nuclei (3) was prepared for electron microscopy by fixation with 0.1% gluteraldehyde essentially as described by Thoma and Keller (18). Unstained chromatin was supported with benzalkonium (Mobay Chemical Corp.) and picked up by the droplet procedure (18) onto carbon grids and rotary shadowed with platinum-palladium. Results

and Discussion

Rizzo tones

and Nooden

in isolated

that

yeast

least

four

similar of Rizzo

did

--G. cohnii

five

major

with

5. cohnii,

and Nooden

methylsulfonyl

chromatin.

not have histones,

of the

ambiguity

(3) have demonstrated

using

fluoride

However,

and then

species

a well

the

later

(6, 19).

lack

of eukaryotic

it

was initially

it

was shown to have at

to repeat

characterized

protease

stages

1330

thought

Due to the possibility

we decided

(PMSF) at all.

his-

of nuclear

the

of a

experiments

inhibitor, and chromatin

phenyliso-

BIOCHEMICAL

Vol. 88, No. 4, 1979

Following

lation. the

addition

essentially

the procedure

of 0.1 mM PMSF to all

any histone-like

proteins

electrophoresis,

by extraction

tin

with

0.2

HCl,

N

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

solutions,

by sodium

the results

of Rizzo

and Nooden

acid

extractable

proteins

in either

that

have

suming

electrophoretic

that

tivated

there

organize

eukaryotes

the DNA into

discrete

is associated

number

depending

nucleosome

upon

contains

of two copies

shown). that

to eukaryotic protease

chroma-

Thus,

we con-

there

are no chromatin

histones,

that

with

for

For example, of polypeptide

is not

asinac-

unit

in the prokaryote

Thermoplasia

material

with

1 shows a time

from -~ G. cohnii

have not performed the

reaction

decrease soluble.

studied.

found

of the

with

an octomer

with

about

40 base pairs

course

of release

by

micrococcal

digestion occur

in the rate

after

utilizing

it

where

where

could

repeating other six

200 base pairs

proteins. copies

of DNA (8)

a "histone-like"

of DNA (9). of ultraviolet

absorbing

nuclease.

of G. -- cohnii

at a constant

structure

However,

in adenovirus

about

does not

An ultraviolet

of DNA, the actual

of chromosomal

but

(7).

The core

(7).

type

acidophilum,

nuclei

extensive

kingdom

which

microscopy

of DNA associated

of histones

is

are associated

Figure

being

of Hl,

nucleosomes

200 base pairs

or analogous

in the absence

is associated

termed

exception

HZa, H2b, H3 and H4 and this

VII

protein

about

the eukaryotic

a repeating

the

or by electron

the organism

a homologous

to be formed

with

units

digestion

140 base pairs

throughout

be possible

acid

or purified

or in isolated

histones,

repeating

each of histones

invariant

marked

not

nuclei

histone

the major

The nucleosome

that

nuclei

similar

is not a specific

by nuclease

or

whole

to detect

(SDS) polyacrylamide

(3) and conclude

nobilities

can be detected

unit

whole

unable

(3) with

by PMSF. In typical

is

sulfate

0.4 N H2S04 (data

firm

and Nooden

we were

dodecyl

of either

or with

of Rizzo

rate

Although

nuclei, but

it

that

we is

clear

there

is

some 13% of the DNA has been rendered

spectrum

of the released

1331

material

was

a

Vol. 88, No. 4, 1979

BIOCHEMICAL

2.0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r

-'5cl cm

5

0.

-IO' -5 I 5

0

I I 15 20 Minutes

I IO

I 25

I 30

3 6 a-"

35

Figure 1: Time course of the release of A26O absorbing material from G. cohnii nuclei during treatment with micrococcal nuclease. Co) A260 ofthe material released from the nuclei into the supernatant. (0) A260 of the supernatant after precipitation with 1.5% TCA. typical

of purified

be noted acid,

that

chromatin

all

the liberated

indicating

that

twenty

residues

(10).

firmed

by the

fact

weight

marker

in an agarose

possibility

that,

--G. cohnii

is

it

it

agarose

gel

electrophoresis

whole

DNA (Fig.

discrete

bands

bottom

of the gel

typical

larger

than

2, lane

enzyme as judged

are

digestion

nucleases

during

digestion

sized

incubated

is

gel

rendered is

than

product

is

is

the DNA is

gradually

the nuclear 2, lane

the

membrane

B.

of

the

DNA by The majority

and co-electrophoreses

with

a smear of DNA with and extending

No significant fdr

electrophoresis acid

to the

without (Fig.

soluble)

reduced

no

digestion

10 minutes

not organized

1332

con-

DNA, we disrupted

of added

2, lane

indicating

that

Our interpretation

not significant.

of --G -cohnii

about

molecular

To eliminate

600 base pairs of DNA).

of less

the nuclear

However,

by either

(0.68%

that

C).

below

when nuclei

the chromatin

eukaryote,

600 base pairs

D) or by TCA solubility

is that

E).

should

in 5% trichloroacetic

a 70 base pair

and analyzed

(( 70 base pairs

by endogenous

2, lane

to nucleosome

is detectable

DNA is detectable

(Fig.

than

It

shown).

released

as shown in figure

is

not

soluble

of the faster

digestion

of the product purified

is

size

gel

the

(data

of polynucleotides

migrates

not permeable micrococcal

material

The small

unlike

after

--G. cohnii

consists

that

nuclei

micrococcal

for

into in size

subunits until

and it

is

Vol.

88,

No.

BIOCHEMICAL

4, 1979

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 2: Acrylamide gel electrophoresis of DNA liberated from G. cohnii after 10 min digestion with micrococcal nuclease. (A) = Hae III-restriction digest of $X174 (New England Biolabs). (B) = extracted from nuclei after micrococcal digestion. (C) = DNA extracted from nuclei without prior nuclease digestion. (D) = DNA extracted from nuclei after appropriate incubation but without added nuclease. (E) = liberated from G. cohnii nuclei after micrococcal digestion. Digestion with micrococcal nuclease was carried out for 10 min. Number of base pairs of the Hae III digest are shown on the left.

rendered

soluble.

susceptible

more

the

acid

to digestion of the chromatin

majority

giving

it

highly

condensed

a

Figure pared chicken

We do not than is

state

of the

in

10% of the chromatin It

is possible

semicrystalline

form

is that

(11)

and accounting

for

the

chromatin. micrograph

of a typical

is organized

some

from digestion

3a shows an electron

to the chromatin

some

the remainder.

of protection

measure

chromatin

know why

into

of -~G. cohnii

eukaryote

(chicken)

repeating

units

1333

which

chromatin (Fig.

3b).

measure

com-

The some

BIOCHEMICAL

Vol. 88, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 3: Electron microscope visualization (b) G. chromatin. Scale bar = 800 i. -- cohnii 115 i in diameter 60 i wide

with

whereas only

additional

thickening

shadowing,

these

either

naked

similar

due to the

dimensions

that

dimensions

The taxonomic they

occasional

are

DNA or DNA uniformly

Thus we conclude of

very

the 5. cohnii

have a nuclear

there

membrane

(a) chicken

chromatin

thickening. supporting

covered

ions

for

of the dinoflagellates

(13),

or other

about some

and to the

of nucleosomes in proteins

or

as a thread

benzalkonium

of 5

1334

appears

allowing

are no nucleosomes

and a relatively

chromatin

After

characteristic

in the chromatin position

of

(12)

and

respectively.

repeating

units

cohnii. is not enormous

clear.

amount

Since of DNA,

BIOCHEMICAL

Vol. 88, No. 4, 1979

including

reiterated

may represent

a class

intermediate survey

sequences,

between

but

involved

in the

genes.

not -~ G. cohnii determine

if

Thus

exhibit

it

appears

At least

would

another

some of these

has intervening

sequences

the mRNA of --G. cohnii

an extensive that

is

of intervening

sequences

are presumably

of heterogeneous

be interesting

they

properties

feature

the presence

and maturation it

Although

that

is

or nucleosomes,

some ancestral

and eukaryotes.

condition

in the processing

[HnRNAl to mRNA.

that

prokaryotes

of the eukaryotic

sequences

have no histones

of organisms

has not been undertaken

diagnostic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nuclear

to ascertain

RNA

whether

or

and HnRNA, and additionally

to

is polyadenylated.

Acknowledgments This tutes

work was supported

of Health

in part

by a grant

from the National

Insti-

[GM24295].

References 1.

Dodge, J.D. (1966) Ltd., London.

2.

Kubai,

3.

Rizzo, P.J:and 402-414.

4.

Hamkalo,

5.

Allen, J.R., 161-169.

Roberts,

6.

Wintersberger, 2, 123-130.

U.,

7.

Elgin, S.C.R. 725-796.

8.

Cordon, J., Engelking, H.M. and Pearson, Sci. (Wash.) 13, 401-404.

9.

Searcy,

D.G.

10.

Cleaver, 124.

J.E.

D.F.

and Ris,

The Chromosomes H.

Nooden,

(1969) L.D.

B. and Rattner,

T.M., Smith,

A.R.

H.W.

(1975)

Ann.

Biophys. 60,

Acta

Acta

(1972)

Biochim.

395,

(1976)

-'349

39-47. L.C. Eur.

Rev. of Biochem.

G.D.

Arnold

508-528.

K. (1973)

Biophys.

1335

96-115,

and Klotz,

P. and Letnansky, H.

pp.

40,

Chromosoma

Loeblich,

Biochim.

and Boyer,

Biol.

Biochim.

(1977)

and Weintraub,

(1975)

J. Cell (1974)

J.B.

of the Algae.

Proc.

Cell

5,

J. Biochem. 44, Nat.

Acad.

535-547. Biophys.

Acta

262,

116-

Vol. 88, No. 4, 1979

L.

BIOCHEMICAL

11.

Lerman,

12.

Olins,

13.

Vollenweider, Sci. (Wash.)

14.

Noll,

15.

Newman,

16.

Blin,

17.

Varshavsky, A-J., Res. 3, 447-492.

18.

Thoma,

19.

Marian,

A.L.

(1973)

Cold

and Olins,

D.E.

H-J., Sogo, 72, 83-87.

M. (1974)

Nature

S. and Noon,

N. and Stafford,

Harbor (1974)

J-M.

251,

D.W.

Th.

and Isenberg,

Symp. @ant. Science

Biol.

183,

and Koller,

Th.

38,

59-73.

330-332. (1975)

Proc.

Nat.

Acad.

249-251.

P. (1978)

Bakayev,

R. and Koller, J.K.W.

Spring

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Nut.

(1976) V.V. (1977) I.

Acid Nut.

Res. Acid

and Georgiev, Cell (1978)

1336

12,

(in Res. G.P.

press). 2, 2303-2308. (1976)

Nut.

Acid.

101-107.

Biochemistry

E,

3825-3833.

The mesokaryote Gyrodinium cohnii lacks nucleosomes.

BIOCHEMICAL Vol. 88, No. 4, 1979 AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 27, 1979 Pages 1329-1336 THE MESOKARYOTE GYRODINIUM COHN11 LACKS NU...
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