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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