Metabolic conversion of thymine-2-C14 and its incorporation into nuclear RNA of endosperm nuclei of Cocos nucifera . Linn

Vol. 14, No. 1, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METABOLIC CONVERSION OF THYMINE-2-C'* AND ITS INCORPORATION INTO NUCLEAR RNA OF ENDOSPERM NUCFEI OF COCOS NUCIFERA, LINN.* R. Roychoudhury Radiochemical Department

Received

October

Laboratory, Bose Institute, Calcutta-9 and the of Botany, University of Kalyani, KalYani, W. Bengal, India.

21, 1963

While hormone lism

studying

indole

acetic

of endosperm

Linn.)

with

supernatant

5 pmole/ml

MgCl2

ucurie/ml)

25'C in

nuclei

The nuclei

the

0.1

for

method'.

tively,

of the plant

on deoxyribonucleic of the it

from

coconut

C14. very

using

that

nuclear

experiments

young

green

fruits

litres

at pH 6.0

containing

and 10 umole/ml

milk

in a small

lo'%

(0.01

were macerated

and RNA and DNA extracted

and diphenylamine

by Kirby's after

colour

at

sucrose, activity 1A.A at

pmole/ml)

with

iso-

volume

10 pmole/ml

thymine-2-C14(sp.

or without

RNA

were

of coconut

resuspended

a Hilger

nucifera

these

RNA and DNA were estimated

orcinol

(Cocos

metabo-

In

were then

The nuclei

growth acid

was observed

of 2-3

with

12 hrs.

a mortar

with

nuclei

by centrifugation

3000xg. of

acid

effect

considerable

endosperm

lated

the

thymine-2-C14,

had incorporated free

and S. P. Sen.

glass

powder

phenol development

at 66Omp and 6OOmu respec-

spectrophotometer.

Radioactivity

* The following abbreviations have been used : IAA,indole-3 acid; DNA, deoxyribonucleic acid; DNase,deoxyribonuclease. RNA, ribonucleic acid; UMP, uridylic acid. 7

meas-

Vol. 14, No. 1, 1964

BIOCHEMICAL

AND BIOPHYSICAL

urements

were performed

with

a Tracerlab

counter.

It

from

Table

found This

its

is apparent way not

is not

only

the

was radiochemically lysis in

nuclear

in which a single

entire

spot

had

into

RNA.

-2-C 14 with -2-C 14 used

of thymine

activity

which

also

of thymine

as revealed

gas-flow

thymine-&*

DNA but

preparation

pure

windowless

I that

due to any contamination

-C 14 since

uracil

into

RESEARCH COMMUNICATIONS

by chromatographic

was found

ana-

to be concentrated

co-chromatographed

with

authentic

thymine. Table-l

Ai

It

is however

extracted part

along

of the

buffer

with

isolated

and incubated

3 umole/ml fuged

and the

dissolved, specific

that

the RNA.

with

for

this

and counted. of

It

Table-II

8

a

redissolved

in

The contents

were centri-

with times is

clear

the RNA remained

ted.

possibility

containing

24 hrs.

several

DNA was

DNase at pH 7.4

precipitated

was washed

activity

-1abelled

To eliminate

20 ug/ml

supernatant

plated

some Cl4

RNA was reprecipitated,

MgC12 at 28'C

The precipitate

the

possible

4 vol.

with from

of alcohol.

80% alcohol, table

practically

II

that

unaffec-

Vol. 14, No. 1, 1964

To provide resided 0,5N

AND BIOPHYSICAL

additional

evidence

exclusively for

tralised

with in cold.

isopropanol

The acid

KOH and the

that

soluble

fraction

was then

: HC1: H20 (78:20:22). The spot

was eluted

in water

with

at 280 rnp and 260 mu was calculated. which

is

with

was, neu-

ratio

in

radioactive

O.OlN

densities

to 0.30

and the

in

by centrifuga-

A single

dissolved

to be close

activity

chromatographed

in vacua,

found

Cl4

was acidified

KC104 was removed

The hydrolysate

was obtained.

the

The sample

24 hrs.

and centrifuged.

RESEARCH COMMUNICATIONS

the RNA was hydrolysed

in uracil,

KOH at 30°C

HC104

tion

BIOCHEMICAL

HCl,

spot

dried

of optical It

was

characteristic

of UMP.

Table-III Treatment

It lated the

acids

endosperm

nuclei

decreased

on chick

material. embryos

indicate

that

the

has been demethyinto

presence

of uracil

and Reichard'

o f thymidine

thymidine

on liver apparently

consider-

into this

The investigations

Linn.)

of thymidine

of wcereus; conversion

UMP of RNA in

(Cocos nucifera

incorporation

cells

to imply

containing

that

C14-activity cpm/mg UMP.

thymine-2-Cl4

of coconut

the

by intact

that

at 280 m at 260 mn

and incorporated

had observed

interpreted

hand

appears

to uracil-2-C14

Sells2 ably

thus

I).D. O.D.

the nucleic was

to some uracil

of Friedkin

&, Xi3

tissue

on the

other

is not

metabolised

BIOCHEMICAL

Vol. 14, No. 1, 1964

to

compounds

which

RNA. Similar

E.

coli

in

the

may contribute

observations

and Salmonella

tjrphimurium

reverse

stei

direction

This

is

Biswas

in

the

full

synthesis

in

tiles

pea internodes.

as also

into

the RNA of coconut

be enhanced motion

in

the

Whether or the

utilisation

of

IAA favours incorporation

which

enzymological

expected

to answer.

also

release

the

metabo1ism12913'14 acid

and protein

merits

serious We are

ties

P32

of oat

been

is over l*

of

in progress

than

and the its

50%. to uracil, are

at present the

is a possibility

questions

are

synthesis

regulation

effects

to

RNA synthesis

thymine

only

found'l

70%. The pro-

for

is more

not

coleop-

of uracil-2-Cl*

in RNA, or both,

by IAA through synthasis

and rice

has also

here

RNAll

a

and Sen 11 concerning

stimulation

IAA favours

of nuclear

indicate

of

demethylation

studies

to

observstions

nuclei

of uracil

conversion

DNA and RNA by IAA.

of thymine-2-C

the

and in

clearly

Incorporation

IAA as reported

coli

7 or Uraci18yg

the earlier

acids

milk

by IAA and the

in presence

Bose,

nucleic

of

investigators.

of nuclear with

incorporation

=J5

although

above

and Roychoudhuri the

&

deoxyuridine

described

composition

in Escherichia

by several

agreement

and Senl’

the

by Wacke#

i.e.,

The investigations of

towards

by Schaechter

has been reported

stimulation

RESEARCH COMMUNICATIONS

were recorded

and Enterococcus

thymidine

AND BIOPHYSICAL

but of cell

on nucleic which

certainly

consideration. thankful

Read of and to Dr.

the

to Dr. Dept.

B.B.3iswas

D.M.Bose,

of Chemistry for

his

ment.

10

Director, for interest

and Dr.

laboratory

facili-

and encourage-

P.K.

Vol. 14, No. 1, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References: 1. Kirby, K.S., Biochem. 2. Sells, B.H., Biochem. 3. Friedkin, M.; Tilson, 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

J. a, 405, (19.56). Biophys. Acta. 4Q, 548,(1960). D.; and Roberts, D., J.Biol.Chem. a, 627, (19.56). Reichard, P.; and Estborn, B., J. Biol. Chem. QJ$, 839, (1951). Schaechter, M.; Bentzon, M.W.; and MaalBe, 0., Nature 153, 1207, (1959). Deut. Akad. Miss., Berlin, Wacker, A., Abhandl. Kl. Med. 66P (1960). Friedkin, M.; and Roberts, D., J. Biol. Chem. 22Q, 653, (1956). Reichard, P., Acta. Chem. Stand. 9, 1275, (1955). Dinning, J.S.; and Young R.S., J. Biol. Chem. a, 3008, (1960). Biswas, B.B.; and Sen, S.P., Nature 183, 1824, (1959). Roychoudhury, R.; and Sen, S.P., Proc. 51st. Indian Science Congress,Part III (In Press). Thimann, K.V., & Fundamental Aspects of Normal and Malignant Growth. W.W. Nowinski, Editor. Elsevier Publishing Co., Amsterdam, Netherlands, 1960. Galston, A.ti.; and Purves, W.K., Ann. Rev. Pl.Physiol. & 239, (1960). Sen Gupta, A.; and Sen, S.P., Bull. Nat. Inst. Sci. India, U, 70, (1962).

11