Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
4983
-4990
Complex
pa e n
o
al e na i e
splicing
gene a es
unusual
di e si y
in
he
leade
sequence
o
he
chicken
link
p o ein
mRNA
Fe enc
Deak1,
End e
Ba a1
2,
Sil ija
Mes ic1+,
Ma kus
Biesold1
and
lbolya
Kiss'*
Ins i u e
o
Biochemis y,
Biological
Resea ch
Cen e
o
he
Hunga ian
Academy
o
Sciences,
H-6701
Szeged,
PO
Box
521
and
2Ag icul u al
Bio echnology
Cen e ,
H-2101
Godillo,
Hunga y
Recei ed
June
5,
1991;
Re ised
and
Accep ed
Augus
27,
1991
ABSTRACT
We
epo
he e
he
isola ion
o
he
5'
end
and
he
p omo e
egion
o
he
gene
o
chicken
ca ilage
link
p o ein,
and
demons a e
ex ensi e
he e ogenei y
o
he
leade
sequence
a ising
om
di e en ial
u iliza ion
o
mul iple
splice
si es
wi hin
he
5'-mos
exon.
The
500-base
pai s
(bp)
exon
1
consis s
o
solely
un ansla ed
sequence
and
is
ollowed
by
an
in on
>33
kilobase
pai s
(kb).
Toge he ,
he
i e
exons
p edic
a
gene
size
longe
han
100
kb.
Mul iple
ansc ip ion
ini ia ion
si es
we e
mapped
34,
46,
56,
66
and
76
bp
downs eam
o
a
TATA-like
mo i .
Sequence
analysis
e ealed
ha
in
addi ion
o
he
non-
spliced
a ian ,
mul iple
mRNA
species
we e
gene a ed
by
al e na i e
splicing
esul ing
in
he
exclusion
o
92,
166,
170,
174
and
263
nucleo ides
(n ),
espec i ely,
om
exon
1.
Polyme ase
chain
eac ion
con i med
he
exis ence
o
a ious
splice
o ms,
and
showed
cell
ype-
and
de elopmen al
s age-speci ic
exp ession
o
one
g oup
o
hem.
Seconda y
s uc u e
p edic ions
indica ed
ha
he
leade s
o
he
splice
o ms
could
o m
s able
hai pin
s uc u es
wi h
di e en
ee
ene gies
o
o ma ion
(up
o
AG
=
-
1
10
kcal/mol),
sugges ing
ansla ional
con ol.
The
splice
a ian
de ec ed
in
he
la ges
amoun
had
he
leas
s able
p edic ed
hai pin
(AG
=
-
31.7
kcal/mol).
INTRODUCTION
Link
p o ein
(LP),
an
abundan
glycop o ein
o
he
ca ilagenous
ex acellula
ma ix,
s abilizes
he
p o eoglycan
agg ega es
ia
simul aneous
binding
o
hyalu onic
acid
and
chond oi in
sul a e
p o eoglycan
(1-5).
Two
o
h ee
o ms
o
LP
ound
in
se e al
species
(6-8),
seem
o
di e
only
in
p o eoly ic
clea age
o
a
sho
pep ide
and
in
he
deg ee
o
glycosyla ion
(7,
9).
The
p ecu so
o
chicken
LP
consis s
o
355
amino
acids
including
a
sec e o y
signal
pep ide
(10).
The
ma u e
p o ein
is
di ided
in o
h ee
s uc u al
and
unc ional
domains
s abilized
GenBank
accession
no.
M35040
by
disul ide
b idges
(11,
12).
The
immunoglobulin-like
domain
in e ac s
wi h
p o eoglycan,
while
he
wo
andemly
epea ed
domains
bind
o
hyalu ona e
(13,
14).
Link
p o eins
may
ha e
ye
o he
unc ions
as
hey
a e
also
ound
in
issues
o he
han
ca ilage
(15-18).
Fu he mo e,
an
al e na i ely
spliced
exon
encoding
an
ex a
segmen
be ween
he
signal
pep ide
and
he
i s
domain
o
a
chond osa coma
LP
has
been
epo ed
(19).
In
chicken
ca ilage,
mRNA
size
classes
o
5.8
-6.0
kb
and
3.0
kb,
di e ing
in hei
3'
un ansla ed
egion
(3'-UTR),
we e
de ec ed
(10).
The
LP
mRNA
species
a e
encoded
by
a
single
gene
pe
haploid
genome
(12).
The
ou
p o ein
coding
exons,
which
e ol ed
by
duplica ion
and
exon
shu ling,
a e
sca e ed
o e
a
genomic
egion
longe
han
70
kb
in
chicken
(12).
The
5'
end
o
he
gene,
howe e ,
has
no
been
isola ed
p e iously.
In
he
p esen
s udy,
we
cha ac e ize
he
i s
exon
and
he
5'- lanking
egion
o
he
chicken
LP
gene.
A
compa ison
o
he
nucleo ide
sequences
o
genomic
and
cDNA
clones,
and
polyme ase
chain
eac ion
(PCR)
p oduc s
e ealed
ha
al e na i e
splicing
wi hin
he
5'-UTR
ga e
ise
o
he
o ma ion
o
mul iple
LP
mRNA
species
in
chond ocy es.
The
po en ial
ole
o
he
he e ogeneous
5'-UTR
in
he
egula ion
o
gene
exp ession
is
discussed.
MATERIALS
AND
METHODS
P ime
ex ension,
cDNA
and
genomic
cloning
All
posi ions
a e
gi en
om
he
i s
nucleo ide
o
he
ansla ion
s a
codon
o
he
LP
p ecu so
(10,
12).
Oligonucleo ides
(Table
I)
we e
syn hesized
by
phospho amidi e
chemis y
(20)
and
labeled
wi h
T4
polynucleo ide
kinase
(21).
RNA
was
p epa ed
om
s e na
o
14-day-old
chicken
emb yos,
and
om
s e na
and
a icula
ca ilage
o
6-week-old
(ju enile)
chicks
as
desc ibed
(22).
Fo
analy ical
p ime
ex ension,
0.1
pmoles
o
end-labeled
p ime
was
annealed
wi h
4
yg
o
poly(A)+
RNA
immedia ely
a e
dena u a ion
wi h 10
mM
me hyl-me cu ic
hyd oxide,
and
elonga ed
wi h
40
uni s
o
M-
MLV
e e se
ansc ip ase
(BRL)
in
50
il
o
50
mM
T is-HCl
*
To
whom
co espondence
should
be
add essed
+
P esen
add ess:
Pli a
Resea ch
Ins i u e,
41000
Zag eb,
I.L.
Riba a
89,
Yugosla ia
..
1991
Ox o d
Uni e si y
P ess
4984
Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
(pH=8.3),
75
mM
KCI,
3
mM
MgCl2,
10
mM
DTT,
supplemen ed
wi h
0.5
mM
dNTP,
40
Ag/ml
ac inomycin
D
and
20
uni s
o
placen al
RNase
inhibi o .
Fo
he
cons uc ion
o
he
p ime
ex ension
lib a y,
0.25
pmoles
o
P3
p ime
and
5
yg
poly(A)+
RNA
we e
used.
The
cDNA-RNA
hyb ids
we e
ailed
using
dGTP
and
e minal
ans e ase
and
annealed
wi h
1.15
pmoles
o
oligo(dC)- ailed
BamHI
linke
in
he
p esence
o
1.5
uni s
o
RNase
H.
The
second
cDNA
s and
was
syn hesized
wi h
10
uni s
o
DNA
polyme ase
I.
The
ends
we e
polished
wi h
T4
DNA
polyme ase
Table
I.
Oligonucleo ide
p ime
sequences
Name
Sequence
Posi ion
PA
PB1
PB2
PC
Fo wa d
p ime s
5'-CAAGTGGTCAGAAGTATCACGT-3'
-605
o
-584
5'-TAGTTCGGGACTGGTGTGCG-3'
-488
o
-469
5'-TTAGTTCGGGACTGGTGTGCGGTGCAGAGC-3'
-489
o
-460
5'-ACTTGGGAGCTCCACACAA-3'
-46
o
-28
Re e se
p ime s
PD
5'-GTCTCTTTGTGGCTCTGGGTGGCAGAGGAC-3'
-
159
o
-
188
PE
5'-GGTGGCAGAGGACCCTAAAAAAGCTCTGCAC-3'
-
176
o
-
188
and
-452
o
-468
PF
5'-CTGCCCCAGCCTCCGTGCCCCTCAGCTTCT-3'
-404
o
-433
PG
5'-GGTGGCAGAGGACTCACACACAGAGTGTCC-3'
-
176
o
-
188
and
-359
o
-375
P2
5'-CTTGTCATCTTCACAGTCAC-3'
8
o
-
12
P3
5'-ACCACAAGTAGGCGGGGTCC-3'
128
o
109
and
liga ed
o
phospho yla ed
SalI
linke .
A e
BamHI
and
Sall
diges ion,
he
cDNA
was
size
ac iona ed
by
Sepha ose
CL-4B
column
ch oma og aphy
and
inse ed
in o
pUC
12
ec o .
Recombinan
plasmids
we e
in oduced
in o
Esche ichia
coli
DH5
cells
(23).
Colonies
we e
sc eened
by
hyb idiza ion
o
end-labeled
P2
and
P3
as
desc ibed
(10).
A
chicken
genomic
lib a y
(12)
was
sc eened
using
he
277-bp
EcoR l-SacI
agmen
( om
posi ion
-312
o
-35)
o
pLP7G12.
Re e se
ansc ip ion
coupled
wi h
PCR
(RT-PCR)
The
p ocedu e
was
simila
o
published
me hods
(24,
25)
and
was
ep oducible
in
epea ed
expe imen s.
cDNA
was
syn hesized
by
ex ension
o
p ime
P3
using
2
jig
poly(A)+
RNA
as
empla e.
Following
alkaline
hyd olysis
o
RNA
and
e hanol
p ecipi a ion,
he
cDNA
was
ampli ied,
as
indica ed,
using
2.5
uni s
o
Taq
polyme ase
(NEBL)
and
100
pmoles
o
p ime s
in
67
mM
T is-HCl
(pH=8.8),
16.6
mM
(NH4)2SO4,
10
mM
2-me cap oe hanol,
6.7
mM
MgCl2,
0.2
mM
o
each
dNTP,
10%
( / )
DMSO
in
a
inal
olume
o
100
1d.
The
las
i e
cycles
we e
ca ied
ou
by
adding
10
i Ci
o
adioac i e
e e se
p ime
o
analysis
on
sequencing
gels.
In
o he
expe imen s
p ime
PD
was
annealed
o
2
jg
poly(A)+
o
20
, g
o al
RNA
a
64°C
and
e e se
ansc ibed
in
a
inal
olume
o
50
yd.
A e
dena u a ion
o
5
min
a
100°C,
4
$1
o
his
eac ion
mix u e
was
added
o
10
mM
T is-HCl
(pH=7.9),
2
mM
MgCl2,
10%
T i on
X-100,
1
y M
o
each
p ime ,
100
y g/ml
bo ine
se um
albumin,
and
200
,uM
o
each
CTGAGGGCTG
TGTGGTGCCA
GTTCGGTAAG
CAAGGCACAC
TCTGTA
GTGTGATG
AGAGAAGCTG
GACCCAGTTC
CTGGAE
iI:CTCPg
j
-692
G!AGGTGAGG
TAAAACAAAA
GCACAAGGGG
ATCCTGTCAG
GTCCAGCTGT
TTCCACCCCT GCACACCCCT
GCCTTTCATG
CATCACTTTC
TCCCTGTCTG
==========e===========_
-
---------------------------------------------------
-592
AAACCACCAC GTTCCCACCC
TTGGAGCTAA
TGGCAGGGTG
GGGGCAAGTA
AGGGGGGGGG
GGGGGGGGGC
TGCGAGCGAG
CAGAGGCAAG
TGGTCAGAAG
I
I
I
1
-492
TATCACGTTT
CTCAGGGTGT
CCCACACC
AT
ATCCCTTCAT
GAGGAACAGT
TCTTTTTTTT
TTTCTTTTTT
TTTCTTTTTT
TTTCTTTTTT
OG1
5S3
-392
TTTTAGTTCG
GGACTGGTGT
GCGGTGCAGA
GCTTTTTTAG
GTGCAGCTCT
GGGAGGGCAG
AAGCTGAGGG
GCACGGAGGC
TGGGGCAGGG
ACTTGGCGGA
--------------------
-
-----------------------~~~~~
8C7
-292
ACAGGACAGC GTGCTGGGAC
ACTCTGTGTG
TGAGTGAGTG
AGACAGACCG
CGTGTGTGGG
TTTATTTTTT
CCTCTCATTC
CTGGGGTCTG
AATGGAAAGC
7G12
-192
ACGTTAAGCT
GTAATTAAGA
CATCTTGGAG
GCGTACTTAA
CTTATGACGT
GTTGCTTTTC
TTTTCCTCTC
GGTGAGAAAG TGGTTCTCTT
TTCCCTCAAT
I1B4
-92
****
*****
****
****
**********
***
**
***
*
***
******
*C*T***A*****
********
*
*
*
*
**
****
CAGGTCCTCT
GCCACCCAGA GCCACAAAGA
GACGCTCGGG ACATAGGCAC
ACACACGCGC
ACACCAACTT
AAGGTGAGCA
CCATAAACTT
CCTCTGCACT
-27
*
********
*************
***
**
*****
TCGCTGTCCT
GTTTCAAACT
GTTGGAGAGT
TCTGAGCGC..
TCTCGACTTG
GGAGCTCCAC
ACAAGg aaa
gaaa aa ca
aac gggg
..In on
1
-26
+1
*******
******
******
****
ccaaaaaa
ga gagcc
c g a g
ga gccc
acagTGAAGA
AGATTCTTGT
GACTGTGAAG
A3CACAAGTC
TACTCTTTCT
...
Exon
2
Me Th Se L
euLeuPheLe
all
clones(
Fig.
1.
Nucleo ide
sequence
o
he
5'
end
o
he
chicken
LP
gene.
Posi ions
a e
gi en
om
he
ansla ion
s a
si e
wi hin
exon
2,
no
including
in on
1
(nucleo ides
in
lowe
case
le e s).
Nucleo ide
sequences
ep esen ed
by
cDNA
clones
a e
indica ed
by
solid
lines.
All
sequences
we e
de e mined
om
bo h
di ec ions.
TATA
and
CCAAT
mo i s
a e
boxed.
Homopu ine
and
homopy imidine
ac s
longe
han
30
n
a e
deno ed
by
double
and
single
b oken
lines,
espec i ely.
A ows
poin
o
he
majo
ansc ip ion
s a
si es.
Nucleo ides
iden ical
wi h
he
human
sequence
(31)
a e
ma ked
wi h
as e isks
abo e
he
sequence.
Physical
map
o
cDNA
(A)
and
genomic
(B)
clones
spanning
he
5'
end
o
he
LP
gene.
Solid
ba s
ep esen
he
ec o s.
Hyb idiza ion
o
p ime s
P2
(-)
and
P3
(=)
is
indica ed
below
he
map
o
each
cDNA.
A
mo e
de ailed
es ic ion
map
o
he
1.7-kb
SalI-EcoRI
genomic
agmen
is
shown
in
an
expanded
o ma .
Open
box
indica es
he
posi ion
o
exon
1.
Ho izon al
a ows
deno e
he
sequencing
s a egy.
Abb e ia ions
o
es ic ion
enzymes:
A,
A aI;
B,
BamHI;
C,
Sacl;
E,
EcoRI;
R,
EcoRII;
S,
SalI;
V,
A al.
b -1
5S3
5S3
8C7
7G12
7G12
8G1,
5S3,
8C7
ilB4
all
bu
IlB4
10/-
Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
4985
dNTP
in
a
inal
olume
o
50
Al.
Ampli ica ion
was
pe o med
wi h
1.25
uni s
o
Taq
polyme ase
using
a
Coy
TempCycle .
DNA
analysis
and
seconda y
s uc u e
p edic ion
The
nucleo ide
sequence
was
de e mined
by
he
me hod
o
Sange
e
al.
(26)
using
M13
subclones
cons uc ed
as
desc ibed
(10).
T ansc ip ion
s a
poin s
we e
mapped
wi h
T4
DNA
polyme ase
on
cloned
genomic
DNA
as
sugges ed
by
Hu
and
Da idson
(27).
F agmen s
sepa a ed
by
polyac ylamide
gel
elec opho esis
we e
ans e ed
o
ni ocellulose
il e s
a e
dena u a ion
by
boiling
o
10
min
in
1
M
ammonium-ace a e,
20
mM
NaOH.
DNA
agmen s
we e
labeled
by
andom
p ime
ex ension
(28).
RNA
seconda y
s uc u e
was
p edic ed
wi h
he
FOLD
p og am
o
Zuke
(29)
included
in
he
Sequence
Analysis
So wa e
Package
o
he
Gene ics
Compu e
G oup,
Uni e si y
o
Wisconsin
(30).
RESULTS
Al e na i e
splicing
in
he
5'-UTR
o
he
chicken
LP
gene
Genomic
clones
o
chicken
LP
isola ed
p e iously
ca ied
he
en i e
p o ein
coding
egion
and
3'-UTR,
bu
no
he
i s
exon
o
he
gene
(12).
In
o de
o
isola e
he
5'
end
o
he
gene,
we
Table
H.
Pu a i e
splice
si es
a
he
5'
end
o
he
LP
gene
dono
si ea
posb
accep o
si eC
posb
cDNA
clone
agTTC
-485
gc agGTG
-451
TAGg gcag
-451
cc caa cagGTC
-
188
pLP
8G1
TGTg gag
-362
cc caa cagGTC
-188
pLP
D48
TGAg gag
-358
cc caa cagGTC
-188
pLP
5S3
TGAg gaga
-354
cc caa cagGTC
-
188
pLP
8C7
TCGg gaga
-220
AAGg gagc
-118
gccc acagTGA
in onI
pLP
1
lB4
AAGg aaag
-26
gccc acagTGA
in onI
all
pLP
clones
excep
1
IB4
A
AGg ag
yyyyyynyagG
consensus
d
amo i s
con aining
AGg
o
g ag
sequence.
:
a
o
g.
bposi ion
o
he
splice
si e
in
he
sequence
as
in
Fig.
1.
Cmo i s
di e ing
by
no
mo e
han
wo
nucleo ides
om
he
sequence
yyyyyynyagG
and
only
a
posi ions
no
unde lined.
y:
c
o
.
dShapi o
and
Senapa hy
(32).
cons uc ed
a
cDNA
lib a y
using
poly(A)+
RNA
pu i ied
om
s e na
o
chicken
emb yos
as
empla e,
and
a
speci ic
p ime
P3
(Table
1),
complemen a y
o
he
LP
mRNA
om
posi ion
109
o
128
ela i e
o
he
ansla ion
s a
si e.
The
lib a y
was
sc eened
wi h
a
mix u e
o
end-labeled
P2
and
P3
oligonucleo ides
(Table
I),
speci ic
o
exons
2
and
3,
espec i ely.
Fi e
o
he
posi i e
cDNA
clones
wi h
he
longes
inse s
we e
sequenced.
The
277-bp
EcoRII-SacI
agmen
o
he
mos
5'-end
clone
pLP7G12
hyb idized
o
RNA
species
o
he
same
mobili y
as
desc ibed
o
LP
mRNA
(10)
(da a
no
shown).
The
same
agmen
was
used
as
a
hyb idiza ion
p obe
o
sc een
a
chicken
genomic
lib a y.
One
posi i e
clone,
XgLP100
was
isola ed
and
cha ac e ized.
Res ic ion
mapping
and
Sou he n
hyb idiza ion
e ealed
ha
he
clone
ca ied
exon
1
alongwi h
800
bp
o
ups eam
sequence
and
19.7
kb
o
he
pu a i e
i s
in on
(da a
no
shown).
E en
hough
he
p e iously
desc ibed
genomic
clone
XgLP12.1
(12)
ex ended
13.6
kb
ups eam
o
he
5'-mos
ansla ed
exon,
named
exon
2
in
his
pape ,
he
es ic ion
maps
o
he
wo
clones
did
no
o e lap,
no
did
he
wo
clones
c oss
hyb idize.
The e o e,
we
concluded
ha
he
i s
in on
is
la ge
han
33
kb.
Since
he
p o ein
coding
exons
including
he
3'-UTR
co e
a
genomic
egion
o
70
kb
o
longe
(12),
hence
his
indica es
a
p ima y
ansc ip
o e
100
kb
in
leng h.
The
nucleo ide
sequence
o
he
i s
exon
and
lanking
egions
was
de e mined
(Fig.
1).
Alignmen
o
he
genomic
and
cDNA
sequences
e ealed
comple e
iden i y
along
exon
2;
howe e ,
only
one
o
he
new
cDNA
clones,
pLP7G12
o e lapped
con inuously
wi h
he
co esponding
genomic
sequence.
Sho e
o
longe
in e nal
segmen s
bo de ed
by
consensus
dono
and
accep o
splice
si es
(Table
H)
we e
absen
om
he
5'-UTR
o
he
o he
cDNA
clones.
This
obse a ion
s ongly
indica es
ha
he
empla es
o
he
cDNA
molecules
we e
gene a ed
by
al e na i e
splicing
e en s
wi hin
exon
1,
de ining
a
leas
i e di e en
splice
a ian s
o
LP
mRNA.
A
homology
sea ch
e ealed
ha
in
he
leade
egion,
wo
ou
o
ou
po en ial
accep o
si es
we e
u ilized,
while
ou
o
he
se en
pu a i e
dono
si es,
six
ha e
con ibu ed
o
he
gene a ion
o
obse ed
di e si y
(Table
II).
Two
sho
open
eading
ames
we e
ound
in
he
5'-UTR
om
-300
o
-276
and
om
-248
o
-122.
Bo h
o
hese
ups eam
ini ia ion
codons
a e
emo ed
by
excision
o
he
al e na i e
in ons
om
he
8C7-,
5S3-
and
8GI- ype
messages.
Polymo phism
was
*****
***
Ex ension
o
PE
°0
°
0
o*00
00o00
0000
0*0
Ex ension
o
PF
V
VV
V
S1
nuc ease
p o ec ion
I
III
1I
1l
I
T4
po yme ase
s ops
-562
-552
-542
-532
-522
-512
-502
-492
-482
-472
| AAAAAT
GATCCCTTCA
TGAGGAACAG
TTCTTTTTTT
TTTTCTTTTT
TTTTCTTTTT
TTTTCTTTTT
TTTTTAGTTC
GGGACTGGTG
TGCGGTGCAG
JT AAJ TTA
CTAGGGAAGT
ACTCCTTGTC
AAGAAAAAAA
AAAAGAAAAA
AAAAGAAAAA
AAAAGAAAAA
AAAAATCAAG
CCCTGACCAC
ACGCCACGTC
Fig.
2.
Composi e
ep esen a ion
o
he
ansc ip ion
ini ia ion
si es
de e mined
by
a ious
echniques.
The
TATA-like
sequence
is
boxed.
Thickness
o
he
ho izon al
a ows
below
he
sequence
e lec s
he
ela i e
equency
o
ini ia ion.
Nuclease
S
I
mapping
o
he
5'
end
o
he
chicken
LP
gene.
Uni o mly
labeled,
single
s anded
p obes,
complemen a y
o
he
mRNA
om
posi ions
indica ed
by
solid
black
ba s
in
he
schema ic
diag am,
we e
made
by
ex ension
o
he
sequencing
p ime
on
M13
subclones.
The
p obes
we e
hyb idized
o
60
g
o
o al
RNA
and
ea ed
as
desc ibed
(43).
The
leng h
o
he
p o ec ed
agmen s,
ep esen ed
by
hin
lines,
a e
gi en
in
nucleo ides.
A,
B
and
C
e e
o
espec i e
p obes.
Lanes
G
and
T,
dideoxy
sequencing
ladde ;
lane
1,
undiges ed
p obe;
lanes
2 and
3,
p obe/RNA
hyb id
ea ed
wi h
nuclease
S1
a
30°C
o
37°C,
espec i ely;
lane
4,
p obe
diges ed
wi hou
RNA
added.
4986
Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
de ec ed
be ween
-370
and
-366,
whe e
some
o
he
cDNA
clones
lacked
one
o
he
epea ed
CT
dinucleo ides.
Sequence
elemen s
o
po en ial
unc ional
signi icance
we e
iden i ied
in
he
5'- lanking
egion
o
he
gene.
Two
TATA-like
(a
-806
and
-562)
and
wo
CCAAT
mo i s
(a
-845
and
-795)
we e
ound
(Fig.
1).
The
sequence
om
-387
o
-370
ma ches
he
consensus
clea age
si e
o
chicken
opoisome ase
(33).
Pu ine
and
py imidine
ich
segmen s
al e na e
h oughou
he
sequence.
The
nucleo ide
sequence
om
posi ion
-
193
o
-27
has
69
%
iden i y
wi h
he
co esponding
human
sequence
(31).
Mapping
o
ansc ip ion
ini ia ion
si es
o
he
LP
gene
Th ee
di e en
echniques
we e
used
o
de ine
he
ansc ip ion
s a
si e
and
he
esul s
a e
summa ized
in
Fig.
2.
S1
nuclease
mapping
using
p obes
o
h ee
di e en
leng hs
(da a
no
shown)
indica ed
mul iple
ini ia ions
in
a
egion
om
posi ion
-529
o
-485.
A
s ong
p o ec ion
was
obse ed
a
he
3'
end
o
a
poly(T)
ac
a ound
-486
and
wo
addi ional
equen
ini ia ion
si es
we e
mapped
a
-
517
-i-1
and
-
508
1
by
each
p obe
(Fig.
2).
ACOG
A
...
.
M13
--
--
-
~
mm
DNA
a
_
_
_
_
Saoi
m
-
1
1-
__
-4.I
a
'I'
k
i
I
LLi
(AP
'A,
.7
AT'A
Pe o ming
SI
p o ec ion
assays
wi h
o he
p obes,
we
could
no ,
howe e ,
de ec
any
ansc ip
o igina ing
om
he
TATA
mo i
a
-
806
(da a
no
shown).
These
esul s
indica ed
ha
he
TATA-
like
mo i
a
-562
may
se e
as
a
p omo e
o
he
LP
gene.
SI
nuclease
sensi i i y
o
he
poly(U)
s e ch
a
he
5'
end
o
he
LP
mRNA
may
ha e
con ibu ed
o
he
S
I
pa e n
obse ed.
The e o e,
we
also
mapped
he
5'
end
using
T4
DNA
polyme ase
(Fig.
3).
A
speci ic
p ime ,
PA,
was
annealed
o
a
single
s anded
an isense
DNA
spanning
he
5'
end
o
he
LP
gene,
and
he
p ime
was
ex ended
wi h
T4
DNA
polyme ase
in
he
p esence
o
hyb idized
RNA.
Mul iple
s a
si es
we e
de e mined
wi hin
he
poly(T)
s e ch
om
-529
o
-487.
The
majo
si es
we e
loca ed
a
-497,
-507
-+1
and
-517
+
1,
and
wo
mino
si es
a
-529
and
a
-487.
The
ex ension
p oduc
ep esen ing
he
las
si e
was
ain
bu
isible
in
he
o iginal
au o adiog am.
In
p ime
ex ension
expe imen s
(Fig.
4)
he
p ime
PE ,
complemen a y
o
he
splice
o m
ep esen ed
by
pLP8G1,
esul ed
in
wo
majo
clus e s
o
ex ension
p oduc s
60
+
I
and
69
±
2
n
in
leng h,
hus
de ining
ansc ip ion
ini ia ion
a
-498
±
1
and
-507
±2,
espec i ely.
In
con a y,
when
PF ,
a
p ime
speci ic
o
all
bu
8G
1- ype
splice
a ian s
was
used,
he
ex ension
p oduc s
indica ed
mul iple
s a
si es
wi hin
a
wide
egion,
be ween
-529
and
-487
(Fig.
2).
The
mos
abundan
ansc ip s
(1
13
n )
ini ia ed
a ound
-517.
The
di e ence
be ween
he
p ime
ex ension
esul s
in
epea ed
expe imen s
sugges s
ha
he
LP
splice
o ms
may
di e
in
he
ela i e
equency
o
u iliza ion
o
he
mul iple
s a
si es.
Howe e ,
due
o
he
complex
na u e
and
high
s abili y
o
he
leade
(see
below)
I.
.1_.
.
a
-P
:'
I0
Fig.
3.
Mapping
o
ansc ip ion
s a
si es
by
T4
DNA
polyme ase.
The
diag am
o
he
le
depic s
he
expe imen al
s a egy.
An
M13
DNA
ca ying
he
RNA-
complemen a y
s and
o
exon
1
and
he
p omo e
egion
was
used
as
empla e.
Solid
ba
wi h
as e isk
ep esen s
he
32P
end-labeled
PA
p ime ,
b oken
a ow
indica es
he
p ime
ex ension
p oduc s.
Lanes
A,
C,
G
and
T,
dideoxy
sequencing
ladde s
o
he
same
empla e
DNA
om
unlabeled
PA
p ime
in
he
p esence
o
[35S]dATP.
Lane
1,
4
ug
poly(A)+
RNA
was
hyb idized
o
he
empla e
be o e
p ime
ex ension.
Lane
2,
p ime
ex ension
in
he
absence
o
hyb idized
mRNA.
The
numbe s
a
he
igh
ma gin
o
he
au o adiog am
deno e
he
nucleo ide
posi ions.
Tha
pa
o
he
au o adiog am
which
ca ies
he
ex ension
p oduc
a
posi ion
-487
is
shown
a e
p olonged
exposu e,
in
an
inle
o
he
igh .
Analysis
o
PCR
p oduc s
on
a
dena u ing
polyac ylamide
gel.
D,
PC/P3
p ime
pai
was
used
in
RT-PCR
unde
condi ions
desc ibed
o
Fig.
6.
A
and
B.
Lane
1,
p oduc s
o
RT-PCR.
Lane
3,
PCR
pe o med
om
I
ng
inse
o
pLP8Gl
as
empla e.
M,
pUC12
HaeIIl
diges
used
as
size
ma ke .
I
a
Fig.
4.
P ime
ex ension
analysis
o
he
5'
end
o
he
LP
gene.
4
pg
poly(A)+
RNA
was
hyb idized
a
60°C
o
5
32P
end-labeled
oligonucleo ides,
comple-
men a y
o
exon
1
a
posi ions
shown
in
he
schema ic
diag am.
Exon
1
is
boxed.
cDNA
was
syn hesized
a
42'C
o
1
h
wi h
M-MLV
e e se
ansc ip ase.
The
ex ension
p oduc s
om
p ime s
PE
(lane
1)
and
PF
(lane
2)
we e
analysed
on
sequencing
gels.
Lanes
A,
C,
G
and
T,
dideoxy
sequencing
ladde s.
Numbe s
a
he
ma gins
o
he
au o adiog ams
deno e
he
leng h
in
nucleo ides.
,
..
.A
Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
4987
which
impeded
he
design
and
ex ension
o
p ime s
speci ic
o
he
dis inc
splice
o ms,
we
could
no
es
his
hypo hesis.
To
sum
up,
he
esul s
ob ained
by
h ee
di e en
s a egies
a e
gene ally
consis en
wi h
he
conclusion
ha
he
LP
gene
is
ansc ibed
om
mul iple
s a
poin s
loca ed
wi hin
he
poly(T)
s ech
34
+
1,
46
+
1,
56
+
1,
66
±
1
and
76
±
1
bp
downs eam
o
he
TATA-like
mo i
(Fig.
2).
Nucleo ide
sequences
a
he
ini ia ion
si es
ma ch
he
YYCAYYYY
consensus
sequence
(34)
desc ibed
o
o he
euka yo ic
genes
excep
o
he
lack
o
A
a
posi ion
4.
The
ini ia ion
si es
a
-507,
-517
and
-497
a e
used
mo e
equen ly
han
he
o he s.
The
majo
disc epancies
in
SI
mapping
( wo
mino
si es
and
inc eased
equency
o
clea age
a ound
-486)
can
be
explained
by
he
SI
sensi i i y
o
he
5'
end
o
he
hyb id.
Indeed,
mo e
accu a e
da a
we e
ob ained
wi h
T4
DNA
polyme ase
as
compa ed
o
he
o he
wo
echniques,
due
o
he
ac
ha
his
analysis
was
nei he
in luenced
by
he
base
composi ion
no
he
seconda y
s uc u e
o
he
RNA.
PCR
analysis
o
he
mul iple
LP
mRNA
species
The
expec ed
agmen
sizes
o
a ious
combina ions
o
o wa d
and
e e se
p ime s
in
RT-PCR
analysis
a e
depic ed
in
Fig.
5.
Using
wo
di e en
p ime
pai s
(Fig.
6A
and
B),
splice
a ian
ep esen ed
by
cDNA
clone
8G1
was
ound
o
be
he
mos
abundan
among
he
LP
mRNA
species
in
emb yonic
s e nal
ca ilage,
ollowed
by
he
8C7/5S3- ype
a ian s.
Bands
co esponding
in
size
o
7G12
and
1
1B4
splice
o ms
we e
no
de ec able
in
PCR
e en
upon
p olonged
o e exposu e
o
he
-
,--
>33
kb
-
>39
kb
-4-11.6
kb-U-
72
kb
-{
s
as
s-
PB1
A
~PB1
7G12
4
-
11B4
I
404
8C7
=
330
5S3
326
D48
322
8G1
_233
Hypl
230.238
Hyp2
141
Hyp3
_1n0168
Hyp4
71
c
P2
PD
7012
331
SC7
e
165
583
ca=161
D48
,
157
801
68
au o adiog ams;
howe e ,
se e al
sho e
p oduc s
we e
ound,
co esponding
in
size
o
he
hypo he ical
messages
Hyp2
-4
(Fig.
6A,
lane
2).
In
o de
o
es
i
addi ional
exons
hidden
in
he
long
i s
and
second
in ons
ha e
con ibu ed
o
he
di e si y
obse ed,
ano he
RT-PCR
was
ca ied
ou
wi h
he
PC/P3
pai
o
p ime s.
A
single
p oduc
o
174
bp
was
moni o ed,
iden ical
in size
o
ha
syn hesized
on
he
cDNA
clone
pLP8G1
(no
shown).
Longe
PCR
p oduc s
we e
no
obse ed
e en
a e
long
exposu e.
The e o e,
we
concluded
ha
no
al e na i e
exons
loca ed
be ween
exon
1
and
exon
3
we e
de ec able
in
chicken
ca ilage.
The
PCR
p oduc s
and
he
cDNA
clones
we e
u he
co ela ed
by
designing
he
PB2/PD
pai
o
p ime s,
each
30
n
in
leng h,
hus
dec easing
he
complexi y
and
minimizing
he
nonspeci ic
backg ound
in
RT-PCR.
Fo y
cycles
p oduced
DNA
agmen s
isible
in
e hidium-b omide
s ained
gels
i espec i e
o
whe he
poly(A)+
o
o al
RNA
was
used
as
empla e
(Fig.
7A).
All
he
bands
co esponding
o
he
a ious
cDNA
clones
we e
obse ed,
excep
o
he
7G12- ype
PCR
agmen .
In
ac ,
hyb idiza ion
o
a
speci ic
p obe
clea ly
indica ed
he
p esence
o
he
7G12- ype
PCR
p oduc
(Fig.
7B,
lane
2)
appea ing
as
agmen s
o
e e -inc easing
leng h.
The
explana ion
o
his
phenomenon
is
he
p esence
o
di ec
epea s,
which
led
o
he
ou -o - egis e
hyb idiza ion
o
compe ing
PCR
p oduc s
a
subsequen
cycles
o
ampli ica ion.
In
addi ion
o
se e al
sho e
epea s,
a
andem
epea
o
23
n
con aining
ou
misma ches
was
ound
a
-338
and
-243.
Simila
a e ac s
we e
de ec ed
by
o he s
when
DNA
empla e
con aining
andem
epea s
was
used
in
PCR
(35).
Su p isingly,
apa
om
he
expec ed
PCR
p oduc s
de ec ed
in
dena u ing
gels
(Fig.
6C),
wo
addi ional
bands
(ma ked
X
A
M32
1
P?
gmen
sizes
s
In
A
.
120
bp
8C7
a
_
5S3
X-
8G1
Hypl
I
/
-'VI
H
yP3
X
Hyp2
0
_
B
M-
-
1
M
C
4
5
6
-
-587
u:_458
-434
-
-298
-
-267
-
-251
8C7
5S3
A
W
D48
M
-
587
*
458
-434
-
-298
w-
267
`
-251
am
-1
74
--174
_
-
102
100
bp
X
-80
Hyp4
8G1-
_
-
-102
PC
P3
all
'-'
174
Fig.
5.
Diag am
o
al e na i e
splicing
in
he
LP
leade
egion.
The
s uc u e
o
he
gene
is
shown
a
he
op
o
he
igu e.
Exons
(E
l
-E5)
a e
numbe ed
om
he
5'
end
o
he
gene
(no e
he
di e ence
om
e .
12).
Open
boxes
ep esen
he
5'-
and
3'-UTRs
and
black
boxes
he
p o ein
coding
exons.
A ows
indica e
he
posi ions
o
he
p ime s
used
in
PCR.
The
PCR
agmen s
assumed
o
be
o med
wi h
a ious
p ime
pai s,
based
on
he
s uc u e
o
he
cDNA
clones,
a e
depic ed.
The
egions
ha bo ed
by
he
cDNA
clones
a e
shown
as
do ed
ba s
o
compa ison.
P ime
pai s
PB1/P2
(A),
PB1/P3
(B),
PB2/PD
(C)
and
PC/P3
(D)
we e
chosen.
F agmen
sizes
expec ed
o
he
a ious
splice
o ms
a e
shown
in
base
pai s
a
he
igh
side
o
he
diag ams.
Fig.
6.
Analysis
o
he
PCR
p oduc s
on
dena u ing
polyac ylamide
gels.
A,
B
and
C
ep esen
p ime
combina ions
as
in
Fig.
5.
A
and
B,
cDNA
sy hesized
om
poly(A)+
RNA
o
chick
emb yo
s e na
was
subjec ed
o
30
cycles
o
ampli ica ion
a
94°C
o 1.5
min,
48°C
o
2.5
min
and
75°C
o
3.5
min
in
he
p esence
o
10%
DMSO.
Lane
1,
P oduc s
o
RT-PCR.
Lane
2,
as
lane
1,
bu
10- old
amoun
was
loaded.
Lane
3,
PCR
pe o med
om
1
ng
inse
o
pLP8G1
as
empla e.
M,
pUC12
HaeIll
diges
used
as
size
ma ke .
C,
To al
RNA
om
emb yonic
s e nal
(lane
4),
ju enile
s e nal
(lane
5)
and
ju enile
a icula
(lane
6)
chond ocy es
was
used
in
RT-PCR
employing
he
PB2/PD
p ime
pai .
30
cycles
o
ampli ica ion
we e
pe o med
a
94°C
o
1
min,
65
°C
o
2
min
and
75°C
o
3
min.
Numbe s
o
he
igh
indica e
he
agmen
sizes
in
nucleo ides.
D
-4
/
-i

4988
Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
N4
;5
&
S
3
I-)
4
B
Fig.
7.
Analysis
o
he
PCR
p oduc s
on
nondena u ing
gels.
40
cycles
o
ampli ica ion
we e
pe o med
in
RT-PCR
unde
condi ions
as
in
Fig.
6C.
A,
UV- luo escen
pic u e
o
he
e hidium-b omide
s ained
gels.
To al
RNA
ob ained
om
emb yonic
s e nal
(lane
2),
ju enile
s e nal
(lane
3)
and
ju enile
a icula
(lane
4)
chond ocy es
was
used.
Con ol
eac ion
was
pe o med
employing
a
cloned
genomic
DNA
agmen
ca ying
exon
1
(lane
1).
B,
Sou he n
hyb idiza ion.
RT-PCR
p oduc s
ob ained
using
RNA
isola ed
om
ju ene
s e al
chond ocy es
we e
sepa a ed
on
a
5%
polyac ylamide
gel
(lane
1),
blo ed
and
hyb idized
o
a
con inuously
labeled
7G12-speci ic
p obe
(lane
2)
ex ending
om
posi ion
-347
o
-188,
and
o
he
5'
end-labeled
oligonucleo ide
PG
(Table
I),
speci ic
o
he
5S3
o m
(lane
3).
M,
posi ions
o
he
pUC
12
HaeLI
agmen s
as
in
A.
and
Y
in
Fig.
7)
we e
isualized
in
non-dena u ing
gels.
The
appa en
mobili ies
o
hese
bands
dec eased
by
inc easing
he
empe a u e
du ing
elec opho esis,
and
hese
bands
hyb idized
o
he
5S3-speci ic
oligonucleo ide
(Fig.
7B,
lane
3).
To
con i m
he
iden i y
o
he
PCR
agmen s,
all
he
bands
we e
excised
om
he
non-dena u ing
gels,
cloned
and
sequenced.
Sequencing
o
he
160
bp
agmen
con i med
he
p esence
o
he
5S3
and
8C7
ypes,
and
in
addi ion,
e ealed
he
exis ence
o
a
new
splice
a ian
(D48),
which
u ilized
he
dono
si e
loca ed
a
posi ion
-362.
Sequence
analysis
also
p o ed
ha
band
X
ep esen ed
he
he e oduplexes
o
5S3/D48
and
8C7/5S3
PCR
p oduc s,
di e ing
in
leng h
by
4
n .
On
he
o he
hand,
due
o
he
polymo phism
o
he
emb yonic
popula ion,
he
2
n
di e ence
esul ed
in
he
he e oduplex
band
Y.
This
band
was
no
obse ed
when
RNA
om
s e nal
o
a icula
ca ilage
o
a
homozygo ic
animal
was
used
in
RT-PCR
(Fig.
7A,
lanes
3
and
4).
The
o ma ion
o
he e oduplexes
can
be
explained
by
compe i ion
o
he
accumula ing
PCR
p oduc s
wi h
he
oligonucleo ide
p ime s
o
annealing
o
empla e
DNA.
To
sum
up,
in
spi e
o
he
di icul ies
due
o
he
complexi y
o
he
5'-UTR,
PCR
con i med
he
di e si y
o
he
LP
leade
sequence
and
e ealed
di e ences
in
he
ela i e
amoun s
o
he
a ious
splice
o ms.
The
68-bp
agmen ,
ep esen ing
he
8G
I
splice
a ian ,
was
he
mos
abundan
p oduc s
a e
a ious
ampli ica ion
egimens.
The
8C7/5S3/D48
splice
o ms,
howe e ,
showed
cell
ype-speci ic
exp ession,
as
being
de ec able
in
signi ican ly
lowe
amoun s
in
ju enile
a icula
compa ed
o
emb yonic
and
ju enile
s e nal
ca ilage,
in
epea ed
expe imen s
(Fig.
6C
and
7A).
Finally,
he
7G12- ype
PCR
p oduc
was
ound
in
e y
small
amoun s
when
ei he
RNA
sou ce
was
used.
5S3
7G12
Fig.
8.
P edic ed
seconda y
s uc u es
o
he
LP
leade s.
The
p og am
FOLD
(29)
was
used
o
p edic
he
mos
s able
s em-loop
s uc u e
in
he
5'-UTR
o
LP
mRNAs
as
shown
om
posi ion
-529
o
+35
o
he
7G12-
and
8Gl- ype
a ian s,
and
om
-529
o
+
128
o
he
5S3- ype.
The
o e all
ee
ene gy
o
o mna ion
(AG)
alues
a e
-150.3,
-71.8
and
-140.4
kcal/mol
o
splice
o ms
7G12,
8G1
and
5S3,
espec i ely.
A,
B
and
A'
ep esen
di e en
domains
wi h
p edic ed
ee
ene gies
o
-110.2
kcal/mol,
-38.8
kcal/mol
and
-31.7
kcal/mol,
espec i ely.
As e isks
ma k
he
cap
o
he
longes
ansc ip ,
solid
ba s
and
hin
lines
show
he
posi ions
o
he
ini ia ing
and
ups eam
AUGs,
espec i ely.
Po en ial
seconda y
s uc u e
o
he
LP
leade s
Fig.
8
shows
compu e
models
o
he
mos
s able
seconda y
s uc u es
p edic ed
o
he
5'
end
o
h ee
LP
splice
o ms.
The
7G12- ype
a ian
appea s
o
possess
he
mos
ex ensi e
seconda y
s uc u e,
which
can
be
di ided
in o
wo
egions:
1/
domain
A
om
posi ion
-477
o
-126
con ains
mos
o
he
leade
including
bo h
ups eam
open
eading
ames
and
is
p edic ed
o
o m
a
e y
s able
s em-loop
s uc u e
wi h
AG=-10.2
kcal/mol;
and
2/
domain
B
om
-120
o
+30
includes
he
ansla ion
s a
codon
o
he
LP
p ecu so
and
has
a
p edic ed
AG=
-38.8
kcal/mol.
The
compu e
gene a ed
hai pin
s uc u e
o
he
8G1
splice
o m
also
in ol es
domain
B
bu
domain
A
is
signi ican ly
unca ed
wi h
a
p edic ed
AG=
-31.7
kcal/mol.
In
con a y,
he
seconda y
s uc u es
p edic ed
o
he
5S3,
8C7
and
D48
a ian s
a e
en i ely
di e en
om
ha
o
7G12- ype.
I
sugges s
ha
dele ion
o
he
leade
sequence
om
posi ion
-358
o
-
189
may
in luence
he
olding
pa e n
a ound
he
ansla ion
s a
si e.
DISCUSSION
The
p esen
s udies
demons a e
ha
he
ansc ip ion
uni
o
chicken
LP
has
a
complex
s uc u e.
E en
hough
he
gene
comp ises
o
only
5
exons,
he
size
o
he
ansc ip ion
uni
is
la ge
han
100
kb,
due
o
he
p esence
o
long
in ons
and
unusually
long
UTRs
(529
bp
o
5
'-UTR
and
4.8
kb
o
3'-UTR).
Long
leade s
a e,
howe e ,
known
o
mRNAs
o
impo an
egula o y
p o eins
in ol ed
in
g ow h
and
de elopmen
(36-40).
The
LP
gene
is
ansc ibed
om
mul iple
ini ia ion
si es
loca ed
be ween
34
and
76
bp
downs eam
o
a
TATA-like
mo i .
Al hough
his
mo i
di e s
om
he
canonical
sequence
o
RNA-
polyme ase
H-dependen
p omo e s
(41),
p elimina y
expe imen s
indica e
ha
a
140
bp
agmen
ca ying
his
mo i
is
indeed
able
o
wo k
as
a
p omo e
in
ansien
exp ession
assays.
The
occu ence
o
mul iple
s a
si es
and
he
high
GC
con en
o
he
AG
Nucleic
Acids
Resea ch,
Vol.
19,
No.
18
4989
LP
p omo e
esembles
o
he
ea u es
o
housekeeping
genes
(42),
bu
di e s
om
hose
in
he
lack
o
he
classical
Spl
binding
si es.
GC- ich
sequences
we e
also
ound
in
he
5'- lanking
egion
o
o he
ca ilage-speci ic
genes
(43,
44).
One
o
he
mos
s iking
ea u es
o
he
LP
gene
is
he
complex
s uc u e
o
he
5'-UTR
(Fig.
5)
a ising
om
al e na i e
splicing
wi hin
exon
1,
ia
di e en ial
u iliza ion
o
six
dono
and
wo
accep o
splice
si es.
The
gene a ion
o
six
di e en
splice
o ms
con i med
by
sequencing
o
cDNA
clones
and
PCR
agmen s,
howe e ,
p o ides
only
a
minimum
es ima e.
Addi ional
splice
a ian s
may
exis
as
sugges ed
by
he
appea ance
o
PCR
agmen s
Hypl-Hyp4
(Fig.
5A
and
6A).
The
eason
why
hese
o ms
a e
no
ep esen ed
by
he
cDNA
clones,
p obably
is
ha
he
cDNA
clones,
ha bo ing
sho
inse s,
ha e
no
been
selec ed
o
u he
analysis.
The
occu ence
o
mul iple
s a
si es,
and
he
u iliza ion
o
dis inc
polyadenyla ion
si es
(10)
may
also
inc ease
he
he e ogenei y.
Fu he mo e,
i
is
possible
ha
al e na i e
splicing
in
he
ansla ed
egion,
al hough
no
de ec ed
in
chicken
ca ilage,
may
con ibu e
o
he
di e si y
o
LP
mRNA
in
o he
species
o
issues,
as
obse ed
ecen ly
in
a
chond osa coma
(19).
Complex
s uc u e
o
he
5'
leade
has
been
desc ibed
in
a
ew
cases
(45
-48).
Al e na i e
splicing
wi hin
he
5'-UTR
is
o en
combined
wi h
al e na i e
p omo e
usage
(47,
48).
In
case
o
he
LP
gene,
whe e
he
mul iple
LP
mRNA
species
a ise
om
he
same
p omo e ,
he
gene
exp ession
may
u he
be
con olled
by
egula ed
splicing
by
p o iding
speci ic
splicing
ac o s.
I
is
possible
ha
some
o
he
LP
splice
o ms
(7G
12,
1
1B4)
may
in
ac
ep esen
p ocessing
in e media es,
which
a e
kep
non unc ional
by
empo a ily
e aining
he
in on.
Examples
o
egula ed
splicing
ha e
been
e iewed
ecen ly
(36).
The
ac
ha
he e ogenei y
is
con ined
o
he
highly
conse ed
5'-UTR
o
he
LP
mRNA
which
shows
cell
ype-
and
s age-
speci ic
exp ession,
opens
he
possibili y
ha
he
a ious
splice
o ms
a e
subjec ed
o
some
kind
o
ansla ional con ol.
The
di e ences
in
he
p edic ed
seconda y
s uc u e
o
he
a ious
splice
o ms
seem
o
suppo
his
assump ion.
The
splice
o m
which
had
he
leas
s able
p edic ed
seconda y
s uc u e
(8G1),
was
ela i ely
mo e
abundan
in
chond ocy es
o
di e en
o igin
compa ed
o
he
o m
wi h
po en ially
mo e
s able
leade
s uc u e
(7G
12;
AG=
-110
kcal/mol).
The
hypo hesis
o
ex ensi e
seconda y
s uc u e
o ma ion
in
he
LP
leade
is
subs an ia ed
conside ing
he
di icul ies
in
p ime
ex ension
and
RT-PCR
analysis
o
he
mRNA,
and
also
in
sequence
analysis.
The
a ious
LP
mRNA
species
may
ha e
di e en
s abili ies.
Al e na i ely,
hey
may
be
ansla ed
a
di e en
a es,
since
hai pin
s uc u es
wi h
p edic ed
ee
ene gies
g ea e
han
-50
kcal/mol,
loca ed
ups eam
o
he
ini ia o
AUG,
ha e
been
p oposed
o
inhibi
he
ansla ion
o
he
mRNA
by
in e e ing
wi h
he
mig a ion
o
he
ibosomal
ini ia ion
complex
(49,
50).
I
has
been
con i med
ecen ly
o
he
mRNAs
o
o ni hine
deca boxylase
and
se e al
p o o-oncogenes
ha
long
leade s,
wi h
ex ensi e
seconda y
s uc u e
and
ups eam
AUG
codons,
impai
ansla ion
(36,
39,
40,
51,
52).
Binding
o
egula o y
p o eins,
as
demons a ed
o
he
leade s
o
e i in
mRNA
and
polio i us
RNA
(53
-56),
may
also
be
in ol ed
in
ansla ional
con ol
o
al e na i ely
spliced
LP
mRNAs.
Sho
open
eading
ames
loca ed
ups eam
o
he
ac ual
ansla ion
s a
si e
may
also
in luence
he
ansla ion
e iciency
o
an
mRNA
(50,
52).
Two
o
he
LP
splice
a ian s
(7G12
and
IIB4)
ca y
ups eam
open
eading
ames
in
e ained
in ons.
Since
all
h ee
AUG
codons,
including
he
ini ia o
codon
o
LP,
occu
in
un a o able
con ex
o
ini ia ing
ansla ion
(50,
57),
he
ups eam
AUG
codons
may
also
ha e
a
egula o y
ole.
Da a
p esen ed
he e
p o ide
a
basis
o
u he
analysis
o
he
ole
o
he
5'- lanking
egions
and
a ious
leade
sequences
in
he
ansc ip ional
and
ansla ional
egula ion
o
he
exp ession
o
LP
in
di e en
cell- ypes
and
du ing
de elopmen .
ACKNOWLEDGEMENTS
We
hank
F.Solymosi
o
aluable
discussion,
L.Szilak
o
he
syn hesis
o
he
oligonucleo ides,
I.Feke e
and
A.Simon
o
excellen echnical
assis ance,
and
A.Bo ka
o
he
a wo k.
This
wo k
was
suppo ed
by
G an
OKKFT
T
1986/296
om
he
Na ional
Founda ion
o
Technical
De elopmen
o
Hunga y
and
G an
OTKA
168
and
896
( o
I.K.)
and
a
special
und
om
he
Biological
Resea ch
Cen e
( o
F.D.).
REFERENCES
1.
Hascall,
V.C.
and
Sajde a,
S.W.
(1969)
J.
Biol.
Chem.
244,
2384-2396.
2.
Heinega d,
D.
and
Hascall,
V.C.
(1974)
J.
Biol.
Chem.
249,
4250-4256.
3.
Oegema,
T.R.,J .,
B own,
M.
and
Dziewia kowski,
D.
D.
(1977)
J.
Biol.
Chem.
252,
6470-6477.
4.
Ha dingham,
T.E.
(1979)
Biochem.
J.
177,
237-247.
5.
Tho n on,
D.J.,
Sheehan,
J.K.
and
Nieduszynski,
I.A.
(1987).
Biochem.
J.
248,
943-951.
6.
De
Luca
S.,
Heinega d,
D.
and
Hascall,
V.
C.
(1977)
J.
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