Queuosine deficient tRNAHis and tRNAAsp from the spleens of young mice, erythroleukemic tumoral spleens and cultured Friend cells

Vol. 109, No. 4, 1982 December

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

31, 1982

QUEUOSINE DEFICIENT

tRNAHis

1140-1147

AND tRNAASP FROM THE SPLEENS OF YOUNG MICE,

ERYTHROLEUKEMIC TUMORAL SPLEENS AND CULTURED FRIEND CELLS R.M. Landin

(2)

and G. PBtrissant

(1)

Institut de Recherches ScienLaboratoire de Physiologie cellulaire, tifiques sur le Cancer, CNRS, BP No 8, 94802 Villejuif, France

(2)

Laboratoire en-Josas,

Received

de Physiologie France

November

de la Lactation,

CNRZ-INRA,

78350

Jouy-

5, 1982

tRNAs were isolated from the spleens of young mice, erythroleukemic spleens and cultured Friend cells. Queuosine (Q) deficient tRNAs were labelled in their anticodon with radioactive guanine using the exchange reaction catalyzed by E. coli tRNA-guanine transglycosylase. tRNAs were then specifically aminoacylated. In both normal and tumoral spleen (TF-PlO), tRNAHis was the main guanine containing (G) isoacceptor as shown by RPC-5 chromatography. In vitro, the tumor derived cell line (TF-PlOc) retained the G-tRNAHiS species while Friend cell line, clone 707 (FLC), did not. A common feature found in both cultured cell lines was the high level of G-tRNAASp. The meaning of the relative abundance of Q-deficient tRNAHIS and of tRNAASP observed in transformed cells of erythroid origin is discussed. In normal

adult

mammalian

tRNATYr)

tissues

families

tRNAASn,

tRNAASP,

of their

anticodon

(Q-tRNA

or Q*-tRNA).

Unmodified

instead

of queuine

(G-tRNA)

are absent

or represent

increase

in the relative

observed

under

age (2)

We therefore

thought

nature Double with

cells

(8)

it

would

labelling

cells

there

is

Cop.vrighl All rights

0 I982 by Academic Press, of reproduction in any form

Inc. reserved.

1140

,

5' end guanine

species.

An can be

of animal

of significant

amounts

the erythropoietic

period

murine

Friend

between

the

G-tRNA isoacceptor was achieved exchange

catalyzed

(7).

erythroerythroid

distribution. by acylation by the

Friend Leukemia virus ABBREVIATIONS : FLV-P : Polycythemia-inducing TF-PlO : in vivo transolantable tumor induced bv FLV-P : TF-PlOc : in vitro cell line established from the TF-PlO tumor ; FLC : Friend : 7-{[(cis-4,sdihydroxy-2leukemia cells, clone 707. Q (queuosine) cyclopenten-1-yl) amino]-methyl]-7-deazaguanosine ; Q* : saccharide derivative of Q (i.e. B-D-mannosyl Q or f3-D-galactosyl Q). 0006-291X/82/241140-08$01.00/0

His

4, 5, 6).

to examine

L3H]-guanine

minor

as a function

a correlation

G-tRNA isoacceptors

and by the

very

the presence

and the

at the

to Q-tRNA isoacceptors

during

be of interest

to see wether

of specific

liver

tRNA

tRNAs containing

(l),

(3,

we have shown in foetal

of viral-transformed [14C]--amino-acid

in tumors

of tRNA (i.e.

or hexosylqueuine

conditions

noted

report,

lsoacceptor

queuine

of Q-deficient

nutritional

and is generally

of G-tRNAHiS leukemic

amount

defined

In a previous

contain

four

;

BIOCHEMICAL

Vol. 109, No. 4, 1982 E.

coli

tors

tRNA-guanine

out by Reverse

The results

reported

i/

The G-tRNA line

erythroid

paper

isoacceptor

is

the

of normal

the permanent

cell

In murine

virus

(RPC-5). :

common marker

young animals

of tRNA isoaccep-

for

the erythro-

as well

as for

the

spleen. is

and is

enhanced

cells,

G-tRNA

related

to cell

by in vitro

transforma-

establishment

of

lines.

erythroleukemic

80 % of the guanine

MATERIALS

Fractionation

show that

in the FLV-P tumoral Asp The G-tRNA isoacceptor species leukemia

RESEARCH COMMUNICATIONS

Phase Chromatography

in this

in the spleens

by Friend

about

His

enzyme.

precursors

ii/ tion

transglycosylase

was carried

poietic

AND BIOPHYSICAL

exchange

ability

His

and G-tRNA Asp account

of crude

for

tRNAs.

AND METHODS

Chemicals All chemicals were analytical laboratory grade. Acrylamide and bisacrylamide were purchased from Eastman Kodak. RPC-5 column packing was prepared by the method of Kelmers and Heartherly (9). Uniformly labelled [14C]-histidine (300 mCi/mmole), and [14C]-aspartic acid (200 mCi/mmole) [8-3Hj-guanine sulfate (10 Ci/mmole) were obtained from CEA (Saclay, France). was obtained from the Radiochemical Center (Amersham, England). Isolation

of unfractionated

tRNAs

tRNA preparations were carried out as previously reported (10) except for the first step (extraction) which was adapted to the different cell types as follows : a) Spleens

of young mice

(24 days old)

Extraction of tRNA from Balb/2 mouse spleen, still retaining erythropoietic function (80 mg weight), was carried out as usual (10) in a V/V mixture of phenol and of 100 mM, Tris buffer (pH 7.5), 1 M NaCl, 5 mM EDTA (pH 7). b) 1~ vivo mouse, tions supplied resulting of pH EDTA, resulting pitated

transplantable

tumor

(TF-PlO)

An erythroblastic cell line derived from a FLV-P infected DBA/2 isolated and maintained in viva by serial intravenous transplantain supralethally irradiated isogenic recipients (11) was kindly by Dr P. Tambourin (Institut Curie - INSERM - Orsay, France). The enlarged spleens were homogenized in a V/V mixture of phenol and 7.6 buffer containing 50 mM Tris, 20 mM MgCl 200 mM NaCl, 2 mM 0.5 % SDS, 0.3 % macaloid. After shaking for2i0 min at 20°C, the cellular extract was centrifuged and the aqueous layer was preciwith 3 vol. of ethanol and stored at 4OC. c)

Tumor cell

line

(TF-PlOc)

TF-PlOc is a permanent cell line originally established in vitro from the TF-P10 tumor (12). TF-PlOc cells were harvested during the exponential growth phase, 72 h after seeding. Cells were pelleted by centrifugation and washed in Phosphate Saline Buffer (PBS), then they were resuspended at 3 x lo7 cells/ml in the same extraction buffer as for the tumor and similarly processed. d) Friend

leukemia

cell

line

(clone

707-17~)

Friend etythroleukemia cell cultures were cultivated as described by Minty and Gros (13) and were a generous gift from Dr M. Crdpin (Institut 1141

Vol. 109, No. 4, 1982

BIOCHEMICAL

Pasteur, Paris). was as described

AND BIOPHYSICAL

The cells were harvested in paragraph c.

RESEARCH COMMUNICATIONS

at 92 hours

; tRNA extraction

Enzyme preparations a) Aminoacyl-tRNA

synthetases

Preparation of crude aminoacyl-tRNA locytes and adult sheep liver was carried paper

synthetases from out as reported

rabbit reticuin a previous

(7).

b) E. cozi

tRNA-guanine

transglycosylase

Our sample was an aliquot of fraction V from the tion procedure of Okada and Nishimura (14) and kindly Dr S. Nishimura (Nat. Cancer Center Research Institut, Preparation of double column chromatography

labelled

original purificadonated by Tokyo - Japan).

(guanylated-aminoacylated)

tRNA and RPC-5

Dialyzed tRNA aliquots were first labelled with guanine, and then acylated. The guanine assay was performed as stated by Okada et al. (3) (10 Ci/ using l-l.2 A250 units of crude tRNA, 1 nmol [S-3H] guanine-sulfate mmol) and 1.5 unit of E. cozi tRNA-guanine transglycosylase enzyme in 100 ~1 of the reaction mixture. The enzyme catalyzes exchange of radioactive guanine with guanine located at the 5' position of the anticodon of tRNAHiS, tRNAASn, tRNAASp, tRNATyr (14). E. cozi tRNA-guanine transglycosylase has been used previously as a reagent to compare the levels of G-containing tRNAs in different cells and tissues (3). Total guanylated tRNA was isolated by phenol extraction, extensively washed in pH 4.5 buffer by successive ethanol precipitations and finally dissolved in adequate volumes of distilled water for subsequent steps. tRNA aminoacylation was carried out as previously described (7). Crude aminoacyl-tRNA synthetases from rabbit reticulocytes or from adult sheep liver catalyze at the same rate the aminoacylation of tRNAHis ; by catalyzed by the contrast, aminoacylation of tRNA Asp is more efficiently liver enzyme. Specific double labelled tRNA samples were fractionated on RPC-5 column as described in a previous paper (7). Fractionation

of isolated

tRNAs by electrophoresis

The polyacrylamide gel reported previously (7).

electrophoresis

(PAGE)

procedure

has been

RESULTS In table in different

I we compare cells

the

of erythroid

total

amounts

origin

before

of histidyl

and aspartyl-tRNAs

tRNA chromatographic

nation. TABLE

FROM : HISTIDINE AND ASPARTIC ACID ACCEPTANCE OF tRNAs NORMAL MOUSE SPLEEN AND FRIEND ERYTHROLEUKEMIC CELLS

I

tRNA

Aspartic

Histidine

source

pmol/AZgo Normal

Spleen

(24 days TF-P10 TF-PlOc FLc tRNA

reported

old)

aminoacyl (7).

acceptance

acid

units

10

23

12 23 30

13.5 18 40

test

was performed

1142

as previously

fractio-

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL a

1.c

4.0

0.2 0.5

29

0.1

‘F.PlO

-\ 0 E ,"

jpleen

c I

&

.-s ;; 2

RESEARCH COMMUNICATIONS

b

.; ,’ : i:;:: ::::’,‘..i I’: ;j‘I/ ” ) : &%L TF-PI0

I

1;

1.c I-

0.5

n 'u L! g '0 4.c .-2 .-: z I -3 e

-9 F-PlOc

I .-

:,:i:: .: (j. -a .cc . i‘,(

2.0

4.0

: :’)’ 4;,i‘.:. )_): ,:.,: &AL ‘\ LC

:t

1.2

:‘\.. ,.80 : &!A ..’ 1io 100:

2.0

2.0

0.5

0

0,i61I

0

Fraction

0.6

., ,: :,..

60

0

100

number

Figure 1. RPC-5 chromatographic profiles of double labelled tRNAs from spleens of young mice and Friend Erythroleukemic cell lines cultivated in vivo and in vitro. The column (0.7 x 130 cm) was loaded in each case with Q 1 A unit of radioactive tRNA sample. Elution was performed with a linear g% a ient (total volume 200 ml) from 0.4 to 0.75 NaCl in equilibration buffer at pH 4.5. The volume of the fractions was 1.3 ml.

Isoacceptor

patterns

of guanylated

G-tRNAHZSisoacceptor Fig. from

1 shows

different

dyl-tRNA tumoral

tracer, spleen

[3H]

sources.

tRNAs after

pattern guanylated

RPC-5 fractionation

: tRNA profiles

By comparison

with

of double

the profile

we observe that the main G-containing (TF-PlO) is the late-eluting histidyl-tRNA

labelled

of the

[14C]

tRNAs histi-

tRNA in normal and peak (Fig. la, lb

respectively). 1?z vitro peak that leukemia (Fig.

is cells

cultured

cells

the guanylatable (clone

707)

from

the

tumor

isoacceptor do not

contain

Id). 1143

(TF-PlOc) (Fig.

lc).

show a single

tRNAHis

By contrast, Friend His such a G-tRNA isoacceptor

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

2

1

3

RESEARCH COMMUNICATIONS

5

4

- 5SRNA

tRNA I - xc Figure

2. Electrophoretic patterns of different tRNAs on polyacrylamide gel. 1 A2s unit of each tRNA sample (excepting 0.5 A260 unit for lane 4) in 20 ~1 oF the loading buffer was electrophoresed at 4OC in a 9-18 % acrylamide gel gradient (200 x 200 x 20 mm) during 40 h with xylene cyanol FF (xc) as tracking dye. After electrophoresis tRNA bands were stained with a methylene blue solution in 0.2 M acetate buffer. I, mouse spleen (24 days old) ; 2-3, independent pools of TF-P10 ; 4-5, identical pools of FLC.

G-tRNAASPisoacceptor pattern By comparison can see that

only

cultured

the guanylated last

Only

the

le, It

cient

If,

eluting for

(Fig.

a major (Fig.

peak

le)

(TF-PlO).

increased

we In

in the Similarly,

that

corresponds

lh). aspartyl-tRNA

in viva

and in vitro

appears

as a

sources

and lg,

coelute

minor isoacceptors, eventually Q-defithe G-tRNA Asp peak and thus contribute to the

with

tritium

counts. However this 15 % of the GtRNA Asp values

PAGE electrophoresis Aliquots

from Fig.

spleen

total

leukemia

Friend

differentiated

II

gel

gradient the pattern

and Friend

distribution

Table

(results

leukemia

of guanylated

level not

was calculated

order

by electrophoresis

to assess

corresponding cells,

to exceed

tRNA sources

fractionated

in

not

shown).

and in vitro

tRNAs were

2 shows

Relative

results

background

from in vivo

9 to 18 % acrylamide ted tRNAs.

our

spleen

and lg respectively).

non guanylatable both

tracer,

from mouse spleen

significantly

species

tRNA from

acid

lh respectively). has to be noted that tRNA W

(151,

tumoral

If

aspartic

tumoral

of FLC exhibits

aspartyl-tRNA

early

is

(Fig.

[14C]

absent in the

isoacceptor

counterpart

common isoacceptor (Fig.

species

tRNA pattern eluted

of the

tRNAASP is

a minor

the homologous

in vitro to the

the profile

G-containing

and represents contrast,

with

:

clone

the purity

in a of the isola-

to tRNA from mouse spleen, 707.

isoacceptors

in

tRNAs from

Erythro-

cells summarizes

on induced cells

the main results rabbit

presented

reticulocytosis

in the erythropoietic 1144

in this

as an example line.

paper.

We include

of the fully

Vol. 109, No. 4, 1982

TABLE II

8lOCHEMlCAL

AND BIOPHYSICAL

: DISTRIBUTION OF G AND Q (Q*) CONTAINING ISOACCEPTORS FOR tRNAHis AND tRNAAsp IN CELLS OF ERYTHROID ORIGIN Total tRNA guanine acceptance

tRNA source

pmol/A260 Rabbit

mouse

Q$;:fRNA tRNAHis

units

10 5 8

~!t~~~?ocytes(b)

Young

RESEARCH COMMUNICATIONS

spleen

TF-P10

(a)

tRNAASP

0.50 0.80 1.10

< 0.05 < 0.05 ?r 0.10

9.00 < 0.05

0.90 3.0

In vitro 12 18.5

TF-PlOc FLc

(a) Relative amounts of the tRNAHis and tRNAASP were calculated after chromatography on RPC-5 column from the corresponding radioactive (b)

fractions.

use reticulocyte

We

glycosylase DISCUSSION In normal tRNAASn, of the

adult

tRNAASP,

be due to the

lack

Friend

solid reported

of G +Q results

tumor

Moreover

for

of erythroid

represented

clone

(7)

acceptance

tRNAHiS, 5' position

(For

cells

are

the

the

are

last this

tRNA from

in agreement

of total

of Friend

reported

cells

(6).

tRNA pattern

tumoral eluted

with

guanine

same as those

specific

and from a Friend is

by total

the amounts

of guanylated

and extending

spleen

shows that

histidyl-tRNA

observation

confir-

to the transformed

origin. spleen

cells

cultivated

species

contrast,

in FLC, unmodified

GtRNAASP

isoacceptor

in vitro,

in agreement

is

that tRNA His

a marker

with

a quantitative patterns

in vitro,

other

a G-containing

is

poorly

observations,

mainly

isoacceptor.

represented

By

or absent.

Friend well

is

cell

lines

illustrating

The cultured

the

pre-

in malignancy (18, 19) and a Q deficiency from Friend cell cultures (6).

estimation

(See Fig.

histidyl-tRNA

is

common to both

sence of new aspartyl-tRNA species in the anticodon of tRNAs obtained Furthermore

(i.e. at the

has been shown to

the 745 A (DS 9) clone

isoacceptor

as a single

chromatographic

N.4: codon-set

Friend

; also, 707 (17~)

survey

spleen

results

In tumoral

(17)

et ~2. using

main G-containing

cells

trans-

modification

guanine

cultured

et at.

from an erythroblastic the

tRNA-guanine

Q*) nucleosides

post-transcriptional

an analytical

ming previous

the

(7).

of new isoacceptors

and in vitro

obtained

by Shindo-Okada

Q (or

concerning

by Itho

incorporation

for

before

16).

The present those

a control

the tRNAs of the contains

The appearance

see Ref.

as

as described

tissues,

tRNATYr)

anticodon.

review,

cells

reaction

of aspartyl-tRNA

1) shows that 1145

crude

species

tRNAs from

in each of

Vol. 109, No. 4, 1982 the

examined

BIOCHEMICAL

Friend

cell

tRNAASP isoacceptor one half young nent

(Fig.

(Fig.

mice

le).

lines

G-tRNAASp

Cell

results is

age and origin

constant

species

transformation

show that

accompanied

value

found

represents

almost

in the spleens

by FLV and establishment to a gradual

the ratio

and clone

The situation isoacceptors specific

(for histone

the change

by alterations

modifications

of the Friend

between

in TF-PlOc

increase

of

of permain the

cell

of tRNAs. lines

might

of G-containing 707 (Table

in viva

to in vitro

extent

Moreover account

and specificity

culture for

to Q-containing

the

status, differences

isoacceptor

tRNAs

II).

described

in this

a survey

see Table

ratios

from

in the

in Friend

paper II)

for

histidyl

and aspartyl-tRNA

is reminiscent

erythroleukemic

of that

cells

from

found

for

different

(20).

Considering G-tRNAHiS

these

isoacceptor

data is

as a whole

a good

we propose for

marker

the

that

cells

in

in vivo

of erythroid

the origin

II).

The sharp divergence noted in vitro with regard to the distriHis of tRNA species in TF-PlOc and FLC could be due to differences

bution in these

two cells

We also induced (our

This

seems to lead

of post-transcriptional

(Table

lh).

of non guanylatable

species.

The present

sources

lg,

RESEARCH COMMUNICATIONS

amounts

similar

aspartyl-tRNA

in vitro,

conditions

found

contain

If,

of the homologous

cell

cell

lines

AND BIOPHYSICAL

noted

in clone

unpublished

reported

as discussed

a lack

of GtRNA

707 but

with

observations),

a decrease

throleukemic

of all

cells

We conclude of cell

lines

above. His

when DMSO cell

the persistence whereas

differentiation of GtRNA Asp isoacceptor

Shindo-Okada

et al.

G-tRNA species

in differentiated

that

GtRNA

using

is

clone

murine

ery-

(6). therefore

transformation

in vitro,

by erythroleukemic

Friend

Asp

1s

a

molecular

marker

virus.

ACKNOWLEDGEMENTS. We are indebted to Mrs M. Charon and P. Bucau-Varlet (Institut Curie, Orsay, France) for providing the TF-P10 and TF-PlOc lines.

cell

REFERENCES 1. Reyniers, J.P., Pleasants, J.R., Wostmann, B.S., Katze, J.R. and Farkas, W.R. (1981) J. Biol. Chem., 256, 11591-11594. 2. Singhal, R.P., Kopper, R.A., Nishimura, S. and Shindo-Okada, N. (1981) Biochem. Biophys. Res. Commun., 99, 120-126. N., Sate. S., Itoh, Y.H., Oda, K.I. and 3. Okada, N., Shindo-Okada, Nishimura, S. (1978) Proc. Natl. Acad. Sci. USA, 75, 4247-4251. J.R. and Beck, W.T. (1980) Biochem. Biophys. Res. Commun., 4. Katze, 96,

313-319.

5. Roe, B.A., Stankiewicz, Pike, D., Chen, C.Y. 673-688.

A.F., Rizi, and Chen, E.Y.

H.L., Weisz, C., DiLauro, M.N., (1979) Nucleic Acids Res. 5,

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Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

M. and Nishimura, S. (1981) Eur. J. Biochem., 6. Shindo-Okada, N., Terada, 115, 423-428. 7. Landin, R.M., Boisnard, M. and Petrissant, G. (1979) Nucleic Acids Res., 1, 1635-1648. 8. Friend, C. (1957) J. Exp. Med., 105, 307-318. A.D. and Heatherly, D. (1971) Anal. Biochem., 44, 486-495. 9. Kelmers, G., Boisnard, M. and Puissant, C. (1971) Biochimie 53, 10. Petrissant, 1105-1109. 11. Tambourin, P., Wendling, F., Moreau-Gachelin, F., Charon, M. and Bucau-Varlet, P. (1980) in "In vivotl and "In vitro" Erythropoiesis, Rossi, G., ed., pp. 127-138, Elsevier, New-York. 12. Mathieu-Mahul, D., Weil-Barbieri, D., Wendling, F., Tambourin, P., Moreau, F., Gisselbrecht, S., Gay, F. and Larsen, C.J. (1980) Biochem. Biophys. Res. Commun., 95, 342-349. J. Mol. Biol., 139, 61-83. q. Minty, A.J. and Gros, F. (1980) 14. Okada, N. and Nishimura, S. (1979) J. Biol. Chem., 254, 3061-3066. 15. Lin, V.K., Farkas, W.R. and Agris, P.F. (1980) Nucleic Acids Res., 8, 3481-3489. 16. Nishimura, S. (1979) in Transfert RNA : Structure, Properties and Recognition, Schimmel, P., Soil, D. et Abelson, J., eds, pp. 59-79, Cold Spring Harbor Laboratory, New-York. 17. Itoh, T., Haruna, I.,and Watanabe, I. (1975) Nature, 257, 327-329. 18. Briscoe, W.T., Griffin, A.C., MC Bride, C. and Bowen, J.M. (1975) Cancer Res., 35, 2586-2593. 19. Gallagher, R.E., Ting, R.C., and Gallo, R.C. (1972) Biochim. Biophys. Acta, 272, 568-582. 20. Levy, S.B., Blankstein, L.A., Vinton, E.C. and Chambers, T.J. (1979) in Oncogenic Viruses and Host CeZZ Genes, Ikawa, Y. and Okada, T., eds, Academic Press, New-York. PP. 409-428,

1147