cDNA cloning of mRNAS which increase rapidly in human lymphocytes cultured with concanavalin-A and cycloheximide

Vol.

129,

June

28,

Nlo. 3. 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1’985

cC~NA CLONING OF mRNAS WHICH INCREASE CULTURED WITH CONCANAVALIN-A Donald Department

Received

of

April

Biochemistry,

R.

RAPIDLY IN HUMAN AND CYCLOHEXIMIDE

619-625

LYMPHOCYTES

Forsdyke

Queen's University, Canada K7L 3N6

Kingston,

Ontario

12, 1985

To induce or "superexpress" genes involved in the entry of cultured G lymphocytes into the G -phase of the cell cycle, the cells were treated f8r 2 hours with a lectin (c A ncanavalin-A) and a protein synthesis inhibitor (cycloheximide). A cDNA library was generated from small quantities of RNA by the high efficiency method of Gubler & Hoffman (1). 30,000 colonies were screened for differential hybridization to cDNA corresponding to treated cultures!, but not to cDNA corresponding to control cultures. 50 recombinants were identified on this basis. One recombinant (#7) corresponded to mRNA (2150 base pairs) which was increased by cycloheximide alone, but was not increased by concanavalin A. Another recombinant (#19) corresponded to 2 mRNAs (980 and 1120 base pairs) one or both of which were increased either by concanavalin-A or by cycloheximide. It is speculated that the latter mRNAs are products of a locus which is activated when the concentration of a repressor is decreased by 0 1985 Academic press, Inc. concanavalin-A or cycloheximide.

Most Unless the

human stimulated

body

for

bacterial

of

for

many

by

increased

transcription

under

repressor

would

orchestrate

explore synthesis

some or

entering

the

be

under

autogenously

chain

of

events

by

The of

model, (cycloheximide).

the

of into

Go lymphocytes

G1-phase

were

The

619

latter

by

and

of

result other

in loci

("activation the

with be

which,

a monoclonal

from

loci

a low

Inactivation

would

treated would

such

(3,4).

in from

a repressor,

(lectin),

latter

persist

with

either

the

(2).

Extrapolating

of

locus

lived

Go-cells

regulated

the

long

cells

control

repressor

products cell

that

activator the

be

cycle.

initiated

a polyclonal from

cell

the

to

"resting"

predicts

be

control.

this

these

would

both

entry

appear

antigen,

theory

would

induction,

(antigen)

protein

demand

products

activatolr

To

without

systems,

repressor

T lymphocytes

a specific

years

their

efficient the

blood

by

model

demand for

peripheral

cell

an

expected

loci") cycle.

inhibitor to

of decrease

0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol.

129,

the

concentration

repressor

of

control.

increase

in

the

products

in

treated

cultures

detection

method

This

Consistent

with

with

and to with

mRNA

species

the

lectin

or

by

of

this

which

concentration

we were

no

from

loci

(5)

a rapid

found

detected

mRNA

(concanavalin-A),

as

increase

possibly

under

their was

because

detected our

sensitive.

from

and human

are

screening

lymphocytes

Using

and

COMMUNICATIONS

transcription

which

However,

cycloheximide. mRNAs

RESEARCH

prediction,

preparation RNA

BIOPHYSICAL

enhance

sufficiently

concanavalin-A the

thus

3 mRNAs

lectin

not

quantities

corresponding

of

AND

(p28,p39,p55).

reports

concanavalin-A

treated

and

was

paper

small

repressor

concentration

translation

from

BIOCHEMICAL

No. 3, 1985

cDNA

we is

rapidly

library

with

from

recombinants

expressed

cycloheximide, which

a cDNA

cultured

probes

differentially

of

of

report increased

in the

made

cultures detection either

of by

cycloheximide.

MATERIALS

AND

METHODS

These were as described (5,6). Enzymes were from BRL Inc., Gaithersberg, MD or Pharmacia-PL Biochemicals Inc., Piscataway, NJ. RNAasin was from AMV reverse transcriptase was from Promega Biotec, Inc., Madison, WI. Nick translations were performed Life Sciences Inc., St. Petersburg, FL. with a BRL kit. cDNA

preparation

Peripheral blood was used as a source of non-cycling GOTlymphocytes which, unlike lymphoblastoid cell lines, should retain all in vivo control elements and associated mRNAs during culture. RNA was isolatedfSom freshly isolated cells after culture for 2h with concanavalin-A (ZOOug/ml) and cycloheximide (100 uM) as described (5). First strand cDNA synthesis using total RNA denatured with methyl mercury hydroxide followed procedures described by Maniatis et al. (7) cDNA

library

Using an unpublished modification of a transfection protocol of D. Hanahan, which routinely produces lo'-lOa ampicillin-resistant colonies per pg of intact plasmid pBR322, a library of 1.7 x lo6 independent apparent recombinants was established in E. coli C600 from 750 ng of dC-tailed, Eco RV-cleaved, pBR322 vector arim of dG-tail cDNA, which had not been size-fractionated (1). Background with vector alone was 2 colonies/rig. Agarose gel electrophoresis of -Barn Hl-digested plasmids prepared from 10 randomly picked colonies demonstrated only 2 with excisable inserts (400 and 900 base pairs). Thus the yield of recombinants with excisable inserts was likely to approach 340,000. A library of this size should contain representatives of all clonable mRNAs (8). Inserts excised with Barn Hl contained a 188 base pair sequence derived from the vector (1). This could be conveniently removed with Sau 3A to generate cDNA inserts which could be gel purified (9). For propagation and further study, selected recombinants were routinely transfected into a recbacterial strain (E. coli DH1;7).

620

Vol.

129,

9lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 3, 1985

Screening

by

Differential

Hybridization

30,000 colonies on nitrocellulose filters were screened by the high density method of Hanahan & Meselson (10). 4 replica filters were made from each master plate and two of these were hybridized with ['2P]-labelled CONA derived from RNA from control lymphocytes cultured for 2h. The other two filters were hybridized with ['2P]-labelled cNDA derived from RNA from cultures which had been treated for 2h with concanavalin-A and cycloheximide. Colonies were scored as positive if, on both pairs of filters, they were labelled much more by cDNA from treated cultures.

RESULTS 50 30,000

recombinants Of

colonies.

moderately.

treated

hybridized with

to

the 1200

examination were

1).

hybridized

differential treated

for

by

a mean not

Blots

of

with

clearly

replating

was 2h with

size

RNA

of

at

cell

low

insert

518

base

observed

concanavalin-A

Size of cDNA (base pairs)

the

Size of (bases)

density

and

varied

from

for

further

agarose

control

gel

RNAs

RNA

and

corresponding and cycloheximide Frequency library

390

2150

0.06

10

320

2150

0.05

19

800

RNA

30

650

not

to in (%)

determined

cDNA sizes were determined by comparing migration of Barn Hl-excised inserts through agarose gels with the migration of DNAtandards RNA sizes were determined as described (BRL, 123 base pair ladder). in Fig. 1. Frequency in library was determined by hybridizing each cDNA insert, labelled by nick-translation, back to the original 30,000 colonies on nitrocellulose filters. Studies with recombinant #30 are preliminary. 621

from 1).

0.13

& 1120 3400

Clear

(Fig.

7

980

electro-

(11).

cycloheximide

cDNA recombinants by concanavalin-A

been

strongly

selected

plasmids

and

not

pairs.

by

between

from

had

sizes

were

fractionated

among

distinguishable which

cDNA of

detected hybridized

Eleven

nick-translated

of

26

lymphocytes

cross-hybridize

the

TABLE 1. Characteristics mRNAs induced in lymphocytes Recombinant -nllmber

still

were

cycloheximide.

did

hybridization

cultures

and

control

purified

with

which

(Table

from

were

strongly

hybridization.

pairs,

4 recombinants

but

and were

signals

hybridized

cDNA

differential base

differential

weakly,

with

colonies

phoresis

21

concanavalin-A

hybridizing repeating

clear

these

3 hybridized

colonies

150

giving

In

Vol.

129,

BIOCHEMICAL

No. 3, 1985

Probe

no.

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

7

Conconavalin-A

-

+

Cvcloheximide

-

+

I

0.6

-

Figure

1. Hybridization of nick-translated cDNA recombinants to blots of RNA from human lymphocytes cultured for 2h in the absence (-) or presence (+) of concanavalin-A and cycloheximide. Glyoxal-denatured total RNA samples (20119) were electrophoresed through 1.4% agarose gels and transferred to Pall Biodyne A membranes, which were hybridized and washed as described (11). Designated plasmid numbers (7,10,19,30) are shown at the top of each pair of tracks. Molecular weights were determined with reference to glyoxylated DNA markers which were electrophoresed in parallel.

the

case

of

several

plasmid

tracks

#19,

two

RNA

were

not

observed

(12)

of

species

were

when

pure

labelled.

inserts

Bands were

used

common as

to

hybridization

probes.

"Cytodots" times

with

probed #I9

various

with (Fig.

alone

combinations

In

the

and

cycloheximide,

the

corresponding

case

RNA. cultures. contol

of

cultures

or The

lymphocytes

inserts

and

7,

RNA was

in

the

case

combination,

the

incubated

was probe

increased

concentration In

of

to

increased

but

increased

experiment for

experiments. 622

shown 4h.

This

for

were

plasmids by

increased

19,

both

and

by

concanavalin-A

concentration

more

rapidly

there

was not

#7

cycloheximide

not

the

was

various

cycloheximide

corresponding

concanavalin-A,

in

cultured

concanavalin-A

probe

However, alone

heximide-treated in

of

plus

alone.

from

cDNA

cycloheximide

concanavalin-A

increase

prepared

nick-translated 2).

and

RNA

seen

in

of cyclo-

a small in

other

Vol.

129,

No. 3, 1985

BIOCHEMICAL

AND

Concanavalin-A

-

Time Cyclo-

Probe no. 7

(h)

COMMUNICATIONS

+

0~~1240~~124 42

heximide -

19

BIOPHYSICALRESEARCH

4 2

1 d c*e?J

*

.*c

x

-

* e

Figure 2. --dot" analysis of the concentration of specific mRNAs corresponding to probe 7 and probe 19 during the first 4 h of culture. Lymphocytes (6x lob/ml) were incubated with various combinations of concanavalin-A and cycloheximide. At the times shown, 0.5 ml cultures were harvested and cytoplasmic RNA prepared as described by White & Bancroft (12). Each RNA preparation (upper dot at each tlime point) and a corresponding 10 fold dilution (lower dot at each time point) were transferred to nitrocellulose and hybridized with purified nick-translated cDNA probes (approx. 0.5 x 10" d.p.m./ml) as described (12). Autoradiographs were developed after 1 week at -70°C with intensifying screens.

DISCUSSION A rapid efficiency

and by

with

this,

DNA,

appear

intense the

a variety to

of

loops

corresponding

protein

regulatory The

use

properties hence

Theoretically, into

4 classes

the

cloning be in

critical

regulatory

cDNA

to

of

the

work

because work

exploits

genes

to

many

Consistent of

transcription

the increase

which

bind

b.y the transcripts

corresponding of

maximum

Such

efficiently

cDNAs

with

(3,4).

(3,13,14). of

this

achieved

loop

synthesis

difficult this

be

proteins,

inhibition For

might

may

a self-regulating

own

involve

(15).

a stimulus

regulatory

their

cycloheximide of

facilitate

important

may

Thus proteins

of

of

autoregulate

short-lived.

to

interruption

autoregulatory

be

response

the

to

low

such

abundance

possible mRNA

should

of

autoregulatory concentration

and

cloning.

our corresponding

differentially to

hybridizing mRNAs

which 623

recombinants increase

in

mRNAs.

response

will

fall to

(i)

Vol.

129,

No.3,

BIOCHEMICAL

1985

cycloheximide

only,

(ii)

the

combination

of

least

the

two

second the

first

class

has

cycloheximide

lectin

and

classes

the

concentration

are

which

BIOPHYSICALRESEARCH

or

lectin, RNA

represented

in

predicted falls

in

lectin

only

"cytodots"

the

for

cells

COMMUNICATIONS

(iii)

cycloheximide.

properties

of

AND

show

library

loci

with

(iv)

that

(Fig.

regulated

treated

or

at

2).

by

The

a repressor

cycloheximide

or

lectin. A number have

of

reported

investigators, rapid

increases

(16-19).

In

expressed

mRNAs,

but

candidates

would

be

which

we

identified

to

play

a role

(~55) thought increased

or

the

in

present

case, do

not

the

in

55

nuclear

kilodalton formation

recombinants to

recombinant

in

lectin-treated

#7

or

c-myc -

and #19

a complex #lO

may

appear

with

relate

to

different

kilodalton

these from

the

mRNAs

with

inducing is

agent

increased

superinduced

kilobase various

by

mRNA, cell

protein

lines

and

(23-24).

the known

rapidly

treated

is

oncogenes,

genes

are

mRNA)

and

in

other

which are

(2.2

protein

and

kilobase

rapidly a 39

Major

appropriate

expression

increases

differentially

acidic

mRNAs

lymphocytes

c-fos

protein)

an

(2.4

spleen

the

lines

with

expression

Similarly,

of

have

together

lymphocytes

for

oncogenes

cell

proteins,

lectin-treated

and

Some

various

known

proteins.

(p28,p39) (5).

to

probes

proliferation

alone,

in

corresponding

proteins

mouse

(16).

mRNAs prepared

the

when

concanavalin-A-treated

involves

known

cell

corresponding

specific we have

basic

particular,

cycloheximide

probes

previously

either

In

in

"superexpressed"

cycloheximide, (20-22).

using

mRNAs to

While corresponding

increase

rapidly

lymphocytes.

ACKNOWLEDGEMENTS

of

I thank Ms. L. Russell Canada and the Leukaemia

and Ms. Research

J.

Dentry Fund of

for technical Toronto for

help support.

and

REFERENCES 1. 2. 3.

Gubler, U. and Hoffman, B. (1983) Gene 4, 263-269. Buckton, K.E., Jacobs, P.A., Court-Brown, W.M. and Doll, R. (1962) Lancet 2, 676-679. Savageau, M.A. (1979) in Biochemical Regulation and Development, Goldberger, R. ed. (Plenum Press, N.Y.) pp. 57-108.

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Vol.

4. 5. 6. 7. 8 9: 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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226, 24.

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 3, 1985

1080-1082.

Mitclnell,

40,

R.L.,

Zokas,

L.,

Schreiber,

R.D.

1209-217.

625

and

Verma,

I.M.

(1985)

Cell,