In, vitro studies of protein synthesis in Drosophila

Vol. 35, No. 2, 1969

8lOCHEMiCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

--IN VITRO STUDIES OF PROTEIN SYNTHESIS IN DROSOPHILA Raymond Rose and Ralph Department Temple

ReceivedMarch

University,

Hillman

of Biology

Philadelphia,

Pennsylvania

19122

17, 1969 Summary

Comparative studies of protein synthesis in Oregon-R wild--in vitro type and A53g mutant Drosophila melanogaster indicate that the A53g genotype has an effect at the translation level. The A53g postmicrosomal supernatant fluid stimulates the incorporatxof cl4 amino acids by cell-free systems using ribosomes prepared from either A53g or Oregon-R wild-type flies. The level of --in vitro charging of Drosophila and calf liver tRNA with Cl4 amino acids by the A53g postmicrosomal supernatant fraction is also greater than the level of charging by the Oregon-R supernatant fluid.

A great mental

deal

of information

biochemistry

the genetic insect.

control

of protein

synthesis

melanogaster. and a series of abnormal

using This

transfer supernatant

using

supernatant

RNA (tRNA)

which

and calf

the

initial

little

--in vitro

level

consists

of a sex-linked

modifiers,

used are:

(fi),

(Hillman,

Drosophila

ribosomes,

fractions;

and 2) --in vitro

fractions.

197

major

gene

the production 1953).

Cl4 amino

endogenous

Cl4 amino

of

in Drosophila

causes

1) a cell-free,

tRNA with

in this

investigations

Abdomen

fly

and develop-

is known about

Abnormal

in the adult

liver

genetics

at the molecular

and autosomal

tergites

techniques

system

and Drosophila

reports

genotype,

abdominal

synthesis

the mutant,

of sex-linked

The --in vitro incorporating

paper

about

but relatively

of Drosophila,

The present

protein

is known

charging acids

acid

messenger, of Drosophila by Drosophila

Vol. 35, No. 2, 1969

--In vitro

BIOCHEMICAL

investigations

have been carried The Tenebrio Ilan

out

system

and Lipmann

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of protein

by Jenny,

et al.,

(1966),

Preparation

and Ilan

of Drosophila

The cell-free Ilan

(1968).

medium, used per pH 7.6;

collected

0.025

suspension

supernatant

M Tris-HCl, at -500C

microsomal

pellets

containing pH 7.6;

0.5 M NH4Cl;

0.035

with

pellets

were Transfer

(Gierer layer

RNA (tRNA)

was separated acetate

of the

The tRNA was precipitated

until

M

the

first

at

The supernatant

overnight

of supernatant by hand 0.01

in

factors.

The

1% deoxycholate

M MgC12;

0.035

containing

M MgC12; for

frozen,

1 M sucrose;

and 0.007

2 hr.

M Tris-

M 2-

Ribosomes

the deoxycholate;

were

the ribosomal

used. from

105,OOOg

by centrifugation was added

Omni-Mixer,

M 2-mercaptoethanol,

0.01

omitting

M Tris-HCl, and 0.005

was dialyzed

at 105,OOOg

was prepared

1956)

0.035

was

The post-microsomal

on a buffer

pH 7.6;

at -5OOC until

and Schramm,

Potassium 2%.

frozen

2 hr.

M 2-mercaptoethanol;

buffer,

One gm of flies

used.

20 minutes.

for

and 0.005

and centrifuged the above

for

of

meal-Karo-agar

and centrifuged

homogenized

M Tris-HCl,

on corn

in a Sorvall

used as a source gently

by

by the method

M 2-mercaptoethanol;

centrifugation

and 0.5 M NH4C1, layered

mercaptoethanol, washed

until

0.007

0.007

at 27,000g

this

prepared

contained:

cheesecloth

pH 7.6,

were

which

at 105,OOOg

from

and stored

HCl,

then

centrifuged

fraction 0.01

buffer

through

and investigated

raised

homogenization

20 minutes,

was then

were

at -500C until

M MgC12;

was filtered

(1965).

Fractions

medium,

Following

20,OOOg for

against

0.01

et al.,

AND METHODS

had been

and stored

M KCl;

phenylthiourea.

fluid

which

3 ml of homogenizing

and Fox,

in Drosophila

(1968).

of Drosophila

flies,

systems

described

Subcellular

fractions

Adult

were

(1962),

has been extensively

MATERIALS I.

synthesizing

its

Drosophila

by phenol

supernatant

fluid.

and extracted

twice

concentration

in

by the addition

198

treatment The aqueous

with

ether.

the solution

of 2 volumes

of cold

was ethano 1.

Vol. 35, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The tRNA was stripped pH 10.0

for

dissolved the

60 minutes

and stored

tRNA was purchased Ribosomal

assuming

24 optical

II.

Cl4 Amino Acid

mg/ml

ribosomal

following

which

trichloroacetic filtered

units

system

acids,

0.06

ti

out

Millipore were

in a liquid

scintillation

Aminoacylation

of tRNA

2.4 mM ATP; 0.2 mg tRNA; Incubations

stopped

by the addition

Protein

concentrations

washed

that

6 ad+l MgC12;

and

0.5

kinase;

0.2 pC Cl4 amino

acid;

2

and 0.2 mg supernatant varying

to 9OoC for

which with

of Ilan

pH 7.6;

by the addition

of time,

lengths

of 5 ml of 5% 20 minutes,

have an average

pore

40 ml of 5% TCA, dried,

diameter and

spectrophotometer.

reaction pH 7.0;

mixture

carried

contained,

10 mM MgC12;

0.1 uC Cl4 amino

were

spectrophotometrically,

60 pg pyruvic

was heated filters

tRNA charging

30 mM imidazole,

of Warburg

mixture

at 30°C for

This

The filters

Ohio.

the reaction

ribosomes;

acid

protein.

ml,

each;

was stopped

The --in vitro

essentially

of 0.20

the reaction

counted

ml:

is

8.0 mM phosphoenolpyruvate;

(TCA).

Falls,

(1951).

30 mM Tris-HCl,

Jl.

of 0.10

et al.,

calf

System

volume

of 0.45

III.

Stripped

measured

at A26o per mg RNA.

of Lowry,

was carried

through

KOH.

were

RNA (r-RNA) of Drosophila

Incubation

over

against

of cold

tRNA concentrations

amino

protein.

2 volumes

by the methods

In a total

19 cl2

with

measured

Incorporating

mM GTP; 2.0 &I ATP;

overnight

were

8 mM dithiothreitol;

46 mM KCl;

pH 6.7 and dialyzed

Chagrin

incorporating

(1966).

tRNA was

Biochemicals,

by the method

The cell-free

in 0.5 M Tris-HCl, reprecipitated

at 4'C in vacua,

density

determined

contained:

buffer,

from General

(1941);

were

Lipmann

The resulting

RNA concentrations

and Christian

then

at 35OC.

by incubation

The tRNA was precipitated

dried,

liver

acids

in 0.05 M phosphate

same buffer.

ethanol,

of amino

acid;

5% TCA.

199

2.4 mM dithiothreitol;

and 0.1 mg supernatant

out at 30°C for

of 5 ml of cold

in total

30 minutes, The mixture

then was then

volume

Vol. 35, No. 2, 1969

filtered cold

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

through

Millipore

5% TCA, dried,

filters,

and counted

which

were

washed

with

40 ml of

as above,

RESULTS AND DISCUSSION A typical

time

TCA precipitable in Figure fluid,

1.

curve

material

for

the

incorporation

by the Oregon-R

Incorporation

is

dependent

as shown by the difference

ribosomal

fraction

Supernatant

fraction

alone

without

alone

(Ore-R) upon

between the addition

has no activity.

of Cl4 leucine wild

type

the addition

the complete

into system

of the system

of supernatant The rate

5 INCUBATION TIME

10

15

( MIN.

fluid.

of incorporation

20

) AT 30bC

Fig. 1. Time curve for the incorporation of Cl4 leucine into hot trichloroacetic acid (TCA) precipitable material by Drosophila (Oregon-R) cell-free system as described in Methods. Complete system ( l ); ribosomes minus supernatant ( 0 ); supernatant without ribosomes ( W ).

200

is

shown

supernatant

and the

t 0

hot

Vol. 35, No. 2, 1969

6;; 0

0 A 0

10

5

INCUBATION TIME

II -1

_----

___A-___________

----

,=.s=====‘--

A

20

15

( MIN.

) AT 30°C

of time curves of incorporation Fig. 2. Comparison of Cl4 leucine into hot TCA precipitable material by cell-free systems of Oregon-R and A53g Drosophila. Oregon-R ribosomes plus Oregon-R supernatant ( l Oregon-R ribosomes plus A53g supernatant ( 0 ); A I' jg ribosomes plus Oregon-R supernatant ( A ); A53g ribosomes plus A53g supernatant (A ). Oregon-R ribosomes only ( 0 ); and A53g ribosomes only ( n ).

by the complete decreasing

system

Supernatant incorporation

for

of incorporation

and A53g flies

incorporation

linear

5 minutes

and then

proceeds

at a

rate.

Comparison type

is

is depicted

is noted fluid into

by extracts

from hot

with

in Figure

regard

the mutant

prepared 2.

to the

TCA precipitable

material

of ribosomes.

201

A marked

source

preparation

from Ore-R

wild

difference

of the

stimulates regardless

supernatant

in fractions

Cl4 leucine of the source

Vol. 35, No. 2, 1969

BfOUiEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1

STIMULATION OF Cl4 AMINO ACID INCORPORATION BY A53g SUPERNATANT FRACTION Incorporation Supernatant A53g

Fraction Ore-R

Ribosomes Ore-R A53g.

A53g

Ore-R

CPM/mg rRNA Phenylalanine

5,723

6,120

3,331

3,343

Leucine

7,389

11,366

3,394

3,161

Proline

4,722

4,508

2,530

2,958

14,767

17,596

9,275

11,042

5,923

7,719

2,319

2,462

Glutamic

Acid

Valine

Incubation at 30°C for 15 minutes as described in Methods. Supernatant fractions from Ore-R or @, 0.2 mg protein, were added to each tube which contained either Ore-R or A53g ribosomes, 2 mg rRNA/ml, as indicated, and 0.2 pC of the Cl4 amino acid to be tested.

The results acids

by this

of incorporation system

by the mutant

are

supernatant

studies

summarized

using

in Table

fraction

five

1.

different

Stimulation.

can be seen for

all

Cl4 amino of incorporation

five

Cl4 amino

acids

tested. Since the

the

stimulatory

supernatant

supernatant tRNA with

fraction, fluids

were

Cl4 leucine,

A53g supernatant tRNA to a greater Comparative

fraction extent studies

effect rather tested

of the mutant than

with

charges than

does

the ribosomes, regard

as described

both

calf

the Ore-R acid

202

wild

confined

ability

Table

liver

is

to

the A53g and Ore-R

to their

in Methods.

of Cl4 amino

system

to charge

2 shows that

the

tRNA and Drosophila type

incorporation

supernatant into

hot

fluid. TCA

Vol. 35, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2

STIMULATION OF CHARGING OF DROSOPHILA AND CALF LIVER tRNA WITH Cl4 LEUCINF, BY A53g SUPERNATANT FRACTION

Incorporation Supernatant Ore-R

Drosophila

tRNA

Fraction A53g

5,031

10,138 (129)

(120) Calf

Liver

tRNA

3,833 (110)

8,746 (109)

Incubation conditions as described in Methods. Supernatant protein, 0.1 mg, from Ore-R or As3g was added to each tube as indicated. Drosophila or calf liver tBNA, 0.2 mg per tube, was used as shown. Numbers in parenthesis represent controls to which no tRNA was added and are expressed as CPM per incubation tube.

precipitable there

is

material a difference

is confined indicate level

by extracts in

of Ore-R

incorporation,

to the post-microsomal that

the A53g mutant

and that

this

effect

of the post-microsomal Work is continuing

is

Acknowledgement.

The authors

assistance

Joseph

of Dr.

and that supernatant

affects

protein

involved

supernatant to further

and A53g flies

with

fluid

define

this

gratefully

the fluid.

the ability

203

shown that

of this

difference

results

at the translational of the constituents

tRNA with

amino

effect. acknowledge

Ilan.

source These

synthesis

to charge

have

the advice

and

acids.

BIOCHEMICAL

Vol. 35, No. 2, 1969

This

work was supported,

Public

Health

Service

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in part,

by Grant

and by the Temple

# lTl-HD-138

University

from

the Ll. S.

Committee

on Research.

REFERENCES Fox,

A. S., Kan, 2059 (1965).

Gierer,

A.,

Hillman,

.I.,

Kang,

and Schramm, Dros.

Inform.

Biol.

Chem.,

.I.,

J.

Ilan,

J.,

and Lipmann,

Jenny, Lowry,

G.,

R.,

Ilan,

E.,

Hicklin,

S. H.,

A.,

Nature, Ser.,

243,

F.,

Acta

O.,

and Christian,

27, 5859

W.,

177,

B.,

J. Biol.

Chem.,

2,

702 (1956).

56 (1953). (1968).

Biochem.

and Leuthard,

0,. H., Rosebrough, N. J., Chem., 193, 265 (1951).

Warburg,

and Wallis,

Pol.,

F.,

Farr,

Helv.

A. L.,

Biochem.

204

l.3,

Z.,

353 (1966).

Chim.

Acta,

and Randall, 310,

384 (1941).

5, R. J.,

2123 J.

(1962). Biol.