Adenylate-rich oligonucleotides of ribosomal and ribosomal precursor RNA from HeLa cells

Vol. 42, No. 6, 1971

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ADENYLATE-RICH OLIGONUCLEOTIDES OF RIBOSOMAL AND RIBOSOMAL PRECURSORRNA FROM ReL_a CELLS H.C. (Biology Chalk Atomic Chalk

Birnboim and B.V. Coakley and Health Physics Division, River Nuclear Laboratories, Energy of Canada Limited, River, Ontario, Canada.)

Received February 2, 1971 Abstract. HeLa cell ribosomal precursor (45s) RNA has been found to contain nucleotide sequences also found in 18s RNA and others distinfound in 285 RNA. Thus, 18s RNA and 28s RNA are readily guishable, whereas 45s RNA is similar to 185 and 28s RNA combined. The nucleotide sequences analysed were those resistant to the combined action of pancreatic and Tl ribonucleases. We have studied cursor (l-3)

RNA's

(45S,32S)

and mature

in a way different

Purified

18S, 28S,

combination tetra-,

had both

kinds

labelled

These data

model of ribosomal To isolate

mental

these

RNA molecules (ii)

provide

(i)

the resultant

into

size

column;

each size components

classes class

mixture (isopliths)

was further

by electrophoresis

at low pH (11). 1169

a

this

treatment, 45s RNA

32s RNA had those support

in eukaryotic the

the appropriate with

with

in 28s RNA.

additional

oligonucleotides,

were digested

was separated (iii)

whereas

(4-10).

Amongst the

survive

only

RNA maturation

scheme was employed:

ribonucleases;

individual

18s and others

of oligonucleotides

in 28s RNA.

current

in

RNA's

were digested

and Tl ribonucleases. which

pre-

been employed

32s and 45s RNA species

only

ribosomal

18s and 28s ribosomal

and hexanucleotides

found

between

than has hitherto

of pancreatic

penta-

some were

found

the relationship

for

the

cells

following

experi-

radioactively

pancreatic

and Tl

of oligonucleotides on a DEAE-Sephadex fractionated on polyacrylamide

into gels

(1,3).

A

Fig. 1 Electrophoresis of ribonuclease-resistant tetranucleotides from a combined pancreatic and Tl ribonuclease digest of RNA (1.8 x 10 dpm); A. 3H-18S RNA (2.3 x 10' dpm) and 14C-28s and 28s RNA (9.3 x 10' B. 3H-32S RNA (1.7 x 10' dpm) and 14C-18 dpm); C. 3H-45S RNA (1.9 x 10" dpm) and 14C-18 and 28s RNA (1.1 x 10" dpm) . 1170

Vol. 42, No. 6, 1971

45S, labelled

BIOCHEMICAL

32S,

with

carried

out

and 14C-285 and 28s RNA;

gel

the

(2)

(3)

electrophoresis.

will

Amongst

455,

the

components 32S,

occurs

found

in

1A is

found

in

in

18s but

Fig.

Three nucleotides

second than

in

Cl -

Five

covered

in

that

(Fig.

3A).

case, by

Fig.

1,

individual

lA-C,

peaks

their

first

distribution

a fourth This

the

peak

seen

component

A fifth

in

is

is

peak

seen

28s RNA; however,

it

is not

are

amongst

quantitative 1.9X,

also

in

Fig.

well

In

of

peaks

seen,

others peak

2B),

in slightly

higher

the the

Of these,

is

component

3X, the

45s RNA (Fig.

in distribution

although

occur

differ-

amounts 2C) close-

18s and 285 RNA.

amongst

some experiments.

third

penta-

18s and 285

32s RNA (Fig.

for

differences

detected

in both

the

is markedly

and the

The profile

striking

The fourth

occur

are present

of a mixture

the

all

18s RNA.

components

are

seen to occur

distribution

is

have been

28s RNA, whereas

lA,

Although

28s as in

The most

molecules

in

lB,C).

only

18s and 28s RNA.

resembles

of the

185 RNA.

in

component

and third in

in

not

2A-C).

their

The second

as frequent

each

separated

shown in Fig.

In Fig.

not

components

2A),

14C-18

1B and C.

(Fig.

RNA (Fig.

of

are shown in

difference

32s and 45s RNA (Fig.

resolved

ent.

tetranucleotides

28s but

In

RNA

(12).

show little

in

were

and quantitation

28s and 18s RNA.

which

mixture

and hexanucleotides

elsewhere

were

3H-18S

and 28s RNA.

The electropherograms

be described

three

(1)

RNA and an equimolar

Characterization

HeLa cells

experiments

mixtures:

RNA and 14C-18

penta-

from

Three

following

3H-32S

3H-45S

tetra-,

2 and 3.

ly

3H or 14C-adenine.

analyse

RNA;

individual

28s and 185 RNA were prepared

either to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

almost

also 1171

between

hexanucleotides first only

is the

the major

(Fig.

incompletely fourth

exclusively labelled

RNA 3Are-

occurs in

18s RNA component

in

Vol. 42, No. 6, 1971

BIOCHEMICAL

I

I

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

I

I

A

I

I

I

I

I

I

I

I

I B

2 Fig. tides.

Electrophoresis of ribonuclease-resistant Samples as in Fig. 1. 1172

pentanucleo-

Vol. 42, No. 6, 1971

Fig. 3 tides.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Electrophoresis of ribonuclease-resistant Samples as in Fig. 1. 1173

hexanucleo-

Vol. 42, No. 6, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

- Fraction number. Ordinate - Dpm per fraction/ Fig. 1-3 Abscissa Total dpm in the enzymatic digest. 3H-nucleolar RNA (14) and ribosomal RNA were prepared from HeLa cells incubated at 7.5 x 10" cells/ml for two hours in the presence of 2, 8-3H-adenine (40 pCi/ml; 3.6 Ci/m mole). 14C-ribosomal RNA was prepared from HeLa cells (5 x lO'/ml) grown for 48 hr in the presence of 8-14C-adenine (0.3 pCi/ml; 51 m Ci/m mole). Individual 45S, 32S, 28s and 18s RNA species were isolated by sucrose gradient centrifugation. Conditions for enzymatic digestion, DEAE-Sephadex chromatography and gel electrophoresis are described elsewhere (11,12). Oligonucleotides were eluted from gel slices in 1.5 ml 0.02M NH40H and counted in a scintillation c0unte.r after the addition of dioxane-based scintillator. The data, recorded directly on paper tape, was quench corrected, normalized and plotted by corn uter. In all (cathode) is on the left. 'H= x f$yes , the origin .

in

325 RNA (Fig.

RNA (Fig.

3B).

1C and 2C, the

As in Fig.

3C) resembles

that

of an equimolar

profile

mixture

for of

45s

18s and

28s RNA. The experiments model

for

synthesis

cells.

By identifying

RNA but

not

show that iate found

in those

precursor, in

bution

in

since

also the

apparently

occurring

in

32s RNA.

All

tion

in

sequences versa,

it

tetra-, also

resembles

that

18s or 28s RNA.

adenine

label

is

residues

in

occur

found

in

45s RNA.

in

185

is

to

its

immed-

Their

of an equimolar found

perhaps

predominantly are

RNA in eukaryotic

and hexanucleotides

were This

current

has been possible

pentafound

the

which

28s RNA are also

No oligonucleotides

radioactive

with

of ribosomal

28s RNA and vice

45s RNA closely

most

consistent

nucleotide

18s and 28s RNA are

found

fully

and maturation

of 18s and 28s RNA. not

are

conserved

in

in not

adenine, during

distrimixture

45s RNA surprising and matura-

(13). REFERENCES

1. 2. 43:

Darnell, J-E., Jr., Bacterial. Rev. 32, 262 (1968). Maden, B.E.H., Nature .E, 685 (1968). Perry, R-P., Prog. Nut. Acid Res. Mol. Biol. 6, 219 (1967). Wagner, E-K., Penman, S., and Ingram, V-M., J. Mol. Biol. -'29 371 (1967). 1174

Vol. 42, No. 6, 1971

5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Weinberg, R-A. and Penman, S., J. Mol. Biol. 47, 169 ('1970). Amaldi, F. and Attardi, G., J. Mol. Biol. 33, 737 (1968). Jeanteur, Ph., Amaldi, F., and Attardi, G., J. Mol. Biol. 33, 757 (1968). Jeanteur, Ph. and Attardi, G., 5. Mol. Biol. 45, 305 (1969). Choi, Y-C. and Busch, H., J. Biol. Chem. 245, 1954 (1970). Quagliarotti, G., Hidvegi, E., Wikman, J., and Busch, H., J. Biol. Chem. 245, 1962 (1970). Birnboim, H.C. and Glickman, J., J. Chromatog. 44, 581 (1969). Birnboim, H-C., in preparation. Willems, M., Wagner, E., Laing, R., and Penman, S., J. Mol. Biol. 32, 211 (1968). Penman, S., Vesco, C-, and Penman, M., J. Mol. Biol. 34, 49 (1968).

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