Grw.

104 (1991)

;c: 1991 Elscvicr

GENE

197-202 Science

Publishers

B.V. All rights reserved.

197

0378-l 119,‘9lK$O3.50

051146

Cloning and expression analysis of a cDNA encoding fumarylacetoacetate tional modulation in rat liver and kidney (Recombinant DNA; Northern/Western ends; nucleotide sequence)

blotting;

gene regulation;

hereditary

tyrosinemia;

hydrolase: post-transcrip-

rapid amplification

of cDNA

Yves Lahelle, Daniel Phaneuf and Robert M. Tanguay Ontog&t%e et Gbn&tique Moltkulaire, Received by S.1. Case: 24 December Revised: 4 April 1991 Accepted: 5 April 1991

Cerltre de Recherche du Cerltre Hospitulier de I’lJniversitt! Laval. Sainte-Fo,y. QuPhec Gl V 4G? (Canada) 1990

SUMMARY

Fumarylacetoacetate hydrolase (FAH) is an enzyme which is deficient in human hereditary tyrosinemia type 1. We have cloned and sequenced a rat liver cDNA encoding FAH. The identity of the clone was ascertained by hybrid-selection experiments and deduced amino acid (aa) sequence homologies with sequenced oligopeptide fragments of the purified rat liver protein. The cDNA codes for a 419-aa protein of 45 946 daltons. We used this cDNA as a probe in conjunction with a specific anti-rat FAH antibody to study the expression pattern ofthe FAH gene in rat liver and kidney. Northern blot analysis indicates that the kidney contains slightly more FAH mRNA than the liver. Western blotting shows, however, that the liver contains about twice as much FAH protein as the kidney. Primer extension experiments suggest that there are no differences in the 5’-untranslated (UT) ends of the FAH mRNA of both tissues. We conclude that synthesis of the FAH protein is in part regulated at the post-transcriptional level in rat liver and kidney, and that this regulation does not appear to be mediated by the 5’-UT sequence of the FAH mRNA.

INTRODUCTION

Fumarylacetoacetate hydrolase (FAH; EC 3.7.1.2) is the terminal enzyme in the catabolic pathway of tyrosine. The

Correspmdmce ro: Dr. R.M. Tanguay, laire,

Centre

Sainte-Foy,

de

Recherche

QuCbcc,

Tel. (418)654-2103; Abbreviations:

CHUL,

et Genetique

Mold-cu-

Boulevard

Laurier,

2705

GlV 4G2 (Canada) Fax(418)654-2748.

aa,

deoxyribonucleoside aceloacetate

Ontogtn&se

du

amino

acid(s);

triphosphate;

hydrolase;

bp,

base

DTT, dithiothreitol;

FAH, DNA (and mRNA)

pair(s);

dNTP,

FAH,

fumaryl-

encoding

FAH;

kb,

kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PA, polyacrylamide; PCR, polymerase chain reaction; Pipes, 1,4-pipcrazinc-diethanesulfomc SDS, sodium UV, ultraviolet.

dodecyl

acid; RACE, sulfate;

rapid

tRNA.

amplification

transfer

of cDNA

ends;

RNA; UT, untranslated;

enzyme is deficient in hereditary tyrosinemia type 1 (Lindblad et al., 1977; Tanguay et al., 1984; 1990; Berger et al., 1987), an autosomal recessive disease characterized by elevated levels of tyrosine and its metabolites, chronic liver failure and renal tubular dysfunctions (see Goldsmith and Laberge, 1989). The incidence of the disease in the Province of Quebec (Canada) is 1: 10000 births. However in a northeastern region of the Province, it reaches 1 : 1850 (De Braekeleer and Larochelle, 1990), possibly due to a founder effect (Laberge, 1969). Human FAH is mainly expressed in liver and kidney (Tanguay et al., 1990). Lower levels are also detected, ranging from 2.5 to 0.01’; of the liver level, in a variety of tissues and cell types: adrenal gland, lung, heart, bladder, intestine, stomach, pancreas, spleen, skeletal and cardiac muscles, placenta, chorionic villi, fibroblasts. lymphocytes, leucocytes and cultured amniotic fluid cells (Kvittingcn

et al., 1983; Berger et al., 1987: Tanguay et al., 1990). The liver and kidney are presumably the only tissues containing the whole set of enzymes necessary for the complete degradation of tyrosine (Lindblad et al., 1977).

1

2

3

4 kDa

Nicole et al. (1986) have purified the FAN mKNA from rat liver and estimated its relative abundance at about 0.14”,, of total mRNA. Berger et al. (1987) find that this value agrees with their estimate that FAH accounts for about 0.2”,, of total rat liver protein. There is at present no information concerning the regulation of FAN gcnc cxpression. In order to gain some insights as to the mechanisms involved in the differential expression of this gcnc. WC have isolated and sequenced a rat liver FAH cDNA clone. The

-

92

-

45

-

FAH

-

30

cxprcssion pattern of the f+l H gene was analysed in rat liver and kidney by Northern and Western blotting. We report that the expression of the FAH protein is post-transcriptionally regulated in these two tissues.

RESULTS

AND DISCUSSION

(a) Isolation of the FAH

cDNA A rat liver cDNA library constructed in the expression vector Agtl 1 (Schwarzbauer et al., 1983) was screened (Huynh et al., 1985) with an affinity column-puri~cd antirat FAH antibody (Tanguay et al., 1990). A 236bp clone was isolated, sequenced and shown to contain a 160-nt coding sequence (corresponding to the last 53 aa of the protein) and 76 nt of the 3’-non-coding sequence. A region coding for a stretch of 11 aa identical to a sequenced peptide fragment of the purified rat liver FAH protein was identified (see Fig. 2). The clone was further authenticated in hybrid-selection experiments using either an enriched rat liver FAH mRNh fraction obtained by polysome iml~lun~~absorption with the antibody (Nicole et al., 1986) or total rat liver poly(A)+ RNA (Fig. 1, lanes 1 and 2, respectively). The results clearly indicate that the clone selects a mRNA which, following in vitro translation and immunoprecipitation with the FAH antibody, yields a polypeptide of ~lpproxilnately J3 000 daltons, the cxpcctcd molecular weight of the rat liver FAH l~ionon~er (Fig. 1). This clone was used to rescreen the cDNA library by hybridization (Mason and Williams. 1985). and although clones up to about 1000 bp were obtained, DNA sequencing indicated that they did not contain the 5’-UT end of the mRNA. ~urtherltiorc. Northern blots showed that the F;4H mRNA is about 1600 nt long (see section b below). We therefore elected to use the RACE (rapid amplification of cDNA ends) protocol recently described by Frohman (1990), with the PCR, to clone the 5’-end of the &LAHmRNA. The primer for reverse tr~~nscripti(~Ii ofthe nnRNA and the gcnc-specific primer for ~~llipli~cati~~?l are shown in Fig. 2 (see legend). Amplification products \vcrc

Fig. 1. Iiybrid

seiection

of FAW mRNA.

iz 236-bp cDNA

was inserted

mto the pT.ZlSR vector and 1OOgg of the supercoiled r~~~rnb~t~~nt plasmid wcrc denatured in 0.4 N NaOH at 1W’C for 5 min, cooled in an and adjusted to I M ammonium acetate. The DN.4 I cm2 Gene Screen nylon membranes (Dupont) previously washed in water and I M ammonium acetate. Membranes were

ice bath, centrifuged tias pipcttcd air-dried

unto

and the DNA denatured

1min and neutralized Membranes

6&l

was

again in 0.5 N N&l

I:‘15 M NaCl for

1 M Tris HCI. pfI 8.0:1.5 M NaCl for 1min.

wcrc W-irradiated

at room temperature rcitlon

in

for 5 mm and washed

3 times with water

and once with water at 100 C for 30 see. Prehybridi-

at 42’C

for 2 h in 50uc, formrrmide~?O mM

Pipes.

pH

mM EDTAI’0.4 M NaCI,‘O. 2”,, SDS:5 p’g per ml tKNA.‘IO 1~6per ml

po1y(A). Hybridization

was carried

except tRN.4 was omitted

or an imrn~~~nol~t~rilicd FJIi present.

Membranes

out overnight

in the same conditions

’ RNA at I mgml

and either rat liver poly(A) mRNA

were washed

fraction

(Nicolc

et al., IYXh) was

10 times in 0.15 M NaCI~O.0 15 M

sodium citrate,,O.S” 0 SDS. 5 times in 2 mM EDTA pH 7.0, and RN.4 wab cluted twice in water at 100-C for 1 min. The cluted RNA ~35 precipitated with ethanol in the presence ofcarrier tRNA, recovered in water nuclcn~c-trcatcd lys31c and translated in ;t rabbit reticulocyte (~~nl~rsh~lli~) ~oilt~itlirl~ immuilctprecii7it:ftcd

I -1~~Slin~thinr~ille. Tr~~n~l~lti(~llproducts

with the anti-rat

ct al., 19X6). separated dried gels were exposed

FAH antibody

in 12”,, PA gels containing to XAR-5 films (Kodak).

ah dcxribed Il. I “,,

were (Nvxle

SDS and the

Results of the second

clution are shown. Lanes I and 2: plasmid with insert hybridized \rith the purified fraction and total poly(A) ’ RNA, respcctivety: Ianes 3 and 4: p&mid

without

insert

hyhridizcd

with total

po1y(A)

’ RNA ;md the

purified fraction. rccpectively. Protein markers arc shoxvn on the right margin (phosphcrylase a. 92: nctin, 45; and cxhomc :mh>drasc, 30 kD:i).

199 C_CC_GCCCAGTGCTCT_CAGC ATG

TCC

TTT

n::

CCG

GTG

GCC

GAG

GRC

TCC

GAC

TTY

ccc

ATC

C4A

AAC

CT;

xc:

ser

9b.e

iLe

pro

val

ala

9-u

asp

ser

asp

phe

313

ile

qi-,

asr

Ic:,

CGG

ATT

GCT

GTG

GCC

ATC

GGT

GAC

CAG

ATC

TTS

GAC

CTG

AGT

STC

A-T

arq

_lr

gly

vdl

d-a

ile

gly

asp

qln

ile

lee

asp

leu

ser

“a_

:le

ccc prc

TAT

GGC

GTT

TIC

TCC

ACT

CA.&

AGC

AAC

CCA

AAG

cc*

1:2

:yr

qly

“al

pne

ser

tllr

q1n

ser

a*>

prc

lYS

pro

i3

AAA

cnc

CTC

TTT

ACC

GGA

CCT

GTC

CTC

TCC

AAA

CAT

CA:

CAT

102

lys

5s

:el.

obe

thy

O!Y

0’0

“al

!el.

SP’

1~s.

:lis

gin

his

69

GCA

AGA

GCR

?CT

TTA

CAG

?‘TA

C?C

TCT

292

OLIGO GTC

XTC

-.a1

phe

as”

qll.

ti:r

ZhI

led

asn

se:

Fhe

‘net

see

AGC

CAA

GCC

CAG

CTC

AGA

GAT

GRC

AAG

GAG

ala

0-c

leu

arq

asp

asp

lys

glJ

Ia

CAT

GAG

se-

ACA

ACT

CTC

AAT

RGC

TTT

ATG

GGC

CTC

GGC

CGA

CCG

qly

le’~

gly

?_r

ala

CTT

CGG

CAG

IST

GCh

leu

arq

qln

ar;

ala

22

1

AAC

GAG

a?a trp

lys

q_”

ala

arq

ala

ser

le,

olr:

AAC a,..*.>.

,e”

_e~

se_ -

10

TTC

ACC

TCC

CRG

GCT

-CT

GCC

ACG

AT’,

CAC

CTT

cc:

SCT

ACC

382

phe

thr

ser

gin

ala

ser

ala

thr

met

his

lex

pro

ala

:.3r

:*O 412

GCA

?‘GG

ATA

GGR

S.AC

‘TAC

ACG

TAC

TTC

TAC

TCC

TCT

CTG

CAG

CAY

GCC

ACT

AAC

GTT

GGC

RTT

ATG

TTC

AGG

GGC

ARG

GAG

AAT

GCG

CTG

YTG

qly

asp

tyr

rhr

asp

phe

tyr

ser

ser

1eu

g1n

ccc

ile

b..;S

ala

tb.r

as”

val

gly

ile

met

phe

arq

qly

1ys

q:u

asr

ala

1eu

leL

FrO

150

RAT

TCG

CTC

CAC

TTRCCT

GTG

GGA qly

TAC tyr

CAT his

GGC gly

CGA arg

GCT ala

TCC ser

TCC ser

GTY va?

GTG val

GTG val

TCT ser

GGT gly

ACC thr

CCA pro

ATT ile

CGA arq

AGA arg

CCC pr3

ATS mew

GGA qly

CAG qln

ATG met

562 1SC

asr- trp

?eu

his

;eu

pro

val

AGA aI9

CCT Pro

GAT asp

ARC TCA .3Sr. SfT

ARG

CCT

CCT

GTG

TAC

GGT

GCC

AGC

AAA

CGC

TTA

GAC

ATG

lYS

PI0

PI0

Vdl

tY=

gly

ala

Se_;

lyS

aZg

162,.

asp

met

GAG‘TTG qlu leu

GAA ql"

ATG met

GCT ala

TTC phe

TTT phe

6-h "al

GGC qly

CCT pro

666 gly

AAC as"

652 210

AGA arg

YTC pk

GGC 41"

GAG &

CCA DTO

ATC 11e

CCC ATT F'S 1-e 0.0

TCC ser

AAG lys

GCC ala

CRC gin

GAG qlu

CAC his

AT: Ile

TTC GGG pr.e qly

ATG Per

GTC val

CTC leu

ATG met

ARC as"

GAC asp

TGG trp

AGT ser

GCT ala

CGA arz

ZAC asp

ATC 1lr

CAG qln

742 240

CAA

TGG

GAG :I"

TAC tyT

GTC Val

CCC pro

CTT le"

CCA pro

TTC phe

CTG ?e"

GGG qly

AAA lys

AG: se:

-TT pie

GGA qly

ACC thr

ACC 5hI

ATC ICC 11'2 Ser

CCA

TGG

GTG

GTG

CCC

ATG

GAT

GCT

C-C

ATG

832

PTC

trp

VaL

val

pro

me?

aSp

ala

iEJ

met

2.10

A:C AAC ~lt, as"

CTG :e~

TCC ser

922 3OC

q:fl tr?

GGG qly

OLlGO

2

CCC

T:T

G-G

GTG

CCA

AAC

CCA

ARG

CAG

GAC

CCT

AAG

CCC

CTG

CCA

TAT

CYC

TGC

CAC

RGC

CAG

CCC

TAC

ACA

pro

phe

val

val

pro

asn

pro

lys

qln

asp

pro

.ys

prc

lee

p-3

iyr

leu

cys

his

ser

gin

pro

tyr

thr

TTY phe

GAT as?

GT? va!

GCT ala

'ITG AAA led :ys

GGA gly

GAA qlu

GGR qly

AYG net

AGC se:

CAG qln

GCA ala

GC: ala

ACC chr

A:C ale

:GC cys

,',GG TCC arq seT

AAC asr

TTT AAG F'le :yS

CAC

A?G

-AC

TGG

ACC

AT': CTG

CAG

CAA

CTG

1012

hi*

Ret

tyr

tT?

thl

lie

le.>

ql”

ql”

le”

,2c

ACA

CAC

^iiC

TCY

G:T

RAT

GGA

TGC

AAT

CTG

AGA

CCT

GGS

GAC

CYC

:TS

GC?

TC:

GGA

ACC

A:C

AGT

GGR

TCA

GAC

CC:

GAP,

AGC

:TT

GGC

1132

tnr

his

cis

ser

val

as"

qly

cys

asri le"

arg

3ro

gly

asp

leL

:e;

a;a

se:

qly

ztr

iLe

ser

g;y

ser

asp

pro

glu

ser

pne

q:y

363

TCC

ATS

CTG

GAA

"TG

TCC

TGG

AAG

GSA

ACA

AAG

GCT

ATC

GAT

G:G

GGS

CAG

&CC

AGG

ACC

TTT

CTT

CTG

GRC

GGR

ser

net

leu

ql-

leu

ser

tr.c lys

gly

thr

lys

ala

ile

asp

"al

gly

qln

GGC, CA.4 qly gin

thr

arg

-hr

ohe

!~II let> asu

aiv

GAT GAA GTC iiso clia vd

1192 39c

ATC w

ATA

ACA

GGT

CRC

TGC

CAG

666

661 qly

GGG

AAA

GTG

gly

GTY val

GCT

gin

CGT arg

TGT

cys

TAC ryr

CAA

his

GGC gly

GG:

gly

GAT asp

T?T

tnr

phe

gly

gin

cys

ala

gly

lys

val

CTG leu

CCT pro

TCG ser

CCA pro

1282 419

AGCTCCAGAATCCAChGRACRCAGCC?.

Fig. 2. The nt sequence chain termination Montreal) and arrows

indicate

The putative

of the rat liver F.4H cDNA

and deduced

aa sequence

peptide fragments

of the protein. (performed

Oligo (OLIGO) primers (I 8 nt long) used in the RACE protocol

direction

used as the gene-spccitic

of synthesis:

oligo 2 was used for reverse

primer for the subsequent

polyadenylation

CTC leu

GCC ala

TGA end

~GCCTTGTGAGGATCATACTGCAACTGCRTGRGTCAGGAATG~G~T~TTTTG*TTGGGG*~******~*~~~

method of Sangcr et al. (1977). Sequenced

are underlined.

GCC ala

signal is underlined

amplification (in the bottom

reaction.

transcription

(b) Expression of the FAH gene In order to determine the copy number of the FAH gene, Southern blot experiments were performed on rat genomic DNA using two probes: one covering most of the coding sequence of the cDNA and the other covering only the 3’-extremity of the cDNA (Fig. 3. panels A and B, respectively). The results show that for each of the six restriction enzymes used. between three and six bands hybridize to the long probe whereas only one band hybridizes to the short

Both strands

were sequenced

by Dr. Van der Rest, Shriners (Frohman,

of the FAH mRNA is deposited

completely

Hospital

1990) for 5’.end extension

in GenBank

by the dideoxy

for Crippled

Children,

ofthe cDN.4 are overlined

from rat liver poly(A)’

Oligo I was also used in the primer extension

line). The sequence

cloned and six independent recombinants were sequenced. This analysis permitted us to add 250 nt at the 5’-end of the FAH cDNA. The complete cDNA is 1386 nt long including 13 A residues of the poly(A) tail. A search in GenBank revealed no extensive significant similarity with stored sequences. The cDNA encodes a 419-aa protein of 45946 daltons. In addition to the sequenced peptide fragment found in the original clone, three regions were found to code for stretches of aa identical to other sequenced peptide fragments (Fig. 2).

1386

RNA and oligo

experiments

under accession

described

number

I was

in Fig. 5.

M37685.

probe. This suggests that there is a single copy of the FAH gene per haploid rat genome. The liver and kidney arc the two major organs where FAH is found. In humans, the liver contains about three times the amount found in the kidney (Tanguay et al.. 1990). Western blot analysis of total proteins from rat liver and kidney using the FAH antibody shows that the situation is analogous in the rat (Fig. 4, panel A): the kidney contains about 459,, the amount of FAH found in the liver. Overexposure of the film reveals a faint band present in the spleen sample (data not shown). The minor band seen just beneath the major band in the liver sample is a truncated form of the FAH protein. Evidence for this comes from transfection experiments using a human FAH cDNA and showing that the presence of this minor band, along with that of the major band, is dependent upon transfection of the FAH cDNA in CV-1 cells (Y.L.. unpublished observation). The same tissues were analysed by Northern blots of total RNA with the FAH cDNA probe. A band at about

EC

Ba

Ps

HI

0g

xb

EC

Ba

Ps

HI

f3g

Xb

Cb

,J 0

23

I .I

Oi

1.1p. 3. Southern digcstcd

blot analysis

in duplicate

a 0.8”,, agaroac a 7’P-labcllcd

ofthe

FAH gcnc. Rat spleen genomic

with the indicated

gel. The DNA was transferred probe (Multiprimc 1, markers

are shown

18724

enzqmes containing

nylon membrane

either

(panel B). Hybridization bet\\een

DNA was isolated

74

CPM - 45 kDa

18 s

IOD

4

143

Western and Northern blot analyals of FAH in rat h\cr, kldncy spleen. Six to seven weeks old male Sprague Dawley rat\ v.ere killed

Fg. 1.

b> dccapltation stored at -40

and the tissues remo\zed. frozen in Ilqmd nitrogen and C. (A) Samples from each tissue were homogenized in

IO”,, (WV) 10 mhl sodium phosphate 2Omin

a1 4-C

I’rotcm

concentrations

and

according

washes

to the supplier’s

were done according

to standard

pH 7 2, centrifuged

the supcrnatants

rccovercd

and

at I2Wl

x gfol

kept at -2O’C.

were determmed

111 to

(panel A) or only the last

methods

(Maniatis

et al.. 1982).

VV;LS puriticd from each tissue sample by the method of C‘hlrgwn et al. (lY7Y). Ahquots mamidc.

(5 h(g) were denatured in 6.5”,, formaldehyde/50”,, forin 1 “,, agarose gels containing 6.5”,, formaldehyde.

separated

transferred

to Zeta-Probe

nylon membranes

mined by cuttiny and counting

accurate

the area of the tiltcr corrcspondlnp

(Packard).

and hybridized

(c) Analysis of the 5’-end of the FAH mRNA It is known that sequences at the 5’-UT end of mRNAs are involved in translation regulation (Kozak, 1986). Furthermore, many examples have been found of 5’.UT sequence heterogeneity in single mRNA species in mammals (see Smith et al., 1989 and references therein). Such heterogeneity can arise by alternative splicing mechanisms

a ‘“P-labelled FAH probe according ct al.. 1982). Prior to hybridization, methylenc blue to confirm equivalent

counter

instructions

1600 nt is detected in both tissues; however, it can be seen that the kidney contains slightly more FAH mRNA than the liver (Fig. 4, panel B). Similar results have been found in six dif’f’crent animals examined, thus they can be considered as being rcprescntative of the expression pattern of the FAH gene in these rat tissues.

(Bradford. 1Y7h). and 25 ~(6 aliquots were separated in 12”,, PA-O. I ‘I,, SDS gels. transferred to nitrocellulose filter:, and rcactcd first \%ith the [rabbit anti-rat t AH nntibod) “‘I-labclled an(ibody as and second with a goat anti-rabbit I& described prcviouslq (Tan~uay et al.. 1900). Radioacti\ 10 \!a\ deterband in a Gamma

(19X7) and 10 /~g wcrc

SPLEEN

KIDNEY

B

and

(BioRad)

and Frlschauf

Bg, BglII and Xb: Xhal) and scparatcd

the two panels

8617

122

of Hermann

Ps: PsrI, Hi: HindIII,

1029 bp (or X2”,,) of the coding region of the FAH cDNA

and membrane

A

LIVER

by the method

(Ba: Brrf>?HI. Ec: EcoRI,

to a %&-Probe

kit, Amersham)

?2 bp (2.6”,,) of the coding sequence BstEll-digested

restriction

(B) For Northern

to each

blots. total RNA

ribosomal warch

RNAs. Bands wcrc quantified

Analysis

integrated

System. Cl.-1000

program,

within a IO”,, error margin. optical

density.

(Bio-Rad)

and hybridized

to

to standard procedures (Maniatis the membrane was stained with sample loading of the 28s and 18s by dcnsitomctric Amersham)

scanning

(Re-

and number5 are

18s: 18s rat ribosomal

RNA: IOD:

201 12

34

In order to determine

whether

such a situation

occurs

with the FAH mRNA, primer extension experiments were carried out with rat liver and kidney poly(A)+ RNA using oligo 1 (see Fig. 2) as primer. The results indicate that the longest 5’-UT end detected is 69 nt long (Fig. 5. band at 3 15 nt). It is not clear whether the lower bands are true 5’-ends or incomplete elongation products. In any cast, the band distribution that is seen in the liver and kidney is identical in terms of both the length and relative proportion of each band (Fig. 5, lanes 1 and 2). Thus, these results

nt

suggest that there is no 5’-UT sequence heterogeneity in the rat FAH mRNA from liver and kidney. and the mechanism accounting for the differential expression of the FAH protcin must be sought elsewhere. (d) Conclusions

258

-

Fig. 5. Primer extension The method

is essentially

analysis

Fig. 2) was 5’.end-labelled activity

of approx.

otide kinasc

ofFAH

mRNA

that ofwilliams with [;‘-“P]ATP

7 x IO” cpm;pg

as described

in rat liver and kidney.

and Mason (1985). Oligo I (cf. (6000 Ci;‘mmol) to a specific

using bacteriophage

by Ausubel

T4 polynucle-

et al. (1987). Primer

(5 fmol) was

mixed with 5 pg of RNA in 0.4 M NaCI/IO mM Pipes, pH 6.4 in a final volume of IO ~1. The reaction and hybridized

overnight

the mixture was adjusted

mixture was sealed in a 25 ~1 glass capillary

at 5O’C. After expulsion to 50 mM Tris

MgC12/25 ng per ml actinomycin mycloblastosis 42’C.

virus

the mixture

formamide/O.O5”,, 95°C

reverse

Lanes I and 2: elongation liver

in parallel (in nt).

and

(Pharmacia),

with ethanol,

cyanoli0.05”,

for 5 min and analyzed

kidney, respectively; from

D/500 mM each dNTP/lO transcriptase

was precipitated xylem

products

bromophcnol of poly(A)

respectively.

was used to determine

tube,

I h at in !95”,,

blue. heated sequencing

to gel.

’ RNA from liver and

reactions A

units avian After

recovered

in a 4”” PAZ M urea

lanes 3 and 4: elongation kidney,

into a microfuge

HCI pH 8.4/‘10 mM DTT:‘6 mM

ofpoly(A)

sequence

the length of the extended

ladder

RNA run

molecules

and/or through the use of different transcription start points. In the case of the mouse z-amylase mRNA, a 5’-UT sequence heterogeneity was shown to be tissue-specific in the liver and salivary gland (Hagenbtichle et al.. 1981).

We have isolated a rat liver FAH cDNA clone and used it in conjunction with a specific anti-rat FAH antibody (Tanguay ct al., 1984) to study the expression pattern of the FAH gene in rat liver and kidney. Our results establish two points: (1) although there is about twice as much FAH protein per mg of total protein in the liver versus the kidney, the kidney contains slightly more FAH mRNA per mg of total RNA than the liver; (2) as shown by primer extension, the 5’ ends of the FAH mRNA are identical in both tissues. Our data suggest the following picture for FAH gene expression: in both rat liver and kidney, mechanisms implicated in transcription and processing result in the production of roughly similar amounts of mRNA. The difference in protein content must therefore occur through a posttranscriptional mechanism. Such a mechanism can involve a more efficient translation of the mRN,4 in the liver and/or a shorter half-life of the protein in the kidney. We have found no 5’-UT sequence heterogeneity by primer extension analysis. However, it is becoming clear that 3’-UT sequences and the poly(A) tail can also influence the translation efflcicncy of mRNAs (reviewed in Jackson and Standart, 1990). We are currently investigating this aspect in the case of the FAH mRNA of rat liver and kidney. We have previously shown that the two clinical forms of tyrosinernia have a different molecular basis and that patients with the chronic form have lower amounts of liver FAH protein than controls (Tanguay et al., 1984; 1990). RNA analysis of these patients using a human l-A/f cDNA (Phaneuf et al., 1Y91) should dctcrminc whether the residual amount observed in the chronic form correlates with a lower amount of FAH mRNA or whether the translation efficiency of the mRNA and/or stability of the protein appear to be involved. Interestingly, Bcrger et al. (198X) have reported differences in the stability of the F.4I-I protein in fibroblasts of patients in both forms of the disease. There are many examples of liver-specific gene expression regulated at the level of transcription through trans-

acting factors (Dcrman et al.. 1981; Kugler et al., 1988; Ochoa et al., 1989). Our results show that post-transcriptional regulation plays an important role in the modulation of the tissue-specific expression of the FAH gene in rat liver and kidney.

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We thank Drs. K. Hynes (MIT) for the rat liver cDNA library and J.P. Valet for the preparation of the affinitypurified anti-FAH antibody, and Drs. P. Morcau and L. Nicole for comments on the manuscript. This Lvork was supported by the Medical Kescarch Council of Canada (R.M.T.). Y.L. and D.P. were supported by Scholarships from le Fonds pour la Formation de Chercheurs et I’Aide in la Rccherche, la Fondation de I’Universit6 Lava1 and le Minist&-e dc I’Education du Qut-bet-Actions structurantes.

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Cloning and expression analysis of a cDNA encoding fumarylacetoacetate hydrolase: post-transcriptional modulation in rat liver and kidney.

Fumarylacetoacetate hydrolase (FAH) is an enzyme which is deficient in human hereditary tyrosinemia type 1. We have cloned and sequenced a rat liver c...
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