Regulation of mRNA levels of rat liver carbamoylphosphate synthetase by glucocorticosteroids and cyclic AMP as estimated with a specific cDNA

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984

Pages 882-888

November 14, 1984

REGULATION OF mRNA LEVELS OF RAT LIVER CARBAMOYLPHOSPHATE SYNTRETASE BY GLUCOCORTICOSTEROIDS AND cyclic AMP AS ESTIMATED WITH A SPECIFIC cDNA C.J.de Groot, A.J.van Zonneveld*, P.G.Mooren, D.Zonneveld, A.van den Dool, A.J.W.van den Bogaert, W.H.Lamersff, A.F.M.Moorman and R-Charles. Department of Anatomy bleibergdreef 15,

and Embryology, University of Amsterdam 1105 AZ Amsterdam, The Netherlands

Central

Laboratory P-0.

wDepartment of Molecular Biology, of the Netherlands Red Cross Blood Transfusion Box 9190, 1006 AD Amsterdam, The Netherlands

Received

September

26,

Service,

1984

SUMMARY. The construction and cloning of a cDNA complementary to the mRNA of rat liver carbamoylphosphate synthetase (ammonia) is described. Using this cDNA, the size of the mature, cytosolic carbamoylphosphate synthetase (ammonia) mRNA is estimated to be 6.0 Kb. The levels of carbamoylphosphate synthetase (ammonia) mRNA in liver are shown to be regulated by glucocorticosteroids and cyclic AMP. By studying mRNA levels of carbamoylphosphate synthetase, albumin and phosphoenolpyruvate carboxykinase, using specific cDNA clones, we show that carbamoylphosphate svnthetase gene expression, like that of albumin liver-specific. Q 1984 Academic Press, Inc. The terminally the capacity appropriate

conditions.

cell-specific the

at which,

expression

is

EC 6.3.4.16)

since

in studies

requirements

to

that

that

serve

are

aim to identify

differential synthetase

exclusively

by

under

proteins

by which

Carbamoylphosphate enzyme is

is characterized

of proteins

such a spectrum,

as parameters

the

this

of cells

spectrum

and the factors

regulated. meets

the liver

state

a unique Within

can serve

levels

cells

differentiated

to synthesize

gene (ammonia)(CPS,

as such a parameter localized

in

liver

for parenchymal

(1,2). The factors

have been using

that

extensively

regulate

CPS gene expression

studied.

Ryall

a specific

cDNA clone,

content

of the diet.

protein regulate

the developmental

that

et al.

CPS mRNA levels

We have studied formation

(3)

at the protein recently

level

reported,

are controlled

in detail

of CPS and have shown the

whom correspondence should be addressed. The abbreviations used are: CPS, carbamoylphosphate PEPCK, phosphoenolpyruvate carboxykinase; pCPS, preCPS; preproalbumin; TMV, tobacco mosaic virus.

by the

the factors

primary

-To

0006-291X/84 Copyright All rights

$1.50

0 1984 by Acudemic Press, Inc. of reproduction in any form reserved.

882

that

synthetase; ppALB,

is

Vol. 124, No. 3, 1984 regulatory

BIOCHEMICAL

factors

enzyme activity

to be glucocorticosteroids levels

In order

to further

CPS-gene

under

to measure

the

were analyze

influence

mRNA levels

to CPS mRNA. Using analysis

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

to reflect

the level

is

to construct

cDNA we could of the expression

(4-9).

enzyme protein

at which

of hormones

and hence

a cloned control

shown

and glucagon

levels

the expression

regulated,

it

cDNA clones

demonstrate

Furthermore, (10).

of the

was necessary complementary

by Northern

blot

of the CPS gene in liver

involves changes in the levels of CPS mRNA. The tissue-specificity CPS gene expression was demonstrated by comparison with albumin

of

and PEPCK gene expression.

MATERIAL AND METHODS Animals. Male Wistar rats, 3-4 months of age, were obtained from the TN0 animal farm in Zeist, The Netherlands. The animals were made diabetic by intraperitoneal injection of 70 mg per kg of streptozotozin after an overnight fast. 36 Hours later, rats were checked for glucosuria and injected intraperitoneally with 10 mg per kg dexamethasone. The animals were killed 16 h later. Preparation of RNA. RNA was extracted by the guanidinium thiocyanate method (11) as modified previously (12), except that the initial homogenate in guanidinium thiocyanate was repeatedly extracted with 0.5 volume chloroform/isobutanol (4:l) (13). The poly(A)+ fraction was isolated by 2 cycles of oligo (dT) cellulose chromatography (type 7, P-L Biochemicals Inc., 10 mg total RNA per 150 mg oligo (dT) cellulose). 200 ug Poly(A)+ RNA, denatured in 20 mM methylmercury hydroxide was size-fractionated in an isokinetic sucrose gradient, prepared exactly as described in (14). The sucrose was buffered with 20 mM boric acid, 2 mhl Na borate and 1 m?d EDTA. Gradients were centrifuged at 38K rpm and 5OC for 18 h in a SW 40 (Beckman) rotor. CPS mRNA was identified by in vitro translation in nuclease-treated rabbit reticulocyte lysate (N90, Amersham) supplemented with 35Smethionine. Proteins were immunoprecipitated with monospecific rabbit antibodies (15) and Staphylococcus aureus cells (13). Proteins were separated by SDS-polyacrylamide gel electrophoresis (16) and visualized by fluorography (17). RNA was size-fractionated on formaldehyde For Northern blot analysis, - containing agarose gels and transferred to nitrocellulose as described in (18). Construction of a cDNA library. Fractions enriched in CPS mRNA were pooled and used as a template for synthesis of double-stranded cDNA essentially as described in (19), except that the DNA-RNA hybrids were passed through Sephadex G50 before alkaline hydrolysis and that on DEAE-Sephacel, a Mung bean nuclease was used. After purification cDNA library was constructed by dC homopolymeric tailing and insertion by RbCl-facilitated into the dG-tailed Pst 1 site of pBR 322, followed transformation of E.coli DH 1 (19). Differential screening was performed as in (20) with radioactively-labeled cDNA's to total kidney RNA or to poly(A)+ fractions enriched in CPS or albumin mRNA. Plasmids of interest were isolated by alkaline lysis or by CsCl density-equilibrium centrifugation (19). Hybridizations. Hybridizations were carried out as described in (18). Ml3 "C" tests and Ml3 hybridization to total RNA were done in 0.25% SDS and 25 mM EDTA for 1 h at 65OC (21). The hybrid-selection and translation procedure was carried out as in (19), using both double stranded pBR recombinant plasmids and their single stranded Ml3 derivatives. The size of CPS mRNA was estimated using the markers described in (22). 883

Vol. 124, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RESULTS

In vitro

translation

treated

with

of high

molecular

fore

decided

fraction

weight

by

represents

RNA was found mRNA) than

a cDNA library

mRNA fractions

only.

these

were

colonies

-hybridized

to each other

plasmids,

designated

and one recombinant albumin

mRNA (as identified

CPS mRNA less from

(not

was

This

there-

synthesis number

RNA (not with

shown). (0.75,

as

(relative

to

shown).

cDNA library

were

the CPS-enriched

of the recombinant

(PBR-ALB)

protein

protein

well

the

hybridization

kb in size

pBR-CPS l-3 plasmid

of total

or unfractionated

The inserts 0.5-1.4

It

in CPS protein.

colonies showed

1).

of the CPS mRNA level

to translate

25 (4%) colonies

(Fig.

2-3s

an underestimation

650 Ampicillin-sensitive

rats

from a sucrose-gradient

2);

was found

poly(A)-RNA

from diabetic

CPS was the predominant

was synthesized

densitometry

probably

screened;

that

in CPS mRNA (Fig.

poly(A)+ albumin

mRNA isolated

showed that

to construct

enriched

as estimated

of liver

dexamethasone,

plasmids

from

and 75% of the

inserts

Three

recombinant

of these

0.9 that

and 1.4 kb,

respectively)

was complementary

. by cross-hybridization

cross-

to

to a previously

-pCPS

-ppALB

0,.1

i F ;

02 Fig. 1. SDS-polyacrylamide gel electrophoresis translation products of 4 ug total liver RNA. pCPS and pp albumin protein is indicated.

of The

in vitro position

Fig. 2. Sucrose-gradient fractionation of poly(A)+ RNA. The drawn line depicts the optical density (260 nm) profile of total RNA in a parallel gradient. The 2 panels depicting the translational products indicate the position in the gradient of albumin-mRNA and CPS mRNA containing fractions.

884

of

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.pCPS

-wALB - PEPCK

C3 Fig. 3. Agarose gel electrophoresis of M13-mCPS1 (lane Z), M13c-CPSl (lane 4), a mixture of M13-mCPS1 and M13-cCPS1 (lane 3), a mixture of M13-cCPS1 and poly(A)+ RNA (lane 5), a mixture of M13-mCPS1 and Poly(A)+ RNA (lane 6) and poly(A)+ RNA only (lane 7). For reference, an EcoRl, Hind III digest of lambda DNA is shown (lane Fig. 4. Gel hybrid-selected

electrophoresis mRNA's.

of mRNA's

the translational were hybrid-selected pBR-ALB (lane 3),

1).

products of by MlS-cAtB M13-cCPS1 (lane

(lane 1). M13-mALB (lane 2), 4 and 5), M13-mCPS1 (lane 6 and 7), pBR-CPSl (lane B and 9), pPCK 10 coding for PEPCK (12) (lane 10 and 11) and were translated. Lanes 5, 7, 9 and 11 depict immunoprecipitated translation products. described

albumin

(Fig.

were

4))

pBR-ALB,

the

cDNA clone amplified.

inserts

either

the

strand

(M13-mCPS1

hybridization latter (lane

5).

respective products (Fig.

in in

(Fig.

5). The hormonal

diabetes, the

that

administration liver liver

a shift

(lane cyclic

4).

1 digestion into

were

Ml3 mpl0.

identified

to poly(A)+RNA

in mobility

is

of the mRNA that

regulation elevates

liver

cyclic

containing

3)

In the

(21).

the cDNA-like

was unambiguously was hybrid-selected

strand

established by the

of the translation

demonstrated

of dexamethasone, 5). Feeding AMP levels

(Fig.

of CPS mRNA was estimated is

Clones

or the mRNA-like

by complementary

seen with

and immunoprecipitation The size

translation

of pBR-CPS 1 and

and M13-cALB)

of the cDNA clones

translation clones

Pst

(M13-cCPS1

and M13-mALB)

The identity

and by hybrid-release

re-inserted

strand

and hybridization only

case,

by in vitro

After

were

cDNA-like

(23)

in Fig.

to be 6 Kb 6. Experimental

AMP levels (24), in combination with induces high levels of CPS mRNA

a protein-free diet, that (24), to an adrenalectomized 885

leads to a decrease rat causes

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

76543-

-pCPS 2-

.28S 8PEPCK ’ ppALB

I-

818s sI-

I 03

I 05

I 07

I 0.9

06

relative mobility Fig.

5. Estimation

of the size of CPS mRNA from rat liver.

Fig. 6. Northern blot in kidney (lane 1 and induction (lane 2 and blots were hybridized and to pBR-CPS3 (lanes

a low level

analysis of CFJSmRNA, PEPCE mRNA and albumin 4) and liver total RNA under conditions of 5) and deinduction (lane 3 and 6). Northern to pPCK 10 and pBR-ALB combined (lanes l-3) 4-6).

of CPS mRNA (lane

shown by the fact

that

(PEPCK) mRNA levels specificity

6).

albumin

are not

affected

of CPS and albumin

by the absence PEPCK gene

is

The specificity

and also

expressed

in both

by this

this

treatment

gene expression

of the corresponding

of

effect

phosphoenolpyruvate

is

carboxykinase (lanes

by the liver

mRNA's in kidney

mRNA

2 and 3). is

extracts,

The

demonstrated while

the

tissues. DISCUSSION

The level mainly upon

of CPS activity

by glucocorticosteroids the assumption

transcriptional

that

level,

rats

mRNA as judged

1% of total percentage

based

as markers,

than

the estimate

based

primarily

which

is known

anomalous

AMP (see

by in vitro

upon

We have estimated size

and cyclic factors

as a rich

mRNA as judged is

in rat

exert

we have routinely

dexamethasone-treated of total

these

and protein

the size

their

is

e.g.

on the

translation

(see (see

e.g.Fig.

e.g.Fig.

mRNA abundance

of CPS mRNA, using

electrophoretic

rRNA mobility

is

high also

mobility

886

in Fig.

0.5% 1) and approx.

6; this

latter

of 7% (25)). mRNA's of known 0.5 Kb shorter appears to be

of ribosomal

in comparison

illustrated

Based

at the

of diabetic,

6 Kb (Fig. 5). This is et al (21). Their estimate

to be relatively

4-9).

of CPS mRNA (approx.

to be approx. of Ryall

controlled

effects

used liver source

by hybridization an albumin

liver

with

RNA's,

mRRA's.

2 where

This

the peak

of

Vol. 124, No. 3, 1984 translational

activity

absorbance size

BIOCHEMICAL

peak,

necessary

translation

is

located

suggesting

that

for

a protein

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

well

of methylmercury

hydroxide

through

was larger

than

285 rRNA (not

size

as measured

with

the estimated Fig.

mRNA-like

As Fig. translation

expected,

the very

repeatedly

clearly

this

procedure

3' non-coding levels

is

of gene turnover

level

of this

capacity

for

(C.J.

CPS protein

is

can identify a hybridworked

and M13-mCPS1

as

failed activity.

are observed

expression

strand

limited

6)

at the transcriptional

also

be excluded.

suggest

et al.,

the rate

in embryonic

upon changes

AMP (Fig.

of RNA cannot

de Groot

to study

mRNA, especially

5).

as the

of the cDNA-like

that

in CPS mRNA levels interest

be of great

(Fig.

and M13-mALB

and cyclic

regulation

It

gels

system,

only.

differential

the transcriptional

in accordance

although

M13-cCPS1

in CPS mRNA levels

changes

is

in CPS mRNA translational

of glucocorticosteroids

developmental

over

M13-cALB

due to hybridization

region

direct

CPS mRNA

reaction

surprise,

with

However,

in mRNA hybridization

hybridization

with

same procedure

failure

although

will

which

control

To our

Kb) rRNA

the minimum

show that

in formaldehyde

a simple

strands.

to show any differences

suggest

level

a very

3 shows,

The differences in the

shown),

elegant

-arrested

to the

did

strand

provides

than

mRNA, fractionated

agarose

of the Ml3 cloning

cDNA- and mRNA-like

Possibly

denatured

low-melting

28S (4.7-5.3

l,(ref.26)).

4 shows the usefulness

reactions. the

the

smaller

of 165 Kd (Fig.

by electrophoresis in size

below

CPS mRNA is

liver,

regulation

manuscript of synthesis where

The at

in preparation). and turn-

the synthetic

(27).

ACKNOWLEDGMENTS. We wish to thank Drs. H. Pannekoek and H.F. Tabak for useful suggestions and excellent advice, Prof. J.M. Tager for critically reading the manuscript, and Ms. J. Husslage and Ms. M.Nouwen for secretarial assistance. These investigations were supported in part by the Foundation for Medical Research FUNGO Grant no.13-53-38. REFERENCES 1. Gaasbeek Janzen, J.W., Lamers, W.H., Moorman, A.F.M., de Graaf, A., Los, J.A., and Charles, R. (1984) J. Histochem. Cytochem. 32, 557-564. 2. Knecht, E., Hernandez, J., Wallace, R., and Grisolia, S. (1979) J. Histochem. Cytochem. 27, 975-981. 3. Ryall, J., Rachubinski, R.A., Nguyen, M ., Rozen, R., Broglie, K.E., and Shore, G.C. (1984) J. Biol. Chem. 259, 9172-9176. 4. Lamers, W.H., and Nooren, P.G. (1980) Biol. Neonate 31, 113-137. 5. Lamers, W.H., and Mooren, P.G. (1980) Biol. Neonate 37, 264-284. 6. Lamers, W.H., and Mooren, P.G. (1981) Biol. Neonate 40, 78-90. 7. Lamers, W.H., and Mooren, P.G. (1981) Mech. Ageing Dev. 15, 77-92. 8. Lamers, W.H., and Mooren, P.G. (1981) Mech. Ageing. Dev. 15, 93-118. 9. Lamers, W.H., -and Moore!, P.G. (1981)_.. -. Mech. Ageing Dev. 15, 119-128. 10. Charles, R., de Graaf, A., Lamers, w.ki., and Moorman, A.FX. (1983) Mech. Ageing Dev. 2, 193-203. 887

Vol. 124, No. 3, 1984 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry Is, 5294-5299. Lamers, W.H., Hanson, R.W., and Meisner, H.M. (1982) Proc. Natl. Acad. Sci. USA 2, 5137-5141. Alvino, C.G., Tassi, V., Paterson, B.M., and Di Lauro, R. (1982) FEBS Letters 137, 307-313. Grivell, L.A., Reijnders, L., and Borst, P. (1971) Biochim. Biophys. Acta 247, 91-103. Charles, R., de Graaf, A., and Moorman, A.F.M. (1980) Biochim. Biophys. Acta, 629 36-49. Laemmli, U.K. (1970) Nature 227, 680-685. Laskey, R.A., and Mills, A.D. (1975) Eur. J. Biochem. 56, 335-341. Meinkoth, J., and Wahl, G. (1984) Anal. Biochem. 138, 267-284. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory Publications. Grunstein, M., and Hogness, D. (1975) Proc. Natl. Acad. Sci. USA 12, 3961-3965. Chandler, P.M. (1982) Anal. Biochem. 127, 9-16. Cimbala, M.A., Lamers, W.H., Nelson, K., Monahan, J.E., Yoo-Warren, H ., and Hanson, R.W. (1982) J. Biol. Chem. 3, 7629-7636. Kloussis, D., Hamilton, R., Hanson, R.W., Tilghman, S.M., and Acad. Sci. USA 76, 4370-4374. Taylor, J.M. (1979) Proc. Natl. Seitz, H.J., Liith, W., and Tarnowski, W. (1979) Arch. Biochem. Biophys. 195, 385-391. Nahon, L.J., Gal, A., Froin, M., Sell, S., and Sala-Trepat, J.M. (1982) Nucleic Acid Res. lo, 1895-19111 Raymond, Y., and Shore, G.C. (1979) J. Biol. Chem. 254, 9335-9338. Lamers, W.H., Zonneveld, D., and Charles, R. (1984) Dev. Biol. in press.

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