The complete sequence of the mouse skeletal α-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region

Abstract

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This
single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron
in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at
codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding
genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids,
Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken,
and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding
region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These
sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat
sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.

Full text

MOLECULAR
AND
CELLULAR
BIOLOGY,
Jan.
1986,
p.
15-25
Vol.
6,
No.
1
0270-7306/86/010015-11$02.00/0
Copyright
X
1986,
American
Society
for
Microbiology
The
Complete
Sequence
of
the
Mouse
Skeletal
oL-Actin
Gene
Reveals
Several
Conserved
and
Inverted
Repeat
Sequences
Outside
of
the
Protein-Coding
Region
MICKEY
CHIEN-TSUNG
HU,
SANDRA
B.
SHARP,
AND
NORMAN
DAVIDSON*
Department
of
Chemistry,
California
Institute
of
Technology,
Pasadena,
California
91125
Received
17
June
1985/Accepted
24
September
1985
The
complete
nucleotide
sequence
of
a
genomic
clone
encoding
the
mouse
skeletal
a-actin
gene
has
been
determined.
This
single-copy
gene
codes
for
a
protein
identical
in
primary
sequence
to
the
rabbit
skeletal
a-actin.
It
has a
large
intron
in
the
5'-untranslated
region
12
nucleotides
upstream
from
the
initiator
ATG
and
five
small
introns
in
the
coding
region
at
codons
specifying
amino
acids
41/42,
150,
204,
267,
and
327/328.
These
intron
positions
are
identical
to
those
for
the
corresponding
genes
of
chickens
and
rats.
Similar
to
other
skeletal
a-actin
genes,
the
nucleotide
sequence
codes
for
two
amino
acids,
Met-Cys,
preceding
the
known
N-terminal
Asp
of
the
mature
protein.
Comparison
of
the
nucleotide
sequences
of
rat,
mouse,
chicken,
and
human
skeletal
muscle
a-actin
genes
reveals
conserved
sequences
(some
not
previously
noted)
outside
of
the
protein-coding
region.
Furthermore,
several
inverted
repeat
sequences,
partially
within
these
conserved
regions,
have
been
identified.
These
sequences
are
not
present
in
the
vertebrate
cytoskeletal
(-actin
genes.
The
strong
conservation
of
the
inverted
repeat
sequences
suggests
that
they
may
have
a
role
in
the
tissue-specific
expression
of
skeletal
a-actin
genes.
The
actins
represent
a
multigene
family
of
highly
con-
served
proteins
found
in
all
eucaryotes.
Differences
in
amino
acid
sequence
among
the
various
actins
have
shown
that
at
least
six
different
isoforms
are
expressed
in
vertebrates
(52,
53).
Two
striated
muscle
isoforms,
skeletal
a
and
cardiac
a
(52),
and
two
smooth
muscle
isoforms
(53)
are
found
in
the
contractile
apparatus
of
muscle
fibers,
whereas
two
cyto-
skeletal
isoforms,
p
and
y,
are
present
in
the
cytoskeleton
of
all
cells
(51).
These
actin
proteins
are
extremely
conserved
in
amino
acid
sequence.
Actin
gene
expression
is
tissue
specific
and
developmen-
tally
regulated
(27,
29,
32,
35).
By
studying
the
structural
organization
of
the
actin
gene
family,
one
can
begin
to
look
for
the
controlling
elements
which
modulate
the
expression
of
these
genes
during
development.
Here
we
present
the
complete
nucleotide
sequence
of
the
single
genomic
copy
of
the
mouse
skeletal
a-actin
gene.
The
coding
region
of
this
gene
is
interrupted
by
five
introns
which
are
located
in
the
same
positions
as
introns
previously
identified
in
other
vertebrate
skeletal
a-actin
genes
(13,
54).
A
comparison
of
the
nucleotide
sequences
of
several
vertebrate
skeletal
a-
actin
genes
reveals
several
blocks
of
highly
conserved
sequences
in
the
5'-flanking
region
and
in
both
the
5'-
and
3'-untranslated
regions.
Interestingly,
the
conserved
se-
quences
in
the
5'-flanking
region
and
within
the
first
untranslated
exon
can
potentially
form
several
hairpin
loops
by
base
pairing
between
adjacent
inverted
complementary
sequences.
These
regions
do
not
correspond
to
the
potential
hairpin
structure
in
the
corresponding
portion
of
the
rat
cytoskeletal
,B-actin
gene
(33).
Furthermore,
it
is
possible
to
form
long
hairpin
loops
within
the
first
intron
and
one
stem
loop
in
the 3'-untranslated
region
upstream
from
the
putative
polyadenylation
signal
ATTAAA.
These
interesting
second-
ary
structures
are
apparently
not
present
in
the
vertebrate
cytoskeletal
P-actin
genes.
To
our
knowledge,
this
is
the
first
description
of
potential
secondary
structures
in
the
first
*
Corresponding
author.
intron
and
among
the
highly
conserved
sequences
in
the
5'-flanking
region
and
both
the
5'-
and
3'-untranslated
re-
gions
of
vertebrate
skeletal
a-actin
genes.
Since
the
species
compared
(avian
and
mammalian)
have
been
separated
for
more
than
250
million
years
(12),
these
results
indicate
a
strong
selective
constraint
to
conserve
these
sequences
and
suggest
that
they
may
have
an
important
role
in
the
tissue-
specific
expression
of
the
skeletal
a-actin
genes.
MATERIALS
AND
METHODS
Materials.
Restriction
endonucleases,
T4
DNA
ligase,
T4
polynucleotide
kinase,
Escherichia
coli
exonuclease
VII,
and
E.
coli
DNA
polymerase
I
large
fragment
(Klenow)
were
purchased
from
Bethesda
Research
Laboratories,
Boehr-
inger
Mannheim
Biochemicals,
or
New
England
BioLabs.
Avian
myeloblastosis
virus
reverse
transcriptase
was
obtained
from
Life
Sciences,
Inc.
Sp6
RNA
polymerase
and
placental
RNasin
were
obtained
from
Promega
Biotec.
Radioactively
labeled
nucleotides
were
purchased
from
Amersham
Corp.
or
New
England
Nuclear
Corp.
Unlabeled
nucleotides
were
obtained
from
P-L
Biochemicals,
Inc.
Synthetic
oligonucleotides
were
synthesized
by
S.
Horvath,
Caltech,
on
an
automated
DNA
synthesizer
(21)
and
purified
by
electrophoresis
through
a
20%
polyacrylamide-8
M
urea
preparative
gel
in
Tris
borate-EDTA
buffer.
BALB/c
genomic
DNA
was
provided
by
T.
Hunkapiller.
The
Drosophila
actin
genomic
clone
DmA2
(Dm5C
in
reference
14)
and
a
3'-untranslated
region
of
rat
skeletal
a-actin
cDNA
(15)
were
provided
by
B.
Bond
and
L.
Garfinkel,
respectively.
Isolation
of
genomic
actin
clones
and
restriction
mapping.
A
cosmid
genomic
library
of
BALB/c
mouse
sperm
DNA,
constructed
and
kindly
provided
by
M.
Steinmetz
at
Cal-
tech,
was
screened
by
colony
hybridization
(49)
by
using
an
actin-coding
region
probe
isolated
from
the
Drosophila
actin
genomic
clone
DmA2
(Dm5C).
From
16
positive
clones,
1
was
tentatively
identified
to
contain
the
skeletal
a-actin
gene
by
hybridization
with
the
conserved
(29),
isotype-specific
(37),
3'-untranslated
region
of
a
rat
skeletal
a-actin
cDNA.
15

16
HU
ET
AL.
45,"
Cosni
pTL5
.,
+~~~~~~~~~~~~~~~~~~~~~~~~!~
3
.zp
46C6f
@if
co~
ltljk~~<4~
.P62
_,
9-
-
,----,,
CAAAT
TATA
4
_
ExI
1000
ATG
41
150
204
267
327
TAli
Poly(A)
4
+
t
s3
Ex2
Ex
3
Ex4
Ex5
Ex6
Ex
7
2000
3000
4000bp
FIG.
1.
Structure
of
the
mouse
skeletal
a-actin
gene.
(A)
Cosmid
clone
containing
the
genomic
DNA
encoding
the
mouse
skeletal
a-actin
gene.
(B)
Restriction
endonuclease
map
of
the
6.8-kb
EcoRI
DNA
fragment.
One
squiggly
arrow
represents
an
SP6
antisense
transcript
of
a
BamHI-EcoRI
fragment
that
was
subcloned
from
the
6.8-kb
EcoRI
fragment.
(C)
Detailed
restriction
map
of
the
mouse
skeletal
aX-actin
gene
and
flanking
DNA.
Fragments
were
subcloned
into
M13
vectors
and
sequenced
by
dideoxy
chain termination
as
indicated
by
arrows.
(D)
Schematic
representation
of
the
structure
of
the
mouse
skeletal
a-actin
gene.
Solid
boxes
represent
coding
exons,
open
boxes
indicate
transcribed
untranslated
regions,
and
solid
lines
coincide
with
introns
and
flanking
DNA.
Numbers
above
the
exons
correspond
to
codon
positions
(Fig.
2).
The
squiggly
line
represents
the
SP6
antisense
transcript
as
mentioned
above.
Southern
blot
(48)
analysis
with
the
Drosophila
actin
probe
localized
the
mouse
a-actin-coding
region
to
a
single
6.8-
kilobase
(kb)
EcoRI
fragment
in
the
cosmid
clone.
This
fragment
was
subcloned
into
the
EcoRI
site
of
plasmid
pSP62-PL
(28),
provided
by
D.
Melton,
Harvard
University.
The
restriction
endonuclease
map
of
the
6.8-kb
EcoRI
fragment
was
determined
by
single
and
double
enzyme
digests.
Subsequently,
the
3.9-kb
BamHI-EcoRI
fragment
was
subcloned
into
the
BamHI
and
EcoRI
sites
of
the
plasmid
pSP62-PL
(see
Fig.
1B)
and
mapped
in
finer
detail
by
digestion
with
more
restriction
endonucleases.
Localization
of
the
promoter
region
of
the
a-actin
gene.
Several
different
restriction
endonuclease
digests
of
the
6.8-kb
EcoRI
fragment
were
probed
with
a
20-base
oligonu-
cleotide
(5'-GCCCAACACCCAAATATGGC-3')
containing
the
sequence
of
the
CAAT
promoter
homology,
highly
conserved
between
the
skeletal
a-actin
genes
of
chickens
and
rats
(34).
The
oligonucleotide
was
5'
end
labeled
with
polynucleotide
kinase
and
[-y-32P]ATP,
and
hybridization
was
performed
directly
in
the
dried
agarose
gel
as
described
previously
(39).
A
linearized
chicken
skeletal
a-actin
ge-
nomic
clone
(provided
by
C.
Ordahl
[13])
and
EcoRI-
linearized
SP6
vector
were
used
as
positive
and
negative
controls,
respectively.
M13
cloning
and
DNA
sequencing.
Appropriate
restriction
fragments
from
the
6.8-
and
3.9-kb
inserts
were
subcloned,
in
opposite
orientations,
into
the
multiple
cloning
sites
of
M13
mpl8
and
M13
mp19
RF
vectors,
transforming
first
into
E.
coli
JM101
for
high
efficiency
and
replating
with
E.
coli
JM109
(recA-)
to
prevent
sequence
changes.
Single-stranded
M13
templates
were
sequenced
by
the
dideoxy
chain
termination
procedure
(41)
with
an
[a-
35S]dATP
(500
Ci/mmol)
label
as
described
by
Biggin
et
al.
(3),
with
the
following
modifications.
(i)
The
synthetic
pentadecanucleotide
(5'-TCCCAGTCACGACGT-3')
and
the
hexadecanucleotide
(5'-GGGTAACGCCAGGGTT-3')
were
used
as
sequencing
primers.
(ii)
The
dideoxy
sequence
reactions
were
carried
out
in
50
mM
NaCI-7
mM
Tris
hydrochloride
(pH
7.4)-10
mM
MgCl2-3
mM
dithiothreitol.
(iii)
The
final
concentrations
of
unlabeled
nucleotides
in
each
sequence
reaction
were
as
follows:
A
reaction,
25
,uM
dCTP,
25
p.M
dGTP,
25
p.M
dTTP,
20
p.M
ddATP;
C
reaction,
8
p.M
dCTP,
32
p.M
dGTP,
32
p.M
dTTP,
50
p.M
ddCTP;
G
reaction,
32
p.M
dCTP,
8
p.M
dGTP,
32
p.M
dTTP,
50
p.M
ddGTP;
T
reaction,
32
p.M
dCTP,
32
p.M
dGTP,
8
p.M
dTTP,
50
p.M
ddTTP.
(iv)
After
electrophoresis,
the
gel
was
imme-
diately
dried,
without
fixing,
for
1
h
at
80°C
and
autoradio-
graphed.
Computer
analysis
of
sequence
homology
was
done
as
described
by
Hunkapiller
et
al.
(21).
Primer
extension
analysis.
Polyadenylated
[poly(A)+]
RNA
from
a
differentiated
culture
of
the
mouse
myogenic
cell
line
BC3H-1
(42)
was
isolated
by
guanidine
thiocyanate
extraction
(9)
and
two
cycles
of
oligo(dT)-cellulose
chroma-
tography
(1).
A
synthetic
42-base
oligonucleotide
(5'
-
AGAGCCGTTGTCACACACAAGAGCGGTGGTCTC
GTCTTCGTC-3')
complementary
to
a
portion
of
the
coding
sequence,
spanning
positions
1038
through
1079
in
exon
2,
was
5'
end
labeled
with
polynucleotide
kinase
and
[-y-
32P]ATP
(3,000
Ci/mmol)
and
used
as
an
extension
primer.
One
picomole
of
the
labeled
oligonucleotide
(106
cpm/p.g)
was
denatured
by
heating
at
80°C
for
10
min
in
40
p.I
of
98%
formamide
containing
1
p.l
of
0.5
M
EDTA.
Five
micrograms
of
poly(A)+
RNA
in
10
of
200
mM
sodium
piperazine-
N,N'-bis(2-ethanesulfonic
acid)
(pH
6.4)-2
M
NaCl-5
mM
EDTA
was
added,
and
the
mixture
was
incubated
at
37°C
for
12
h.
The
nucleic
acids
were
ethanol
precipitated
from
ammonium
acetate
and
reprecipitated
from
sodium
acetate.
The
dried
pellet
was
suspended
in
25
of
100
mM
Trishydrochloride
(pH
8.3)-40
mM
KCI-20
mM
MgCIz-10
1
kb
Cosmid
PTL5
+
(A)
5
200
bpS6
q
(b)
5
(C)
(D)
.ex
OIA
f
r
C4Q
VA!
MOL.
CELL.
BIOL.

VOL.
6,
1986
MOUSE
SKELETAL
oa-ACTIN
GENE
17
-753
CTCGGGGCAGTCTAGCTGTCATTTTCAGCCTGCCAGCCTTATCTCCCCCTCCATGCGCATA-634
-633
CACGAACTCCGCGCGCTTGGTATGGTAGGCATTTGTGGGGTCAGGGAGAAGATCATTGGTA-514
-513
TTTCTCTCTGGCAGCCCCGTACGCAAGTCTCCGCTACTAGCTGATTATAGACTATGGGATT-394
So
l
-393
GGAGCGATGGGCAGGGAACGGAACTCACGTATTGGATCGCACGCAGATACTGCCGAACCTC-274
-273
GTACTGACATGGGGCGCGTGGTCCCAAAGCTGCGTCACACGGCGGATCCCTCTGTATCGAA-154
-153
CCGGGGCCGCGGACGCAGAGGCCATCCAAC
~
GCTGAGGACAATTCGGGTTGGGGTCGGC
-34
-33
TTATAA
ACCTGTGCAAGGGGACAGOG
T-AC-IGACGTAAGC
CTCACTTCCTACCCTCGGCACCCAGGGCAGAGTCAGAGCAGCATGGAGGGACGG
8
Exon1
-33
TATAAA;Q
CGGT
C
AGGGTGGAGTGAGTACGG
7
88
AGCCGAAAACATACTGTGATACCGGTTGATTTCACCCTCGCATTTATGCTTGAAGCGGGCG
207
208
ACTTTCTACCCCGCCTCTCCCTCGGACAACAGCTCATACACGTTCATACAAGACTTGTGTA
327
326
ATCTCGCTTATCCTCCTTTTTCGGTAACGGCCTTAGAGGTGCGACCCCGGTCCCGTCGGG
447
448
CTATGAAGGTGAAATAGCGTTAGTGGCATCTTAACAGTTCAAGCGACCGCATTCCGATGAA
567
568
8AGGAGGTCTTATTGAGTTATTCAACTTCTGTGGCAACCTCCGGAATCCACTTAAGCGCGT
87
808
CCGAGCATGAGTATTACAAAACCCCTGGCCGCTACGAACTCCGCCCTCAGAGGAGGAAGGT
927
-2
met
Cys
Aso
Glu
2
928
TTTGGATCTTCAAAGTGAAGATGGGTTAAGCGGACCTCAACCTAACCCCCCCCCATCACATATACACAGCACTCAACACCTTGTCTTTGCAACAAACC
ATG
TGC
GAC
GAA
1043
IC
aAA
IA
3
Asp
Glu
Thr Thr
Ala
Leu
Val
Cys
Asp
Asn
Gly
Ser
Gly
Leu
Val
Lys
Ala
Gly
Phe
Ala
Gly
Asp
Asp
Ala
Pro
Arg
Ala
VIl
Phi
Pro
32
Exo
2
1044
GAC
GAG
ACC
ACC GCT CTT
GTG
TGT
GAC
AAC
GGC
TCT
GGC
CTG
GTG
AAA
OCT
GGC
TTT
GCC
000
OAT
OAT
0CC
CCC
AGG
GCT
GTG
TTC
CCA
1133
33
Ser
Ile
Val
Gly
Arg
Pro
Arg
His
Gin
41
1134
TCC
ATC
GTG
GGC
CGA CCC
COT
CAC
C
AG
iCAGGCTGCT
GGCAGGGAAAGATAGGCTCTCTGAATCCAGCCAATGTTCTCCTCACCCCTGGCCGTAGTAACAAGTGTCTGAT
i244
42
Gly
Vai
met
Vai
Gly
Met
Gly
Gin
Lys
Asp
Sir
Tyr
Vai
Gly
Asp
Glu
Ala
Gln
Sir
Lys
Arg
Gly
Ile
Leu
Thr
Leu
67
1245
GTCTCTATTC
GOT
GTC
ATG
GTA
GOT
ATG
GOT
CAG
AAG
GAC
TCC
TAC
GTG
GOT
OAT
GAG
0CC
CAG
AGC
AAG
CGA
GOT
ATC
CTG
ACC
CTG
1336
68
Lys
Tyr
Pro
Ilie
Glu
His
Giy
Ilie
Ilie
Thr
Asn
Trp
Asp
Asp
Met
Giu
Lys
Ilie
Trp
HiS
His
Thr
Phi
Tyr,
Asn
Glu
Liu
Arg
Vai
Ala
97
1337
AAG TAC
CCC
ATT
GAA
CAT
GGC
ATC
ATC
ACC
AAC
TOO
GAC
GAC
ATG
GAO
AAG
ATC
TOG
CAC
CAC
ACC
TTC
TAC
AAT
GAO
CTG
COT
GTG
0CC
1428
Exon
3
983
Pro
Glu
Glu
His
Pro
Thr
Leu
Leu
Thr
Glu
Ala
Pro
Leu
Asn
Pro
Lys
Aua
Asn
Arg
Glu
Lys
Met
Thr
Gin
Ilie
Met
Phi
Glu
Thr
Phi
127
1427
CCT GAG
GAG
CAC
CCG
ACT
CTG
CTC
ACC
GAG
GCC
CCC
CTG
AAC
CCC
AAA
OCT
AAC
COG
GAO
AAO
ATG
ACT
CAA
ATC
ATG
TTT
GAO
ACC
TTC
1516
128
Asn
Vai
Pro
Ala
Met
Tyr
Val
Ala
Ile
Gin
Ala
Vai
Leu
Sir
Leui
Tyr
Aua
Sir
Gly
Arg
Thr
Thr
0
150
i517
AAC
GTG
CCT
0CC
ATG
TAT
GTG
OCT
ATC
CAG
GCG
GTG
CTG
TCC
CTC
TAT
GCC
TCC
GGC
COT
ACC
ACC
0
iAAGCGCTCACACATGGCCCACGCTGGCC
1613
150
ly
Ile
151
1614
CTGAGTGTCAATCGCCCTTATCCGATTCGCTTCGCGTGCCTCCCCTCGCCCCCTGCACAiGC
ATC
1728
152
Vai
Leu
Asp Sir
Giy
Asp
Giy
Vai
Thr
His
Asn
Val
Pro
Ilie
Tyr
Glu
Gly
Tyr
Ala
Leu
Pro
His
Ala
Ile mit
Arg
Leu
Asp
Liu
Ala
081
1729
GTG
TTG
OAT
TCT
GGG
GAC
GOT
GTC
ACC
CAC
AAC
GTG
CCC
ATC
TAT
GAG
GGC
TAT
0CC
CTG
CCA
CAC
GCC
ATC
ATG
COT
CTG
GAC
CTG
0CC
1818
Exon
4
i
182
Gly
Arg
Asp
Leu
Thr
Asp
Tyr
Leu
Met
Lys
Ile
Leu
Thr
Giu
Arg
Gin
Tyr
Sir
Phi
Val
Thr
Thr
A
204
1819
GOT
CGC
GAC
CTC
ACT
GAC
TAC
CTG
ATG
AAA
ATC
CTC
ACT
GAG
CGT
GGC
TAT
TCC
TTC
GTG
ACC
ACA
G
TCGGTGCTCC
CAACCTGCTGAGGGTGGGC
1915
204
ia
Glu Arg
Giu
Ile
Vai
Arg
Asp
Ile
Lys
Giu
Lys
Leu
Cys
217
1916
GGGCAGAGGGTGAGCACACGCCCAGCCTTCGCCTGAGGCTCCTCACTGCTTTTGCTCTTGCAG
CT
GAA
COT
GAG
ATT
GTG
CGC
GAC ATC
AAA
GAO
AAG
CTG
TGC
2019
218
Tyr
Val
Ala
Leu
Asp
Phi
Glu
Asn
Glu
Met
Aua
Thr
Ala
Ala
Sir
Sir
Sir
Sir
Liu
Glu
Lys
Sir
Tyr
Glu
Leu
Pro
Asp
Gly
Gin
Val
246
ExoP
5
2020
TAT
GTG
0CC
CTG
GAC
TTC
GAG
AAT
GAG
ATG
0CC
ACC
OCT
GCC
TCT
TCC
TCC
TCC
CTG
GAG
AAG
AGC
TAT
GAG
CTG
CCT
GAC
000
CAG
GTC
2109
247
Ilie
Thr
Ilie
Gin
Asn
Glu
Arg
Phe
Arg
Cys
Pro
Giu
Thr
Leu
Phi
Giln
Pro
Sir
Phi
Ile
G
287
2110
ATC
ACC
ATC
GGC
AAT-GAG
COT
TTC
CGT
TGC
CCG
GAG
ACG CTC
TTC
CAG
CCT
TCC
TT
T
ATC
G
jGAGC
CGCCGGATCCGCTGGTGTGCOGGGATCAGTTT
2208
267
ly
Mit
Glu
Sir
Ala
Gly
272
2209
TCCCTCGCCCCACACCACAGAGTACGGGGTCTCCACCGCCGGTCCCTTAGCCCGACTCTGCGGTTGCTCACACTGCCTCTCTCCCGGA
CACC.i
GT
ATG
GAG
TCT
GCG
oGGG
2319
aom
Hi
273
Ile
His
Glu
Thr
Thr
Tyr
Asn
Ser
Ile
Met
Lys
Cys
Asp
Ile
Asp
Ile
Arg
Lys
Asp
Leu
Tyr
Ala
Asn
Asn
Val
Mit Sir
Sly Sly
Thr
302
Exi
6
2320
ATC
CAT
GAG
ACC
ACC
TAC
AAC
AGC ATC
ATG
AAG
TGC
GAC
ATC
GAC
ATC
AGO
AAG
GAC
CTG
TAC
0CC
AAC
AAC
GTC
ATG
TCA
000
SOC
ACC
2409
303
Thr
mit
Tyr
Pro
Gly
Ile
Ala
Asp
Arg
Mit
Gin
Lys
Giu
Ile
Thr
Ala
Leu
Ala
Pro
Sir
Thr
Mit
Lys
Ile
Lys
327
2410
ACC
ATG
TAC
CCT
GOT
ATC
GCT GAC
CGC
ATG
CAG
AAG GAG ATC ACA
OCT
CTG
OCT
CCC AGC
ACC
ATG
AAG
ATC
AAG
TGGOATGACGTGCCTGGTGT
2504
328
Ile
Ile
Ala
Pro
Pro
Glu
Arg
Lys
Tyr
Sir
Vai
Trp
Ile
Sly
Gly
Sir
343
2505
GGGTGGAGACCAGGGGCGGGGGAACACGAGGCACGTGACACTCTTGT
CTTGCi
ATC
A
TC
0CC
CCC
CCT
GAG
CGC
AAG
TAC
TCA
GTG
TOG
ATC
GOT
GGC
TCC
2606
344
Ile
Leu
Ala
Sir
Leu
Sir
Thr
Phi
Gin
Gin
met
Trp
Ile
Thr
Lys
Gin
Giu
Tyr
Asp
Sbu
Ala
Sly
Pro
Sir
Ile
Vai
His
Arg
Lys
Cvs
373
Exon7
2607
ATC
CTG
0CC
TCG
CTG
TCC
ACC TTC
CAG
CAG
ATG
TOG
ATC
ACC
AAG
CAG
GAS
TAC
GAC
SAG
OCT
GGC
CCC
TCC
ATT
GTG
CAC
CGC
AAA
TGC
2696
374
Phi
Tnm
374
2697
TTC
TAO
CCCGACGGTGGTTTTCCGAGCACAATGGCGGTGAGTGCCTCCGCTGTCTGCCATCG
2814
2815
CGCCTTTTAGGAAAATATGTTCTATTTATTCACAGCTTGAGAAGAACTAGWTT-A'GCGCTCGT2934
3'UT
2935
TTCCATACqgq6TTTTTCGGGAGATGCGCAGACGCCACCCCGTAAATGTACCTAGAGAGTG3054
t
3175
GCAGGACACCTGCTGCGAACTGGCGGCGCTGGCCGTGGTGCTGCCCGAAGCGCCTCTTGCCAGGGCTGGAAATTGGATCC
3254
FIG.
2.
Nucleotide
sequence
of
the
mouse
skeletal
ct-actin
gene.
Numbers
in
the
left
and
right
margins,
respectively,
refer
to
the
first
and
last
nucleotides
or
amino
acids
in
each
line.
Negative
numbers
indicate
nucleotide
positions
upstream
from
the
transcription
start
site.
The
deduced
amino
acid
sequences
encoded
by
the
exons
are
indicated
above
the
nucleotide
sequence
in
the
three-letter
amino
acid
code
and
numbered
as
described
previously
(23,
52).
An
asterisk
indicates
an
"extra"
serine
residue
between
codons
234
and
235,
which
has
been
desigznated
position
234a
(23,
52).
A
hatched
box
represents
a
sp)lice
junction
border,
and
a
vertical
arrow
denotes
the
cap
site
which
is
assigned
as
nucleotide
number
1.
The
CAAAT,
TATA,
and
putative
polyadenylation
signal
ATTAAA
are
indicated
by
the
boxes.
The
5'-
and
3'-untranslated
gene
regions
are
underlined,
and
a
G+T-rich
stretch
downstream
from
the
putative
polyadenylation
site
(16,
26)
is
underscored
with
a
broken
line.
The
restriction
sites
used
for
the
exonuclease
VII
mapping
described
in
the
legend
to
Fig.
3
are
marked
and
underlined.
Abbreviations:
Trm,
termination
codon;
3'UT,
3'-untranslated
region.

18
HU
ET
AL.
94S6-
6682-
a
436.-
2322
--
2027
-
353
VB'2
--
603--
3
--
7_2
4
Spr-
T
i
-~
~
.
;7
r):
c
"
8
?,i
g'
353
9
330
W
3
-'i2
78
--
8'2
,7
.
so
-mom
--
622
---
58
'-,
-1
---
6-03
_
-
403
As;
_ma.-
-
3
0
_m
-
278
B
-1AEIL
B)m
HI
-
-
U.Eml-
1750
nt
.--
.-.
-
Poe
.40C,
rt
-
--
-_
_
_-
>=
.rl
C
F
ogmert
93*
nt
t-oetc.Ir,g
mner
.8
E
r
_D
e
F-.et
Fgqmer'
FIG.
3.
Exonuclease
VII
mapping
of
the
mouse
skeletal
a-actin
gene.
Total
RNA
(50
p.g)
from
a
differentiated
culture
of
BC3H-1
cells
was
hybridized
at
60
or
65°C
with
approximately
S
ng
of
each
probe
as
shown
in
the
schematic
diagram
(lower
panel).
The
hybridized
samples
were
treated
with
3
U
of
exonuclease
VII
(1
h,
45°C),
electrophoresed
on
an
alkaline
agarose
gel
(A)
or
a
5%
polyacrylamide-8
M
urea
sequencing
gel
(B, C),
and
autoradiographed.
(A)
Lanes:
1,
size
markers,
HindIII
fragments
of
y
DNA;
2,
size
markers,
HaeIlI
fragments
of
(X174
DNA;
3,
undigested
probe
of
1,750-nucleotide
BglII-BglII
fragment;
4
and
5,
protected
products
from
samples
hybridized
at
60
and
65°C,
respectively.
(B)
Lanes:
1,
size
markers,
HaeIlII
fragments
of
4X174
DNA;
2,
size
markers,
HpaII
fragments
of
PBR
322;
3,
undigested
probe
of
580-nucleotide
SphI-BglII
fragment;
4
and
5,
protected
products
from
samples
hybridized
at
60
and
65°C,
respectively.
(C)
Lanes:
1
and
2,
protected
products
from
samples
hybridized
at
65
and
60°C,
respectively;
3,
undigested
probe
of
391-nucleotide
BamHI-BamHI
fragment;
4,
size
markers,
HaeIII
fragments
of
(X174
DNA;
5,
size
markers,
HpaII
fragments
of
PBR
322.
nt,
Nucleotide.
mM
dithiothreitol
and
heated
to
65°C
for
10
min.
This
heating
step
substantially
improved
the
resolution
of
exten-
sion
products
over
background,
as
observed
by
Fornwald
et
al.
(13).
An
equal
volume
of
a
solution
containing
deoxynu-
cleoside
triphosphates
(1
mM
each),
750
U
of
RNasin
per
ml,
and
40
,ug
of
actinomycin
D
per
ml
was
added,
and
the
primer
extended
with
avian
myeloblastosis
virus
reverse
transcriptase
(500
U/ml)
at
42°C
for
1
h.
The
reaction
was
terminated
by
the
addition
of
EDTA
to
10
mM,
and
the
RNA
was
degraded
by
treatment
with
DNase-free
RNase
(50
,ug/ml)
at
40°C
for
1
h.
After
ethanol
precipitation,
the
reactions
were
suspended
in
formamide-dye
buffer,
dena-
tured
by
boiling
for
3
min,
and
electrophoresed
on
a
6%
polyacrylamide-8
M
urea
sequencing
gel
in
Tris
borate-
EDTA
buffer.
DNA
sequencing
reactions
were
used
as
size
markers.
Extended
products
were
detected
by
autoradiogra-
phy
of
the
dried
gel.
Exonuclease
VII
mapping.
For
exonuclease
VII
mapping,
actin-gene-containing
plasmid
DNA
fragments
were
isolated
and
labeled
with
either
polynucleotide
kinase
and
[y-
32P]ATP
(3,000
Ci/mmol)
for
5'-end
mapping,
or
E.
coli
DNA
polymerase
I
large
fragment
(Klenow)
and
[a32P]dCTP
(400
Ci/mmol)
for
3'-end
mapping.
DNA-RNA
hybridization
was
performed
as
described
above
in
the
primer
extension
analysis
by
using
50
,ug
of
total
RNA
from
a
differentiated
culture
of
BC3H-1
cell
line
and
about
5
ng
of
labeled
(ca.
106
cpm/,ug)
DNA
fragment.
Hybridizations
were
carried
out
at
60
or
65°C
for
3
h.
Each
hybridization
mixture
was
diluted
into
10
volumes
of 30
mM
KCI-10
mM
Tris
hydrochloride
(pH
7.4)-10
mM
EDTA,
chilled
on
ice,
and
incubated
at
45°C
for
1
h
with
10
U
of
E.
coli
exonuclease
VII
per
ml
(2).
After
ethanol
precipitation,
the
exonuclease
VII-resistant
material
was
electrophoresed
on
a
5%
polyacrylamide-8
M
urea
sequencing
gel
or
alkaline
agarose
gel
(24)
and
autoradiographed.
In
vitro
transcription
with
SP6
RNA
polymerase
and
RNase
mapping.
Synthesis
of
the
complementary-strand
SP6
probe
and
RNase
mapping
were
carried
out
as
described
by
Melton
et
al.
(28).
RESULTS
Isolation
and
mapping
of
the
mouse
skeletal
at-actin
gene.
A
cosmid
clone
containing
the
skeletal
ax-actin
gene
was
iso-
lated
from
a
BALB/c
genomic
cosmid
library
as
described
in
Materials
and
Methods.
The
location
and
orientation
of
the
gene
within
a
single
6.8-kb
EcoRI
fragment
were
established
by
restriction
endonuclease
mapping
with
5'
and
3'
frag-
ments
of
the
Drosophila
actin-coding
sequence
and
the
rat
a-actin
3'-untranslated
sequence
as
probes.
The
6.8-kb
EcoRI
fragment
containing
the
entire
skeletal
a-actin-coding
and
flanking
sequences
was
isolated
from
the
41-kb
insert
of
the
cosmid
clone
(Fig.
1A)
and
subcloned
into
the
EcoRI
site
of
the
plasmid
vector
pSP62
(Fig.
1B).
Subsequently,
the
putative
promoter
region
of
the
gene
was
(A)
3,
--
664W
6
35--
872
622
__
6-ro
a,
t.t
.
Bg:Z
lm
L-
lmmm
mmL
MOL.
CELL.
BIOL.
a
46
40
-W
--,
.'
-1.
-W
:"
am
4*0
'Aft

MOUSE
SKELETAL
a-ACTIN
GENE
19
localized
to
about
100
base
pairs
within
the
SmaI-SstI
region
(Fig.
1C
and
D)
by
hybridization
with
the
20-base
oligonu-
cleotide
probe
containing
the
sequence
of
the
CAAT
pro-
moter
homology
which
is
highly
conserved
between
the
skeletal
a-actin
genes
of
chickens
and
rats
(34)
(data
not
shown).
This
result
confirmed
that
the
subcloned
EcoRI
fragment
contained
the
5'-flanking
sequence
of
the
gene.
Also,
it
suggested
that
the
position
of
the
transcription
initiation
site
should
be
close
to
the
SstI
site.
The
detailed
restriction
endonuclease
map
of
the
actin-coding
and
flanking
regions
in
Fig.
1C
was
used
to
choose
the
DNA
fragments
to
be
subcloned
into
M13
and
mpl8
and
mpl9
vectors
for
sequencing.
From
these
subclones
we
deter-
mined
the
linear
sequence
of
4,007
nucleotides,
on
both
strands
independently
(Fig.
2).
Amino-acid-coding
region
of
the
mouse
skeletal
a-actin
gene.
The
complete
nucleotide
sequence
of
the
mouse
skel-
etal
a-actin
gene
with
the
5'-
and
3'-flanking
regions
is
shown
in
Fig.
2.
Exons
within
the
protein-coding
region
and
the
introns
separating
them
were
initially
assigned
mainly
by
homology
with
the
rat
skeletal
a-actin
genomic
sequence
(54)
and
in
part
by
comparison
with
the
partial
cDNA
sequence
for
the
carboxy-terminal
portion
of
the
mouse
protein
(29).
This
assignment
was
supported
by
the
fact
that
the
sequences
at
the
presumed
exon-intron
junctions
are
in
accordance
with
the
consensus
sequence
for
splice
sites
(7).
In
addition,
the
expected
lengths
of
exons
2,
3,
4,
and
5
were
confirmed
experimentally
within
an
accuracy
of
±+3
nucleo-
tides
by
hybridizing
an
SP6
anti-sense
transcript
of
a
BamHI-EcoRI
fragment
(Fig.
1B)
to
poly(A)'
RNA
from
differentiated
BC3H-1
cells
and
RNase
mapping
(data
to
be
presented
in
the
Ph.D
thesis
by
M.
C.-T.
Hu
at
the
Califor-
nia
Institute
of
Technology).
The
translated
amino
acid
sequence
for
this
interpretation
of
the
structure
of
the
mouse
skeletal
a-actin
gene
is
identical
to
that
of
rats,
rabbits,
and
chickens.
(The
amino
acid
sequence
of
mouse
skeletal
a-actin
has
not
been
directly
determined.)
The
coding
sequence
begins
with
codons
for
two
amino
acids,
Met
and
Cys,
which
are
absent
from
the
mature
protein.
They
are
followed
by
the
codon
for
Asp
(GAC),
the
known
N-terminal
residue
of
striated
muscle
actin.
The
same
two
codons
preceding
the
codon
specifying
the
N-terminal
amino
acids
are
found
in
the
human
(20),
rat
(54),
and
chicken
(13)
skeletal
oa-actin
genes
and
in
the
human
(19)
and
chicken
(8)
cardiac
a-actin
genes.
Interestingly,
these
two
codons
are
also
found
in
all
six
Drosophila
actin
genes
(14)
and
sea
urchin
actin
genes
(10,
43),
but
the
Cys
codon
is
absent
in
vertebrate
cytoskeletal
P-actin
genes
(22,
33,
38).
It
has
been
suggested
that
the
Met-Cys
dipeptide
is
removed
by
posttranslational
processing
(54).
Although
the
derived
sequence
of
the
primary
skeletal
a-actin
is
377
residues,
we
have
numbered
the
amino
acids
in
Fig.
2
in
conformity
with
the
numbering
system
suggested
by
Lu
and
Elzinga
(23)
and
Vandekerckhove
and
Weber
(52),
which
yields
374
positions.
Of
the
three
additional
positions,
two
are
the
Met-Cys
dipeptide
at
the
N-terminus,
while
the
third
is
an
"extra"
serine
residue
between
positions
234
and
235
which
has
been
designated
234a
(23,
52).
The
coding
region
of
the
mouse
skeletal
a-actin
gene
is
split
by
five
introns
(Fig.
1D)
at
codons
specifying
amino
acids
41/42
(IVS
2),
150
(IVS
3),
204
(IVS
4),
267
(IVS
5),
and
327/328
(IVS
6).
These
intron
positions
are
identical
to
those
for
the
corresponding
genes
of
chickens
and
rats
(13,
54).
The
length
and
positions
of
the
exons
and
introns
may
be
deduced
from
Fig.
2.
Previously,
Zakut
et
al.
(54)
have
reported
that
a
potential
splice
site
(CAG/GTA)
is
present
32
PE
C
G
T
A
(nt)
r
!20
6-
-
4;-
r.~
~
i.
B
mm
2
3
4
5
Ex
IVSI
Ex
2
5
E
=Z-v
_
3
Genanic
DNA
-
5
t /
/
3'
m
RNA
118
nt
Extension
Product
~-'Prirmer
42nt
FIG.
4.
Identification
of
the
5'
end
of
the
mouse
skeletal
a-actin
mRNA
by
primer
extension.
The
5'-end-labeled,
42-base
oligonu-
cleotide
complementary
to
the
coding
sequence,
from
positions
1038
to
1079
(Fig.
2),
was
hybridized
with
5
,ug
of
poly(A)+
RNA
from
a
differentiated
culture
of
BC3H-1
cells
and
extended
by
using
reverse
transcriptase
as
described
in
the
text.
The
extension
products
were
electrophoresed
on
a
6%
polyacrylamide-8
M
urea
sequencing
gel
and
autoradiographed.
The
diagram
(lower
panel)
shows
the
product
expected
from
full-length
elongation
of
mRNA.
Lanes:
1,
primer
extension
products;
2
through
5,
sequencing
ladders
used
as
size
markers.
nt,
Nucleotide.
base
pairs
downstream
from
the
CG/GT
splice
site
at
codon
150
in
the
rat
skeletal
ax-actin
gene.
Our
results
do
not
reveal
this
potential
splice
site
in
the
mouse
gene.
This
is
not
surprising,
because
use
of
the
extra
splice
site
in
the
rat
gene
would
produce
an
actin
with
an
insert
of
11
amino
acids,
and
no
such
product
has
yet
been
detected.
Sequence
of
the
5'-untranslated
region
of
the
mouse
skeletal
a-actin
gene.
Although
the
actin
amino
acid
sequence
data
and
cloned
cDNA
partial
sequence
(29)
could
be
used
to
identify
the
translated
regions
of
the
gene,
independent
means
were
required
for
delineating
the
5'-untranslated
region.
The
5'
borders
of
the
first,
untranslated,
exon
and
the
second
exon
were
approximately
determined
by
exonucle-
ase
VII
mapping
(Fig.
3A
and
B).
In
addition,
the
precise
assignment
of
the
transcription
initiation
site,
ACAC,
was
VOL.
6,
1986

20
HU
ET
AL.
MOL.
CELL.
BIOL.
(A)
100
50[
oXl
I
0
*|
*I
I
I
I
I
f
:~~~
:
"
'I
II
II
II
#I
5'~
~~~~~~
~~~~
-'
I
_
i3
10
5
Ex
E2
Ex2
Ex
3
Ex
4
Ex
5\
Ex
6
--,Ex
7
(B)
)O
-
Mouse
vs
Chick
PAM1
I
v0
500
1000
1500
2000
2500
3000
3500
Nucleic
Acids
(bp)
FIG.
5.
Percent
homology
profiles
for
the
mouse,
rat,
and
chicken
skeletal
a-actin
genes.
In
this
analysis,
the
sequence
search
string
was
25
nucleotides,
and
gaps
inserted
in
the
sequences
for
alignment
purposes
were
scored
as
regions
of
0%
homology.
(A)
Mouse
versus
rat
skeletal
a-actin
genes;
(B)
mouse
versus
chicken
skeletal
a-actin
genes.
The
structure
of
the
mouse
skeletal
a-actin
gene
is
shown
between
the
two
homology
plots.
Vertical
arrows
indicate
two
inverted
duplications
as
described
in
the
text.
bp,
Base
pairs.
confirmed
by
a
newly
developed
mapping
technique
by
using
T4
DNA
polymerase
(M.
C.
T.
Hu
and
N.
Davidson,
sub-
mitted
for
publication)
and
assigned
to
be
nucleotides
-1/1.
This
site
was
identified
1,031
nucleotides
upstream
from
the
initiator
ATG
codon
(Fig.
2).
We
assigned
the
5'
border
of
the
first
intron
to
nucleotides
58/59
by
matching
the
length
of
the
primer
extended
product
(Fig.
4)
with
the
positions
determined
above
for
the
5'
borders
of
the
first
and
second
A
MOUSE
-311
GGTCAAAGCAGTGACCTTTGGCCCAGCACAGCCCTTCCGTGAGCCTTGGAGCCAGTTGGGAGGGGCAGACAGCTGGGGATACTC
TCCATATACGG
CCT
-214
RAT
-300
..
....A...A
.....C...
A.
C.A........
.......
..G
.......
C.........
...........
..C-207
CHICK
-319
T.
.CCCC.GAG.C.TCC.CC
.TCA.G.CT.GG
.G
.
CTCC.TGGCTCC.GCACG.CCCT..
AGAGGCC.C
CACCGCT.G
...T.
2.
G.C
-225
1
MOUSE
-213
GGTCCGGTCCTAG
CT
ACCTGGGCCAGGG
CAGTCCTCTCCTT
-173
RAT
-206
.
.......
T.AGC
..
............C....T
..
-165
CHICK
-224
.
.
G.CG.ATTATTTCGGCCCCGGCCCGGGGGCCTCTAGACGCTCCTTATACGGCCCGGCCTCG..C.........
GC.
.CCA.
AAGCAG
-127
a
a'I
2
2'
3
MOUSE
-172
CTTTGGTCAGTGCAGG
AGACCCGGGCGGGACCCAGGCTGAGAACCAGCCGAAGGAAGGGACTCTAGTGCCCGACACCCAAATATGGCTTGGGAAGGGCA
-74
RAT
-164
.........:.G
.......A.ACA7AGCTGAGG
-70
CHIC -126
4
......G
...C
..G
..C
.G
..G
.........G
G
.G
...........A
-70
CHICK
-126
.
G....
....
G
C.
.G.
.CC.G.
.G
NNCGCC
.G
.....................NNGC.G
....................
GC.
.CCG
.,
.TC
-64
b
b
3'
4
MOUSE
-73
GCAACATTCTT
CGGGGCGGTGTGGGGAGAGCTCCCGGGACTATATAAAAiCCTGTGCAAGGGGACAGGCGGTCACACG
RAT -69
A
.
C.
TC
......A.
A
.A.GCTA
.
CHICK
-63
.
C.GTCGC
....
C.G.C.
.T.G
..
CC...
GC.G.CG..CG..C..
5
5
3
c
4'
HUMAN
I
ACCGCAG
CGGAC
AGCGCCAAGTGAAGCCTCGCTTCCCTCCCGCGGCGACC
A
GGGCCCGAGCCGAGAGTAGCAGTTGTAGCTACCCGCCCAGA
92
MOUSE
1
AC.
..
A.
TA.T
..
...
C.
A
T
C.
59
RAT
I
....
.......A....TA
...
........A..
..C..
....
60
CHICK
I
...
.CT..C...
GG
..
G..G...T..G..A
..G.C.C..
.T
,C.
....
62
C'I
*
HUMAN
93
AA
CTAGACACAATGTGCGACGAA
MOUSE
60
..
......
C
...
RAT
61
.....T
G
CHICK
63
C.G.C..
..AC
....
T
-G
115
82
83
85
FIG.
6.
Comparison
of
the
nucleotide
sequence
of
the
5'-flanking
and
the
5'-untranslated
regions
of
vertebrate
skeletal
ax-actin
genes.
(A)
Alignment
of
the
5'-flanking
region
sequences
of
mouse,
rat,
and
chicken
skeletal
a-actin
genes.
(B)
Alignment
of
the
5'-untranslated
region
sequences
of
human,
mouse,
rat,
and
chicken
skeletal
a-actin
genes.
Dots
indicate
identity
with
the
first
sequence
listed.
Blanks
indicate
that
gaps
have
been
introduced
during
the
alignment
for
maximal
homology.
The
CAAAT
box
and
TATA
box
are
highlighted
with
solid
bars.
A
broken
bar
indicates
the
transcription
initiation
site,
and
an
asterisk
indicates
the
initiation
codon
ATG.
Horizontal
arrows
above
the
sequences
represent
the
adjacent
inverted
complementary
sequences
of
rodents
which
are
indicated
numerically,
whereas
horizontal
arrows
underneath
the
sequences
represent
the
adjacent
repeats
of
chicken
sequences,
which
are
indicated
alphabetically.
I
ls
vs
It
B
1'

MOUSE
SKELETAL
a-ACTIN
GENE
21
(A)
(C)
TTTT
TC
TAA
GC
TA
AT
5
G
TT
AG(25°)KcoaVncle
-21.2
(B)
at
51
G
AC
LG(250)KIca/mole
-604
-32.2
-32.1
-31.6
G
3'
CGCAGT
-64.8
-494
G
TA
TA
AGCATAEA
GT2
A
.
A
Iv
CAGCC
TGAAGcR3
AG(250)14d/nrle
-8.4
(D)
9TGTTT
TGrTAT
.
-A
CG
T
TA
d
t
^A
Id'
TA
AA
5'
A
TTA
CTTCAAGAAGCf
,G(250)Kcdx/roke
-8.2
FIG.
7.
Predicted
inverted
repeat
structures
in
the
5'-flanking
region
and
the
5'-
and
3'-untranslated
regions
of
the
mouse,
rat,
and
chicken
skeletal
a-actin
genes.
(A)
Four
potential
configurations
are
shown
in
the
5'-flanking
and
the
5'-untranslated
region
of
the
mouse
skeletal
a-actin
gene.
Similar
configurations
can
be
found
in
the
rat
gene.
(B)
Three
potential
configurations
are
shown
in
the
5'-flanking
and
the
5'-untranslated
region
of
the
chicken
skeletal
a-actin
gene.
(C)
A
potential
stem-loop
is
demonstrated
in
the
3'-untranslated
region
of
the
mouse
skeletal
a-actin
gene.
A
similar
structure
can
be
found
in
the
rat
gene.
(D)
A
potential
stem-loop
is
shown
in
the
3-untranslated
region
of
the
chicken
skeletal
a-actin
gene.
The
indicated
free
energy
values
for
the
base-paired
regions
were
calculated
by
the
method
of
Tinoco
et
al.
(50).
Note
that
the
free
energies
for
the
base-paired
regions
in
the
5'-flanking
region
were
estimated
by
the
same
method,
assuming
that
the
stacking
energies
of
DNA
base
pairs
are
similar
to
those
of
RNA
base
pairs.
CAAAT,
TATA,
and
putative
polyadenylation
signal
ATTAAA
are
indicated
by
the
boxes.
exons.
The
sequences
at
the
determined
borders
of
the
first
intron
are
in
agreement
with
the
consensus
splice
site
sequences
(7).
Thus,
the
first
intron
is
961
nucleotides
long
and
interrupts
the
5'-untranslated
region
12
nucleotides
upstream
from
the
initiator
ATG
codon.
A
canonical
promoter
sequence
TATATAAA
(5)
was
identified
at
nucleotides
-33
to
-26
(Fig.
2),
and
a
CAAAT
sequence
(12)
was
located
91
nucleotides
upstream
from
the
transcription
initiation
site.
The
positions
of
these
regulatory
sequences
in
the
promoter
region
correspond
well
with
those
of
similar
sequences
found
upstream
from
the
5'
cap
site
of
other
eucaryotic
genes
(7).
Sequence
of
the
3'-untranslated
region
of
the
mouse
skeletal
a-actin
gene.
The
location
of
the
polyadenylation
site
was
VOL.
6,
1986

22
HU
ET
AL.
MOUSE
I
TAGGCGCACCGCATCTGCGTTCGCGCTCTCTCTCCTCAGGACGAC
AATCGACAATCGTGCTGT
GGTTGCAGGGTGGCCCCGTCCTCCGCCGTGGCTCC
RAT
I
.AA
TGTACTTT.............
..
..C.
-A
GG.A...A
CHICK
I.
..AA.ATGTTTACATGATCACTTT..CAA.CACA.
...
-T-
T...A
...C..
CT.
TGA.GA.CTG
HUMAN
i
..
AA
..
.TC..C.CA
.A.G.
GA..T.
.......G..
T.CCAA.G.GG
..C..
TGA.
98
98
77
69
MOUSE
99
ATCGCCGCCACTGCAGCCGGCGCC
TGT
TTTTG
AC
GTGT
136
RAT
99
.
6..........................
.........
1
6
CHICK
78
...
A
TGCAACTT
CA
A..
TA..AATTTCTGGTTACTGTTGCTGCAAAGCCCATGTGACACAGTGTATGTAAA
....
161
HUMAN
70
T.T
..AC.C.GCA.TC..C
......
C
C..
C..G.
CCATC.T
..
.C
.......T.
140
5
5S
MOUSE
137
ACATAGATTGACTCGTTTTACCTCATTTTGTTA
TTTTTCAAACAAA
GCCCTGTGGAAAGGAAATGGAAAACTT
GAAGCATTAAAGC
CAGCCAT
229
RAT
137
.
.CGS
...
...
228
CHICK
162
.
A...
AT.TA
G.
TG
.
C.
T
CAT
CAA.
TAT.
T
260
d
.
d
I
MOUSE
230
TCTGTTTTGCTCCAA
RAT
229
.............
CHICK
261
.....
CACA.C
..CC
244
241
275
FIG.
8.
Comparison
of
the
nucleotide
sequence
of
the
3'-untranslated
regions
of
vertebrate
skeletal
a-actin
genes.
Alignment
of
the
3'-untranslated
region
sequences
of
mouse,
rat,
chicken,
and
human
(partial)
skeletal
a-actin
genes.
Symbols
are
as
described
in
the
legend
to
Fig.
6.
The
putative
polyadenylation
signal
ATTAAA
is
highlighted
with
a
solid
bar,
and
the
termination
codon
TAG
is
indicated
with
an
asterisk.
identified
approximately
at
nucleotide
2944
(±5
nucleotides)
of
the
sequenced
gene
-(Fig.
2)
by
exonuclease
VII
mapping
(Fig.
3C).
The
sequence
ATTAAA
was
located
25
nucleo-
tides
upstream
from
the
polyadenylation
site.
In
addition,
the
sequence
TGTGTGTGG
was
found
4
nucleotides
down-
stream
from
the
polyadenylation
site,
in
agreement
with
the
suggestion
that
a
G+T-rich
stretch
downstream
from
the
polyadenylation
site
is
required
for
the
correct
3'-end
for-
mation
of
mRNA
(16,
26).
Another
potential
polyadenylation
signal
AATAAA
was
also
found
at
nucleotides
2942
through
2947,
about
22
nucleotides
downstream
from
the
end
of
the
putative
polyadenylation
signal
ATTAAA
identified
above.
We
have
no
evidence
that
this
second
potential
signal
functions
in
the
mouse.
Thus,
the
3'-untranslated
region
of
the
mouse
skeletal
a-actin
mRNA
is
about
245
nucleotides
long
[excluding
the
poly(A)
tail].
It
is
about
the
same
size
as
the
3'-untranslated
regions
of
the
rat
(241
nucleotides
[54])
and
human
(253
nucleotides
[37])
skeletal
a-actin
mRNA
but
much
shorter
than
that
of
rat
cytoskeletal
,-actin
mRNA
(670
nucleotides
[33]).
The
length
of
skeletal
a-actin
mRNA
in
mammals
is
about
1,650
nucleotides
(31,
37,
44),
including
the
poly(A)
tail.
Comparison
with
the
total
length
of
transcribed
se-
quences
in
the
mouse
and
rat
genes
(about
1,450
nucleotides)
would
suggest
that
the
poly(A)
tail
is
about
200
nucleotides
long.
Copy
number
of
the
mouse
skeletal
a-actin
gene.
Southern
blot
analysis
of
BALB/c
genomic
DNA
digested
with
four
different
restriction
endonucleases
(PstI,
BglII,
HindIII,
and
SstI)
demonstrated
that
genomic
and
cosmid
fragments
hybridizing
with
the
skeletal
a
3'-untranslated
region-
specific
probe
comigrate
(data
not
shown).
This
result
sug-
gests
that
the
gene
is
present
in
single
copy
in
the
mouse
genome,
in
agreement
with
previous
reports
(29,
30).
DISCUSSION
Strong
homology
and
interesting
inverted
repeat
structures
in
the
5'-flanking
region
and
both
the
5'-
and
3'-untranslated
regions
of
vertebrate
skeletal
a-actin
genes.
We
have
aligned
the
nucleotide
sequence
of
the
mouse
skeletal
a-actin
gene
with
those
of
the
rat
and
chicken
by
using
percent
homology
profiles
(Fig.
5).
All
alignments
depend
on
introduction
of
gaps
for
maximal
homology,
and
areas
of
high
and
low
homology
between
two
sequences
are
displayed
as
peaks
and
troughs,
respectively.
The
coding
sequences
show
a
very
high
degree
of
homology
(-90%),
as
expected
since
the
proteins
themselves
are
identical.
In
comparison
of
rat
with
mouse,
the
intron
sequences
between
coding
exons
are
about
75%
homologous,
except
for
intron
3,
for
which
the
lengths
differ
by
49
nucleotides.
The
corresponding
introns
of
the
chicken
are
much
more
divergent
in
length
and
sequence.
In
comparing
rat
with
mouse,
the
long
intron
following
the
5'
untranslated
exon
1
shows
sharp
peaks
of
conserved
and
nonconserved
regions.
Figures
5
and
6A
show
very
high
conservation
(-85%)
in
the
5'-flanking
region
between
the
cap
site
and
300
nucleo-
tides
upstream
of
rat
and
mouse
skeletal
a-actin
genes.
The
homology
between
chicken
and
mouse
in
the
same
region
is
also
rather
high
(-60%).
Nudel
et
al.
(32)
have
found
a
similar
degree
of
homology
between
the
rat
and
chicken
skeletal
a-actin
genes.
We
have
also
found
considerable
homology
in
the
5'-untranslated
region.
By
introducing
gaps
for
best
alignment
(Fig.
6B)
there
is
(i)
a
high
degree
of
homology
between
rat
and
mouse
in
the
5'
untranslated
region,
(ii)
a
rather
high
degree
of
homology
between
human
and
rodents,
except
for
three
long
inserts
in
the
human
gene,
and
(iii)
a
moderate
degree
of
homology
between
chickens
and
rodents.
Conserved
sequences
between
chickens
and
rats
around
the
CAAT
box
and
about
46
to
59
nucleotides
downstream
from
the
cap
site
have
been
previously
recog-
nized
by
Ordahl
and
Cooper
(34).
A
similar
comparison
of
the
5'-untranslated
regions
of
nonmuscle
3-actins
(human
cDNA
and
rat
genomic
sequence)
also
shows
a
high
degree
of
sequence
conservation
(38).
We
find,
however,
no
cross-
homology
between
a-
and
j-actin
5'-untranslated
regions.
A
number
of
studies
have
suggested
that
the
sequence
and
structure
of
the
mRNA
in
the 5'-untranslated
region
have
an
important
role
in
regulation
of
translation
(11,
36,
40,
46).
The
fact
that
there
are
conserved
sequences
in
the
5'-
untranslated
region
in
all
of
the
vertebrate
skeletal
a-actin
genes,
but
a
different
set
of
conserved
sequences
in
the
cytoskeletal
,-actin
genes,
suggests
that
there
may
be
de-
velopmentally
specific
translational
regulatory
mechanisms
in
muscle
versus
nonmuscle
cells.
It
is
striking
that
a
number
of
inverted
repeat
structures
exist
in
the
5'-flanking
and
the
5'-untranslated
regions
of
the
rodent
and
chicken
genes.
These
are
indicated
as
inverted
repeats
in
Fig.
6
and
as
remarkably
stable
hairpin
structures
MOL.
CELL.
BIOL.

MOUSE
SKELETAL
a-ACTIN
GENE
23
for
a
single
strand
in
Fig.
7A
and
B.
Some
of
these
inverted
repeat
structures
have been
conserved
between
chickens
and
rodents.
The
species
compared
(avian
and
mammalian)
have
been
separated
for
more
than
250
million
years
(12),
indicating
a
strong
selective
constraint
to
conserve
these
sequences
and
suggesting
that
the
sequences
may
be
biolog-
ically
significant.
If
the
primary
transcripts
actually
initiate
at
the
cap
site,
the
structures
shown
in
Fig.
7A
and
B
would
not
occur
in
the
RNA.
These
sequences
could
function
as
duplicated
transcription
factor
binding
sites,
with
the
bound
factors
(presumably
proteins)
having
opposite
orientation
at
the
two
members
of
an
inverted
repeat
as
postulated
by
McKnight
et
al.
(25)
and
by
Giniger
et
al.
(18).
Alternatively,
some
single-strand
DNA
regions
may
be
opened
up
during
formation
of
a
transcription
bubble,
and
these
hairpins
could
then
form
in
the
DNA
as
indicated
in
Fig.
7.
Potential
hairpin
structures
have
also
been
found
in
the
5'-flanking
region
and
the
first
untranslated
exon
in
the
rat
cytoskeletal
1-actin
gene
(33).
They
differ,
however,
from
those
found
in
the
skeletal
a-actin
genes.
For
example,
whereas
the
TATA
box
is
presented
within
a
loop
of
the
rat
P
gene,
it
is
found
between
stem-loop
structures
in
the
a
genes.
The
existence
of
such
differences
between
the
cytoskeletal
and
muscle-
specific
actin
genes
raises
the
possibility
that
the
conserved
inverted
repeats
in
the
a
genes
are
important
for
tissue-
specific
expression.
To
our
knowledge,
this
is
the
first
description
of
inverted
repeats
and
possible
secondary
struc-
ture
formation
among
the
highly
conserved
sequences
in
the
5'-flanking
and
the
5'-untranslated
regions
of
vertebrate
skeletal
a-actin
genes.
A
long
sequence
of
about
110
nucleotides,
including
the
putative
polyadenylation
signal
ATTAAA,
is
highly
con-
served
in
the
3'-untranslated
region of
vertebrate
skeletal
a-actin
genes
(Fig.
8).
It
is
noteworthy
that
two
blocks
of
these
highly
conserved
sequences
in
the
3'-untranslated
region
can
form
a
stem-loop
structure
with
estimated
stabil-
ities
of
-8.4
kcal/mol
for
mice
and
rats
(Fig.
7C)
and
-8.2
kcallmol
for
chickens
(Fig.
7D).
These
structures
are
imme-
diately
upstream
from
the
ATTAAA
polyadenylation
signal.
It
has
been
suggested
that
the
inverted
repeat
at
the
3'
end
of
sea
urchin
histone
mRNA
is
important
for
the
generation
of
the
histone
mRNA
3'
termini
(5, 6).
This
putative
structure
does
not
act
as
a
DNA
cruciform,
but
exerts
its
function
at
the
level
of
the
RNA
transcripts
(4).
It
is
conceivable
that
the
potential
hairpin
structure
upstream
from
the
putative
polyadenylation
signal
in
skeletal
a-actin
plays
a
role
in
the
correct
3'-end
formation
of
skeletal
a-actin
mRNA.
Interesting
features
in
the
first
intron
of
vertebrate
skeletal
a-actin
genes.
Rat
and
mouse
skeletal
a-actin
genes
have
a
long
first
intron
compared
with
chickens
(i.e.,
976,
961,
and
111
nucleotides
for
rats,
mice,
and
chickens,
respectively).
There
are
several
highly
conserved
sequences
in
this
intron
between
rats
and
mice,
but
the
introns
are
quite
divergent
in
other
regions
(Fig.
SA).
The
chicken
first
intron
is
quite
G
+
C
rich
(82%)
compared
with
rat
introns
(53.5%)
and
mouse
introns
(52%).
We
have been
unable
to
find
any
sequence
homology
between
the
chicken
and
rodent
introns.
There
are
two
inverted
duplications
within
the
rodent
first
intron.
Both
occur
within
the
conserved
sequences
(arrows
in
Fig.
5A).
Furthermore,
an
inverted
repeat
can
also
be
found
in
the
chicken
first
intron
(shown
as
a
hairpin
in
Fig.
9C).
The
remarkably
stable
hairpin
structures
in
the
vertebrate
first
intron
(Fig.
9)
may
form
in
the
primary
transcript.
If
the
splicing
apparatus
tracks
along
the
intron
in
search
of
splice
sites,
it
may
be
able
to
pass
along
the
base
of
such
hairpins.
The
hairpins
would
effectively
shorten
the
intron,
thereby
(A)
ri9
(Tr
IVS
I
TE
s
GCT
<!56)
Emon
GAGCA
W
(25*)K
al/rn
x
-
31.5
Exon
2
-34.6
(B)
49,
14S5)
(913
(8
(239)
TA~~~~~~~~~~~~IT
A88
jA
T
~~~~~~~~G,
IvS1I
TA
VS
I
CG
5
GG
TnCGG
2
Exo
I
GAGCA
G
nI
TT
AT
82
/(1036)
Exon
2
AG(2)KCIATo*
-43.7
-55.1
(C)
cc
GCT
clrk
C
A
(87)
e"(157)
Exa
(61462)
21n
CO
G
10
(171/72)
Exn2
AG(25')1IX/mde
-56.2
FIG.
9.
Predicted
inverted
repeat
structures
in
the
first
intron
of
rodents
and
chicken
skeletal
a-actin
genes.
(A)
Two
potential
configurations
in
the
first
intron
of
mouse
skeletal
-actin
gene.
(B)
Similar
configurations
in
the
first
intron
of
rat
skeletal
a-actin
gene.
(C)
One
potential
hairpin
loop
in
the
first
intron
of
chicken
skeletal
a-actin
gene.
The
indicated
free
energy
values
for
the
base-paired
regions
were
calculated
by
the
method
of
Tinococ
et
al.
(50).
Numbers
within
parentheses
indicate
the
numbers
of
nucleotides
downstream
from
the
transcription
initiation
site.
VOL.
6,
1986

24
HU
ET
AL.
expediting
its
excision
by
splicing.
A
similar
mechanism
has
been
proposed
as
one
way
to
explain
intermolecular
splicing
between
two
RNAs
base
paired
through
their
intron
se-
quences
(47).
Alternatively,
we
speculate
that
these
inverted
repeats
may
play
a
role
of
transcriptional
enhancement
in
the
regulation
of
tissue-specific
gene
expression
because
it
has
been
proposed
that
a
tissue-specific
transcription
enhancer
element
is
located
in
the
intron
of
a
heavy-chain
immuno-
globulin
gene
(17).
In
fact,
the
putative
"core"
sequence
in
the
heavy-chain
gene
intron
is
present
as
an
inverted
repeat
(17).
In
conclusion,
we
have
found
several
conserved
and
inverted
repeat
sequences
outside
of
the
protein-coding
region
of
the
skeletal
a-actin
genes.
It
would
be
interesting
to
investigate
whether
these
conserved
inverted
repeat
se-
quences
serve
as
regulatory
elements
in
differentiated
mus-
cle
cells.
ACKNOWLEDGMENTS
We
are
grateful
to
Michael
Steinmetz
for
generously
providing
a
BALB/c
genomic
cosmid
library, to
Charles
P.
Ordahl
for
the
chicken
skeletal
a-actin
genomic
clone,
to
Leonard
I.
Garfinkel
for
a
rat
a-actin
cDNA,
to
Beverley
J.
Bond
for
the
Drosophila
actin
DmA2
genomic
clone,
and
to
Tim
Hunkapiller
for
BALB/c
genomic
DNA
and
help
with
the
computer
analyses.
We
also
thank
Joan
Steitz
for
valuable
discussions
and
David
Solnick,
Bruce
J.
Nicholson,
Terry
P.
Snutch,
Robert
Little,
and
Nevis
L.
Fregien
for
their
helpful
comments
on
this
manuscript.
This
work
was
supported
by
a
Public
Health
Service
research
grant
from
the
National
Institutes
of
Health.
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