Translation of homologous RNA injected into the ciliate Stylonychia mytilus

BIOCHEMICAL

Vol. 80, No. 4, 1978 February 28,1978

TRANSLATION

OF HOMOLOGOUS RNA INJECTED

CILIATE

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 897-904

STYLONYCHIA 1 , H.J.

J.K.C.

Knowles

Tnstitut

fur

versitgt

Tiibingen,

11ecember

7,1977

MYTILUS

Lipps

Biologie

III,

INTO THE

2 and A. Neck Abt.

Zellbiologie,

D - 74 Tiibingen,

Uni-

W - Germany

Abstract:

Evidence is presented that RNA isolated from becomes translated when injected back into cells and that the RNA, synthesized about er interaction of Stylonychia with Con A contains sequences specifically concerned with the first regeneration steps after Con A damage. The use of microinjection combined with a homologous system is discussed,

Translation

of

lation

systems

locyte

system

isolated such (2)

providing

evidence

messenger

RNAs.

the

mit

proteins usually gical ted

give

ciently ly,

while

this

viral

messenger

It

RNAs are

and avian duck

respect

proteins.

into

of translating

with

globin

may perof it

has been

(3)

culture

messenger RNA (5)!.

and,

In

these

biolo-

very

effi-

more

recent-

are

capable

as well

as

experiments

1) Present address: Institute of Animal Genetics, burgh, Scotland 2) To whom correspondence should be send

897

not

demonstra-

cells RNA (4)

the

does

to the

translated

oocytes tissue

of these

technique

message,

reticu-

technique

content

by a particular

many messenger

envelope

information

trans-

or the

(1)

identification

of these

vitro

to be a powerful

any information

human

germ

and subsequent

when injected

that

the

RNAs by in

wheat

has proved

However,

for

function that

as the

of

synthesis coded

messenger

Edin-

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 80, No. 4, 1978

the

information

We have living

content

used cells

function liate

technique

of

injection

in

an attempt

to

identify

RNA synthesis tin

be involved surface

cell

untreated

pellicle

the

repair

RNA from control

cells

we present

plant

into

and that

required

with

actinomyRNAs might

to the

we have

cell

prepared

(Con A RNA) and

RNA) and injected with

that

translated

these the

in

the-

Con A and in which

inhibited.

become

for

binding

an increase

cells

evidence

cells

after

hypothesis

strongly

by Con A treated

information

ci-

the

the

synthesized

treated

In

cells

newly

(control

had been

other

after

studies

Con A treated

Stylonychia

paper

one hour

of Con A damage

this

RNA synthesis this

with

of Stylonychia

these

To test

se RNAs into

biological by the

treatment

Inhibition

that

in

(6).

whole

about

occurs.

D suggested

the

A (Con A).

shown that the

after

known.

of RNA into

of RNA synthesized

mytilus

Concanavalin

of Con A to

RNAs was already

the

Stylonychia

has been

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

of new classes

lectin It

BIOCHEMICAL

RNAs synthesized when injected

RNAs provide

first

regeneration

specific steps

Con A damage.

MATERIAL

AND METHODS

RNA from whole cells was prepared essentially by the methods of Parish and Kirby (7) and Loening (8). Control RNA was isolated from logarithmically growing cells and Con A RNA from similar cells treated for 45 to 60 min. with 4 ug / ml Con A. The RNA was separated on 2.4 % polyacr 4 lamide gels according to the method of Loening (8). As shown in Figure 1 the densitometer tracings of both RNA preparations are very similar. RNA for injection was dissolved in neutral Pringsheim solution (9) at a concentration of 2.5 mg / ml. Cells for injection were treated with 75 ug / ml actinomycin D for several hours. Pulse label ex b eriments showed that this treatment is sufficient to inhibit more than

898

BIOCHEMICAL

Vol. 80, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

: Densitometer tracings of control RNA (Fig. 1 a) A from Con A treated cells (Con A RNA) (Fig. a b). RNA was prepared and separated on polyacrylamide gels as described in material and methods,

w

90 96 of RNA synthesis. St lon chia survives at this concentration without apparen wfects for at least 48 hours. Just prior to injection cells were treated with 10 /ug / ml Con A for 30 min. to make sure that all Con A receptors are saturafed after that time. Each cell (ceQ8volume about 100 /um ) was then injected with 2.5 x IO mg RNA of either Con A RNA (three different preparations) or control RNA (three different preparations) using the microinjection technique of Knowles (10). Surviving of cells after injection was observed in Pringsheim medium containing 25 /ug / ml actinomycin D.

RESULTS

AND DISCUSSION

Con A treated hibited, tion I)

die of

the

cells, after

in which some hours

Con A and actinomycin injected

RNA is

RNA synthesis depending D. If

translated

it

has been

on the is

899

concentra-

assumed

and 2) the

in-

that

RNA synthe-

BIOCHEMICAL

Vol. 80, No. 4, 1978

sized

3.0 to 60 minutes

with

recovery

with

Con A RNA would

similar

from

cells

The results

cant

in

individual

(Fig.

There

is,

time

injected

test tion,

but

cells

after

with

20 hours

but

injected

were dead

dependent

all

1 o-8

mg per

rent

RNA preparations

2 d). control that later

RNA occured of

cells

(Fig.

cells

maintained

ter about

injection half

at

survival

rate their (Fig.

of the

control

after

the

RNA and The grea-

after

injec-

still

a

more

injected This

6 hours

2 c).

4 to 5 96 of than

and un-

difference below

between

was 0.5

x

the

two diffe-

significant

(Fig.

injected

with

injection

but

Con A RNA was about

2 hours

correlation

diffe-

3).

was noted: natural

4 a) before cells

is

for

both

of cells

with

(Fig.

between

addition,

rate

morphological

in

In

difference

2 a - c, Fig.

rence

buffer

of RNA injected;

injected

The following

uninjected

6 hours

15 hours.

death

of cells

2 a - c).

was no longer

The maximum

is no signifi-

there

cells,

after

the

are

but

Con A RNA survived

amount

cell

experiments

survival

with

observed

3).

control

on the

than

difference

injection

of 11 96 (Fig.

injected longer

with

injected

(26 %) is

injected

of

Con A RNA (Fig.

12 hours

difference

time

injected

cells

with

difference

of

RNA and treated

of

concerned

RNA,

a significant

survival

survive

sets

the

or cells

however,

of

those

2 b),

cells

2 and 3. There

between control

to

control

figures

is

treated

be expected

three the

with

cells

Con A damage,

with

difference

injected

Con A treatment

injected of

summarized

after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to this

Almost form they

injected

with

900

for

all

control

some hours

lysed

af-

whereas

Con A RNA became

Vol. 80, No. 4, 1978

BIOCHEMICAL

AND BtOPHYSlCAL

RESEARCH COMMUNICATIONS

rate of injected cells. Cells for intreated as described in material and methods. Fig. 2 a - c show the survival rate of cells injected either with three different Con A RNA preparations (o0) or injected with three different control RNA preparations ). To exclude the possibility that cells die ( because of injection treated but not injected cells were included (Fig. 2 b x-----x) as well as cells injected with buffer alone (Fig. 2 c, x-----x). Fig, 2 d showSthe survival rate of cells injected with about 0.5 x 10 of either Con A RNA (the same preparation as in Fig. 2mE) or control RNA, The number of injected cells was: Fig. 2 a: 92 cells injected with Con A RNA and 96 cells injected with control RNA, Fig. 2 b: 80 cells injected with Con A RNA and 90 cells injected with control RNA, 48 cells were treated with Con A and actinomycin D but were not injected, Fig. 2 c: 104 cells injected with Con A RNA and 120 cells injected with control RNA, 60 cells were treated as described and injected with buffer alone, Fig. 2 d: 82 cells injected with Con A RNA, 78 cells injected with control RNA.

rounded cells

and shed survived

normal. tm

In

cells

ded pellicle

the able

their

cirrae

much longer course to

(Fig. than

those

of regeneration

synthesize

and become

4 b).

round,

RNA shed

which

remained

after

Con A dama-

their

Con A loa-

so reducing

901

The rounded

external

cell

BIOCHEMICAL

Vol. 80, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 3 shows the increased survival rate of cells inJetted with Con A RNA relative to cells injected with control RNA. The standard deviation is calculated from the experiments summarized in Fig. 2 a - c.

hFiionurewi 4:h

a) A micrograph of a cell 2 hours after injeccontrol RNA. This cell shows still a "normalfl form (x 425), b) A micrograph of a cell 2 hours after injection with Con A RNA. The cell has shed most of its cirrae and become rounded as do cells treated with Con A but not with actinomycin D. A new membrane is clearly visible at this stage (x 425),

surface

(11).

RNA behave Analysis

In

in of

this

an analogous

the

likely

that

cells manner

RNA preparations

DNA and no detectable fore

respect

the

amounts agent

injected

with

to regenerating showed only

of protein.

responsible

902

Con A

traces

It for

cells.

is the

thereincrea-

of

Vol. 80, No. 4, 1978

BIOCHEMICAL

sed resistance

to Con A damage

RNA, Although control

the

RNA are

be explained treated

jected

with

Cells

injected

nerate

their

complete

injected

control

RNA.

with

actinomycin

after

but

also

new pellicle

Our results

suggest

liate

Stylonychia

codes

for

feel

that

a homologous useful tion case,

tool

the

of

technique system in

the

of messenger the

that

individual

not

Con A was

more

expected

to

20 hours

in non-

than

inhibited

Con A application able

regeof a

cells

not

in-

resynthesis

with survive

to regenerate

some RNA synthesized early

which

ges of regeneration

just

a

(6).

mytilus

proteins

can not

Con A RNA and cells

Furthermore,

are

Con A RNA and

results

since

takes

D one hour

treatment

complete

with

entirely

(11).

of

injected

RNA concentration

Con A RNA are

pellicle

the

of new r - RNA to

injected

cells

cells

our

addition the

indeed

pattern

similar,

new pellicle

inhibited

this

very

since

in

is

densitometer

by the

cells

identical

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are the

after

required

cell

by the

Con A treatment in

the

early

from

Con A damage.

of microinjection

combined

as described

here

identification

can serve

of the

RNAs in particular RNA species

not

staWe with

as a

biological

when, have

ci-

func-

as in yet

this

been

isola-

ted. This work was supported by the Deutsche Forschungsgemeinschaft

VW - foundation

and the

REFERENCES Marcus, A. (1974) Methods Enzym. 30, 749 - 761 2) Pelham, H.R.G. and Jackson, R.J. (1976) Eur. J. Biothem. 67, 247 - 256. 3) Gurdon, J.B., Lane, C.D., Woodland, H.R. and Marbaix, G. (1971) Nature a, 177 - 182. 1)

903

Vol. 80, No. 4, 1978

4)

Stacey,

D.W.

BIOCHEMICAL

and Allfrey,

AND BfOPHYSfCAL RESEARCH COMMUNKATJONS

V.G.

(1976)

Cell

53, 725

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Stacey, D.W., Allfrey, V.G. and Hanafusa, H, (1977) Proc. Nat. Acad, Sci. (Wash.) 74, 1614 - 1618. H,J. and Bessler, W.G. (1977) Cell Diff. 6, 6) Lipps, 187 - 197. J.H. and Kirby, K.S. (1966) Biochim. Biophys. 7) Parish, Acta 129, 554 - 562. U.E. (1967) Biochem. J. 102, 251 - 257. 8) Loening, G., v. Berger, L. and 9) Ammermann, D., Steinbrtick, Hennig, W. (1974) Chromosoma (Berl.) 45, 401 - 429. (1974) Exp. Cell Res. 88, 79 - 87. 10) Knowles, J.K.C. Frisch, A., Bessler, W., Lipps, H.J. and Ammermann, D. 11) (1976) J. Protozool. 23, 427 - 430, 5)

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