Induction of α2u globulin mRNA by phenobarbital in rat liver: Characterization of a cDNA clone

Vol. 134, No. 3, 1986 February 13, 1986

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS Pages

INDUCTION OF "2u

GLOBULIN mRNA BY PHENOBARBITAL

CHARACTERIZATION M.L.

OSORIO-ALMEIDA",

Laboratorio

December

IN RAT LIVER:

OF A cDNA CLONE

C. SINOGAS, M. LUDOVICE and M.C. LECHNER**

de Bioquimica,

Instituto

2781 OEIRAS, Received

1182-1189

Gulbenkian

de Ciencia,

Portugal

5, 1985

A clone has been selected from a cDNA library previously constructed from phenobarbital pre-treated rat liver polysomal poly(A) RNA, which was reverse transcribed. The double-stranded cDNA was inserted by GC homopolymeric tailing in the Pst I site of PAT 153, and further cloned in E.coli HBlOl. This clone, called 2A9, corresponds to a mRNA whose concentration is increased five fold 16 h after phenobarbital treatment. Its length is 1200 nucleotides as revealed by RNA dot and Northern blot analysis respectively. The two strands of a 450 bp fragment from the 2A9 580 bp double-stranded cDNA insert have been sequenced and proven to correspond to c1 globulin mRNA. It shows one single bp difference from the sequence previo 3 ?ly published by Unterman et al. (1981, PNAS, 78, 3478). Thus, "2u globulin, a hormone regulated gene product, is inducible by phenobarbital. 0 1986 Academic Press, Inc.

The

adaptive

phenobarbital

response

(PB1 - the

I - is complex,

group reticulum

liver

cell

to

of Cytochrome in an intense

associated

c reductase,

the

prototype

resulting

membranes,

cytochrome

of

to

cytochrome

chemical

aggression

P450 mono-oxygenases proliferation

a characteristic

of the increase

P450 and related

by

inducers endoplasmic

in

mono-oxygenases

the

NADPH

activities

(1). Concomitantly rat

liver

capacity

(21,

*

**

observed

a PB rat

Visiting Caparica, Institut

by in

induction

of

a general

increase

vivo

liver

in

of in situ

poly(A)RNA

450 b and e in

hepatic

analysis

acids

protein

colony

cDNA library,

should

be addressed.

Inc. reserved.

‘1182

synthesis

incorporation

(3).

hybridization hybridized

the Universidade Nova de Lisboa - 2785 Recipient of a short-term FEBS Fellowship Universite Paris VII (1985).

$1.50 0 1986 by Academic Press, of reproduction in any form

isocytochromes

amino

polysomal

correspondence

the

and in vitro

by statistical

Fellow from Portugal. Jacques-Monod,

To whom all

0006-291X/86 Copyrighf All rights

the

PB produces

as evaluated

We recently using

with

with

Monte da in the

Vol.

134,

No. 3, 1986

homologous

and

products

with liver

This

clone

to identify

globulin

membrane-bound induced

clone

represents

characterization a2u

C3*PlcDNA

at different

the well

we selected

that

AND

known

BIOPHYSICAL

- that

degrees, change

RESEARCH

COMMUNICATIONS

a significant

number

of

by PB treatment

(4).

in the

protein

and establishment

are

positively

pattern

This

gene

fact

is

PB phenotype.

In order liver,

heterologous

are affected

consistent of the

BIOCHEMICAL

genes 2A9 from

which

the previously

a mRNA species

of the

2A9 pDNA insert,

- a secreted polysomes

clearly

under

constructed induced

performed

protein the control

regulated

which

by the

by PB in

cDNA library barbiturate.

by DNA sequencing, is

synthesized

of numerous

hormones

in

the (5). The

revealed male

liver

- is markedly

by phenobarbital.

MATERIALS AND METHODS Restriction endonucleases were obtained from Boehringer-Mannheim and from New-England Biolabs. E.coli DNA polymarase I (Klenow fragment) and T polynucleotide kinase from Boehringer-Mvheim, bacterial alkaline phos@atas$ (BAFP) was from Cooper Biomedical, Ca- PldATP (3000 Ci/mnole and Cy- PIATP (6000 Ci/mmole) were purchased from Amersham. Male young Wistar rats (300-350 g body weight), from the Gulbenkian Institute Animal House have been used. Phenobarbital (PB) treated animals received 80 mg/kg b.wt. in an aqueous solution by intragastric administration. The rats were sacrified by decapitation after a 18-20 h starving period (1). The 2A9 cDNA clone has been selected from $ library of cDNA, previously constructed from rat liver polysomal poly (A) mRNA, under PB treatment (16 h). The dscDNA was inserted in the PstI site of pAT153 by homopolymeric GC tailing, transfered into E.coli and cloned (5). The time course variation in the concentration of 2A9 mRNA in rat liver polysomes, during the onset of the response to PB was performed by preparing polysomal RNA from rat liver at different times (O-24 h) after one single administration of PB which was subsequently denatured and fixed on nitrocellulose sheets by fittering through in a Schleicher and SchGll Minifold II apparatus (6). For the mRNA size measurement, the Northern blot technique has been used Total cytoplasmic RNA extracted from rat liver by the guanidine (7). thiocyanate method (8) was submitted to electrophoresis on 1.5% agarose gel containing 10 mM methymercury (II) hydroxyde, and subsequently transfered to nitrocellulose filters. Nick-translated 2A9C3*PlpDNA was used as the probe for hybridization with the immobilized RNAs on the nitrocellulose support The autoradiographic signals of the hybridized c'32PlpDNA were obtained by exposing the filters to DuPont-Cronex-4 films at -7O"C, using two DupontCronex Hi-Plus intensifying screems. The autoradiographs of the dots were scanned in a densitometer GS300, Hoefer Scientific Instruments. For the size analysis and restriction endonuclease mapping of the cDNA insert in clone 2A9, approximately 1 ug samples were cleaved with various restriction endonucleases according to the supplier's instructions, and the cDNA fragments resolved in either 0.8-l% agarose or 8% polyacrylamide gels. 1183

Vol. 134, No. 3, 1986

BIOCHEMICALANDBIOPHYSICALRESEARCH

COMMUNICATIONS

Terminal DNA labelling at 5' protruding en.&, produced by restriction endonuclease cleavage, was performed by using Cy- PIATP and T4 polynucleotide kinase (9). Thj23' end labelling of the complementary strand was performed by addition of Ccr- PldATP by the DNA polymerase I (Klenow fragment) (10). Sequencing of both cDNA strands was performed according to Maxam and Gilbert (11) with minor modifications. The identification of the 2A9 recombinant dscDNA has been achieved by screening for homology the sequenced 448 nucleotides in GENBANK, using a CITI 2 computer program.

RESULTS AND DISCUSSION The

variation

in

response

to

PB in

polysomal

RNA extracted

chemical.

Figure

graphic

in

rat

liver,

is

results

In order

to characterize

analysis

of

rat

mobility

shown in

2.

figure

The digestion full

length

31, while

The

of

a single

produced

analysis

of

administration

scanning

of specific

of

the

of this

the

doted

in

autoradio-

RNAs with

mRNA sequences

2A9

present

to 2A9 pDNA insert. of

mRNA is

the

liver

approximately produced

fivefold

in

liver

polysomes

in rat

RNA was

restriction

the

were

labelled

are

of both of

the with

approximately

I,

released

clone 2A9

revealed

in the

the existence

an

nucleotides,

a DNA fragment

site

Northern

mRNA revealed

1200

580 bp as shown

a unique

2A9,

in

as

representing figure

insert

3 (lane

(lane

21.

of two Sau 3AI sites,

in 2A9 dscDNA.

map and

has been performed,

The

to

is

by Eco RI revealed mapping

in

performed.

which

insert,

restriction

digestion

mRNA represented

corresponding

the

DNA sequencing

ends)

mRNA,

by dot

of the

an induction

two Bst NI and no Sau 961 sites,

Eco RI

after

hybridization

of 2A9 pDNA by -Pst

digestion

Further

which

evaluated

of the quantity

that

2A9

PB treatment.

electrophoretic

the

been

active

densitometric

of the corresponding

16 h after

blot

the

homologous

reveal

of

times

after

an index

the RNA preparations,

concentration

has

at various

obtained

This

The

concentration

1 represents

signals

C32PlpDNA.

the

the

strategy

summarized strands

pDNA,

the

of

in figure

was performed two

resulting

20 uCi of [a3*P]dATP. 1184

nucleotide

sequence

analysis

4. from

the

Eco RI site.

fragments

(6 pmoles

For the

5' end labelling,

After of 3' the

134,

3,1986

No.

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

A

COMMUNICATIONS

B

-Origin -20

01

02 0

4

6

12

16

-

s

10-

24 h

Figure

1.

scanning of RNA dot hybridization. 10 pg of oolvsomal RNA from rat livers at various times (O-24 hl after one ;ingle P8 administration were dotted & fittering through a nitrocellulofe sheet, hybridized with [ PJ nick-translated 2A9 pDNA (4 x 10 cpm/pg) and autoradiographed.

Figure

2.

Northern blot analysis of 2A9 dNA. Total RNA from rat livers, (A) - control and (B)-phenobarbital (16 h) was fractionated by electrophoresis in 1.5% agarose-methylmercury (10 mM) ~$1, transfered to nitrocellulose filters and hybridized with [ P] nick-translated 2A9 pDNA. The size of the mRNA revealed is 1.2 Kb as compared to 28s and 185 rRNA markers run in the same gel.

RI

digestion

fragments

gel.

Each fragment

two

Eco

Densitaetric

polyacrylamide

were

previously

(14

pmoles

electroeluted of 5'

ends)

from

a 6%

was labelled

with

100 pCi of CU-~*PIATP. In

both

fragments

were

two fragments

regions

from

isolated

hundred

by the

A and 6 in figure

4.

The

by searching shows

99.8% homology

Dawley

rat

The 2A9 recombinant

the

messenger

a2u-globulin

liver insert

cleavage

have

been

sequenced

in a sequence

bank

with

with

Pst

I,

the

in 6% polyacrylamide

chemical bp

It

digestion

by electrophoresis

fifty

Sprague

of the

secondary

and

identified program.

after

sequenced

Four

(11).

cases,

method

which

and the

correspond

(Appendix

(GENBANK) using

a region

gel

of Maxam and Gilbert

sequenced fragment

resulting

the CITI

of the a2u-globulin

I)

has

to been

2 computer mature

mRNA

(12). expands

RNA. The region

which

mRNA coding

region,

from

nucleotide

has been analysed

1185

covering

the

55 to nucleotide corresponds

596

to 84% of

mRNA sequence

from

Vol.

134,No.

3, 1986

BIOCHEM1CALANDBlOPHYSlCALRESEARCHCOMMUNiCATlONS

-4363 -3602 -2528 -1857 -1461 4% -9929 -125

41 -Q11 c 15

-616

.--

-3383

03

Eco RI

Figure

Pst 1 15

A

B

.100

Size analysis of the 2A9 dscDNA insert. Agarose gel (0.8%) electrophoresis of the pDNA purified from 2Ag clone, after Pst I, Eco RI + Pst I in lanes 2, 3 and 5 digestion by : Eco RI, respectively. Size markers obtained by digesting pBR322 and pAT153 with combined restriction endonucleases in lanes 1, 4 and 6. The gels were treated by ethidium bromide and visualized under U.V. light. The size of the markers (bp) is represented on the right side of the figure.

3.

4.

nucleotide

BstNI #l-

0I Figure

Sau3AI

,-.--

Restriction map of the 2A9 dscDNA insert. The Eco RI, Sau 3AI and Bst NI sites were mapped by either single or double digestion of the pDNA. Sequence analysis of the inserted cDNA was perfomed from the Eco RI site. Plasmidic DNA was digested by Eco RI. The two resulting fragments were labelled at the Eco RI site (at either 5' or 3' ends) and subsequently digested by Pst I to obtain single end-lebelled fragments. The arrows indicate the direction and the extent of sequencing, performed on both strands.

55

up

to

nucleotide

502,

32

nucleotides

upstream

its

termination

codon. The perfect,

except

a base in

homology for

change

position

arginine tridimensional

in 39

The

with

of

fact by

the

sequence

nucleotide both

161

strands,

lysine, structure

this

which lack

which

are and

as

in

codon

in

our

of

homology

both

basic

function

by

indicated

affecting

cf2u -globulin that

published

of 1186

figure

AGA

cDNA

is

protein.

al.

et 5.

replaced in acids,

(121

Actually

corresponding

results amino

the

Unterman

we found to

by

is

arginine

AAA.

the

replacement

may

not

affect

of the

Vol. 134, No. 3, 1986

. ..55

BIOCHEMICAL

SSTXGNLDVAKLNGDWFSIV AGT 'i-CC ACA AGA GGG AAC

AND BIOPHYSICALRESEARCH

CTC GAT GTG GCT AAG CTC AAT GGG GAT TGG TIT *

VASNKREKIEENGSMKVFMQ GTG GCC TCT AAC AAA AGA GAA AAG ATA GM H CAC

I D V ATC GAT GTC Tk

GiG

CRELYLVAYKTPEDGEYFVE TGC AGG GAA CTA TAT TM; YDGGNTFTILKTDYDRYVMF TAT GAC GGA GGG MT

COMMUNICATIONS

A!T

GAG MT

L TZA Gk

l&

G!l-I GCC TAC AAA

ACA 'ITT

ACT ATA CTl-

HLINFKNGETFQLHVLYGRT CAT CTC ATT AAT TTC AAG AAC

G4X

114

G-l-T TlT

ATG

CAG

174

T?C AtG

Tk

E GAA A!T

G GGA G:G

234

ACG CCA

GAG GAT GGC GAA TAT TIT

GTI? GAG

294

AAG ACA

GAC TAT GAC AGA TAT GTC Al-G

GAA ACC TPC

GGC AGC ATG AAA

TCT ATT GTC

C:T

'ITT

354

GTG CTC TAC GGC AGA ACA

414

CTA TGT GAG GCG CAT GGA ATC

474

CAG CTG An;

KDLSSDIKEKFAKLCEAHGI AAG GAT CTG AGT TCA GAC ATC AAG GAA AAG TIT

GCA AAA

I K ATI'MG

R D N I I D L T ACT AGG GAC AAT AT-C ATT GAT CTA ACC A... T

Appendix

I.

Sequence

the

450

molecular

genes

forms

isoelectrically

are

of

known to

of

the

2A9 cDNA

insert.

are

rat

liver

coding

This CDNA

strand. mRNA

for

immunologically

different

identical

but

(13).

difference

(Wistar),

in

which

distinguishable

in our work

be active

a2u-globulin,

The base pair

may be related

observed

which

is distinct

to the rat

from the one studied

strain

used

by Unterman

(12)

Dawley).

It is known that mammalian

liver,

strongly

is

repress

o.2u -globulin under

the

globulin

in the adult

The o?2u -globulin

regulated

o12u-globulin

present

work

Group

molecule,

revealed

a

when compared

estrogens

while

androgens,

and insulin

as reviewed liver

in

Actually,

protein, hormone

which,

by Kurtz

induce

CX~~-

and Feigelson

is

predominantly

under

hormonal

we report

for

the

first

time

inducibility

agent,

phenobarbital,

the

prototype

of

of

liver

I.

The mRNA represented liver,

family

(17).

mRNA by a chemical

enzyme inducers

growth

in the

control

(14,151.

this

male liver,

mRNA level

transcriptional In the

of

hormone,

by a multigene

control

synthesis

thyroid

synthesis

is encoded

multihormonal

glucccorticoids,

(16).

nucleotides

sequence has been verified by sequencing the complementary Numbering according to the sequence of the a*,,-globulin previously published by Unterman et al. (12).

Several

(Sprague

of

502

in

base

pair

the

clone

change

2A9, in

to the "2u -globulin 1187

the

extracted

from

informational

mRNA from

uninduced

PB induced

rat

region

the

rat

of liver

(12)

Vol. 134, No. 3, 1986

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

5’

3’

140

150

’ kA& Go TG

-la

: cc4 3

Figure

The

5.

hypothesis

which

has been

ruled

out.

and Gilbert sequence analysis of dscDM fragment. Part of the nucleotide sequence corresponding to strand (+I of 2A9 cDNA is shown on the leftppanel. The right panel shows the complementary sequence (strand(-)). Base pair 161, indicated by an arrow in both panels, is non homologous to the a -globulin mRNA sequence Numberiilf according to the same published by Unterman et al. authors.

Maxa

that

PB induces

previously

a a2u-globulin

characterized

in

gene, normal

male

different rat

from

liver,

the

one

cannot

be

ACKNOWLEDGEMENTS The authors acknowledge Dr R.L. Pictet from Institut University Paris VII, for receiving one of us (M.L.O.A.) in where the last stage of the sequencing work has been performed. CIT12 computer facilities are also acknowledged. 1188

Jacques-Monod, his laboratory, The use of the

Vol. 134. No. 3. 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Lechner,

M.C., and Pousada, C.R. 11971) Biochem. Pharmac. 20, 3021-3028. G.P., Ghanayeb, Y., Ryan, D., Reik, L., Thomas, EE., Levin, W., and Walz, F.G., Jr. (1982) Biochemistry 21, 789-798. Kato, R., Loeb, L., and Gelboin (1965) BZchem. Pharmac. 14, 1164-1166. Lechner, M.C., Sinogas, C., Osorio-Almeida, M.L., Freirc M.T., ChaumetRiffaut, Ph., Frain, M., and Sala-Trepat, J.M. (1985) Submitted to Eur. J. Biochem. Sinogas, C., and Lechner, M.C. (1985) Ciencia Biologica (Portugal), in press. F.C. (1982) J. Biol. Chem. 257, 8569-8572. White, B.A., and Bancroft, Thomas, P.S. (1980) Proc. Nalt. Acad. Sci. USA 77. 52On205. Harding, J.D., Przybyla, A.E., McDonald, R-J., Pictet, R.L., and Rutter, W.J. (1978) J. Biol. Chem. 253, 7531-7537. Maxam, A.M., and Gilbert,-. (1977) Proc. Natl. Acad. Sci. USA -74, 560564. Molecular Cloning, A Laboratory Manual CSH (1982) T. Maniatis, E.F. Fritsch and Sambrook J. eds. Maxam, A.M., and Gilbert, W. (1980) Meth. Enzym. 65, 499-560. Unterman, R.D., Lynch, K.R., Nakhasi, L.H., Dolan, K.P., Hamilton, J.W., Cohn, D.V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. USA -78, 34783482. Laperche, Y., Lynch, K.R., Dolan, K.P., and Feigelson, P. (1983) Cell -'32 453-460. Kurtz, D.T. (19811 J. Mol. Appl. Gen. 1, 29-38. Dolan, K.P., Unterman, R., McLaughlin,-M., Nakhasi, H.L., Lynch, K.R., and Feigelson, P. (1982) J. Biol. Chem. 257, 13527-13534. Kurtz, D.T., and Feigelson, P. (l-578) Biochemical Actions of Hormones, G. Litwak ed., Vol. 5, p. 433. Kulkarni, A.B., Gubits, R.M., and Feigelson, P. (1985) Proc. Natl. Acad. Sci. usA 82, 2579-2582.

:: Vlasuk, 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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