Incorporation of thymidine into chloroplasts of Spirogyra

Vol. 1, No. 3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMLJNICATIONS

Sept. 1959

INCORPORATION OF THYMIDINEINTO CHIOROPLASTS OF SPIROGYRA C. R. Stocking and E. M. Gifford, University

of California,

Jr.

Davis, California

Received August 15, 1959 One area of uncertainty interaction synthetic tigate

in a consideration of the nuclear cytoplasmic

in green plant cells is the possible role of nucleic acids activities

of chloroplasts.

Three methods have been used to inves-

the nucleic acid content of chloroplasts:

spectrophotometry (Frey-Wyssling, staining methods (Metzner,1952 isolated chloroplasts

4 Littau,

1958);

micro-

(2) cytological

(3) chemical analysis of

(Jagendorf and Wildman, 1954 ; Sisakjan and Odintsova, Although serious objections have been

raised against each method, small but significant

preset

(1) ultraviolet

Ruth, and Berger, 1955);

.1956 ; Chiba and Sugahara, 1957).

ribonucleic

acid (DNA) and ribonucleic

in chloroplasts

amounts of both deoqy-

acid (RNA) have been reported to be

by Metzner (1952); Frey-Wyssling,

Ruth, and Berger

(1955); Sisakjan and Odintsova (1956); and Chiba and Sugahara (1957). Investigators

have failed

(Jagendorf and Wildman, 1954; Littau,

With the ready availability

tissue of plants (Taylor, concentration

of tritiated

on the incorporation

on animal tissue (Friedkin,

thymidine,

1958).

numerousstudies have

of this nucleoside into DNA. Reports

Tilson, and Roberts, 1956) and on meristanatic Woods, and Hughes, 1957) indicate that in low

C14-labeled and H3-labeled thy&dine

is utilized

effectively

the biosynthesis of DNAwithout appreciable diversion of radioactivity other compounds. We have used H3-thy&dine

filaments were cultured Inthe 159

in

Into

to study the possible synthesis of

DNAin chloroplasts.

ActiwlpgrowingSpirogyra

Other

to shcm the presence of appreciable amounts of

nucleic acids in chloroplasts

been made recently

in the

Ughtinhalf

Vol. 1, No. 3

strength lengths

BlOCHEMlCAt

Hoagland's

solution.

The filamentswere

of time in nutrient

solution

ml. (Schuarr Laboratories, 3x10-%thymidine.

propionic

Inc.).

The label

the molecule.

exposure,

the slides

Harris's

hematoxylin.

of %thymidine concentration

indicate

to the slides,

contamination

to the absorption

of thy&line.

Spirogyra

in which bacteria

would be trapped,

under oil

failed

were inbedded radiograms

to reveal

in paraffin

staining

bacterial

and sectiaaed

separated froathe activity

observations

The autorsdiographs

prior

eentrated

cells.

to autoradiography,

uas not confined

millimeters

Fbrther,

wall

contained

the

of

in which the protoplast

had

shwed the radio-

fntothe it

cytoplasmrather

ir evident

in contacttiththe

of the experl.wnt than Into the nuclei

that the radioactirity

fiikuas

(Fig.

or about one third

Is con-

1, Table 1).

approximately

(0.32 111 x 0.05 em) while that of the chloroplast

contained

the auto-

and not in or on the walls.

with the chloroplasts

ly 0.005 square milliwters ohloroplast

of whole cells

filaments

to the cell

contamination

showed that under the conditicms

in assooiation

aperage cdlst~faoe

When the algal

thefixatiarprocess

the thymid3.1~ was incorporated of Spirogyra

obsercontribute

was present in the protoplasm

cellwallduring

only in tbs protoplasm

the follwing

does not have the type of sheath

contamination.

but the radioactivity Simile

was made with

did not significantly

surface as would have been the case if bacterial

Spirogyro.

a two week

and a study of the prepared material

showed that the radioaotitity

radioactivity,

and water,

and after

Post development

that bacterial

of

Kodak (AR-lo) auto-

--Resulta andDiscussion Although the alga was not grown in sterile culture, vations

of about portion

with alcohol

with Haupt's adhesive.

were developed.

per

were fixed in formaldehyde-

washed repeatedly

was applied

for various

in the pyrimidine

the filaments

and mounted on microscope slides film

10 p

This gave a final

fixative,

stripping

incubated

containing

was contained

After exposure,

acid-alcohol

radiographic

Sept. 1959

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the surface.

between 81 to 94 par cent of the total

The 0.016

square

was appraximateHowever, the radioactivity

Vol. 1, No. 3

BIOCHEMICAL

found in the cell

Sept. 1959

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Table 1). Table 1

Intracelltllar

Dietributicm

Spiro-

of Radioactivity

Cells Treated with I$-thynidine

lVu&er ofsilvergrainereducedin emulsion wer cell part indicated

cell NO.

ITzzr HhJQwLdine

Chloroplasts

Honplaetiid cytoplasm

XE* 22 hrs: 22 bra.

;ii 392 4l8

li%

1 :

4

in

ii

The nuclei do not becomepreferentially

"

Nuclei

% of total activlt.7 found in chloroplaets

8 12 0 9

labeled even after

2 E 95 hours of expo-

mre of the filaments to thymidirre.

. .:..:: ::. : . ..: 6 . ‘.., :.. .: ‘... .‘. ‘..

0’

‘.

.

.x5;. .:..

.;, ‘. .‘10, . . .:.. ..o. .

..,p ,’ .,.I.’ . 9. .& .‘:’ .

B

.

Fig. 1.

Tracing of a photograph of parts of two Spirogyra cells treated with H3-thymidine for 24 hours and then for 22 hours in unlabeled thymidine. Pyrenoids in the spiral chloroplasts are indicated as clear circles, the nucleolus is shaded with diagonal lines, and the radioactivity is indicated by small dots in the positions where silver grains appeared cm the autoradiograph. In order to test the nature of the thymidine uptake, large aelled

Spirogyra filamnts

were transferred

after incubating in nutrient diffusible nutrient

to half etrength Hoagldte

solution containing H%hymidine.

fhymidine in the cells was determjned by radioactive solution with a scintillation

counter (Fig. 2). After 161

eolutia The freely assay of the 30 minutes the

Vol. 1, No. 3

filemente (3 x 10%)

BIOCHEMICAL

AND BlOPl$YSlCAL

were transferred

to nutrient

RESEARCH COMMUNICATIONS

solution

and the exchangeable thy&dine

containing

Sept. 1959

unlabeled

thymidine

detemdned.

20I!! 2 iz e :!

CXOIWWEABLE

TtlYYDIYE

--a --0

YETABDLIZED

0

TIML IN h WAOLAND’S 0 IO 20 40 III I I

III h4::”

!‘2’l%i!&“D’S

SDLN.+

UNLABELED 00 MIN. I

THYMIDINE TMYYIDIWE 24 MS. 1

SOLN.

Fig. 2. Relative amounts of free thymidine, readily exchangeable thymidine, and nonexchangeable thymidine in Spirogvra treated with 10 )ac d-thymidine per ml. for 48 hours.

Figure 2 shows that apprmlmately corporated

into the filamente

half

of the radioactivity

and not loosely

was not exchangeable with unlabeled

thymidine

metaboliced

into nualeic

not readily

removed b the ione in the rmttient

that wao in-

held in the apparent free space and preeumably had Men

acid while about half wae held in a form that wae solution

but wae easily

exchangedwithunlabeledthymidine. One interesting

feature

of Spirogyra

with the usual nuclear Feulgen stain for

is that it does not readily deomiboee

lb2

. Since this

stain

investiga-

Vol. 1, No. 3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sept. 1959

tion was undertaken, Drachet (1959) has reported that short time exposure of the unicellular

green alga Acetabularia

to thymidiue results in the incOrpora-

tion of thymidine “into the cytoplasm (apparently this alga also, no detectable

in the chloroplasts).n

amount of Feulgen positive

In

material was found.

Plaut and Sagen (1958) report that the nucleus of Amoebaproteus gives only a faintly

positive

Feulgen reaction and in this organism thymidine ia in-

corporated into the cytoplasm, The failure

of the nuclei of Spirogyra to stain readily

test (under conditions positive

reaction)

in which nuclei of higher plant cells give a strong

and the incorporation

rather than specifically nucleic acid metabolism

of thymidine into the chloroplaats

into nuclei of Spirogyra could point to a peculiar in this alga.

Although the specificity

of thymidine for DNAsgnthesis has been demon-

strated in higher plants and in aniarlr, thymidine as an absolute specific primitive

one must use caution in regarding

DNAprecursor when dealing with these more

organisma. Before one can conclude positively

specifically

with the Feulgen

formed in the chloroplasta

that DNA is

of Spirogyra further

carried out on the nature of the radioactive

work must be

material that is accumulated

in these chloroplasta when the cells are fed ff&hymidine.

Brachet, J.

(1959).

References New observations on biochemical interactions

nucleus and cytoplasm in &noeba and Acetabularia.

Exptl.

between

Cell Research

Suppl. 6:78-96. Chiba, Y. and Sugahara, K.

(1957).

The nucleic acid content of chloroplasts

isolated from spinach and tobacco leaves.

Arch. Biochem. and Biophys.

71:367-376. Frey-Wyssling,

A., Ruth, F., and Berger, X.

(1955).

Monotrope Plastiden-

Metamorphose. Protoplasm 45:97-114. Freidkin,

M., Tilson, D., and Roberts, D.

(1956).

Studies of desoxyribo-

nucleic acid biosynthesis in embryonic tissues with thymidine-C 14. J. Biol.

Chem.202:627-637. 163

Vol. 1, No. 3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Jagendorf, A. T. and Wildman, S. G. VI.

Centrifugal

fractionation

(1954).

Virginia

C.

higher plants. Metzner, H.

(1952).

(1958).

st udy of the chloroplasts in some

Cytochmnische Untersuchungen iber das Vorkommenvan

(1958).

plasm of Amoebaproteus.

Biol.

Zentr. 71:257-272.

Incorporation

of thymidine in the Cyto-

J. Biophys. Biochem. Cytol. 4:843-846.

Sisakjan, N. M. and Odintsova, M. S.

(1956).

changes during plant development.

J. H., Woods, P. S., and Hughes, W. L.

duplication

29:270-279.

Am. J. Botan. 45:45-53.

Plaut, W. and Sagen, L. A.

Taylor,

Plant Physiol.

A cytochanical

Nukleisacren in Chloroplasten.

and its

The proteins of green leaves.

of tobacco leaf homogenatesand some

properties of isolated chloroplasts. Littau,

Ribonucleic acid of plastids Biokhimiya 2l:598-605. (1957).

The organization

of chromosomesas revealed by autoradiographic

tritium-labeled

Sept. 1959

thymidine.

hoc.

studies using

Nat. Acad. Science 43:l2.2-128.

164

and