Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes

Abstract

Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.

Full text

MOLECULAR
AND
CELLULAR
BIOLOGY,
JUIY
1986,
p.
2409-2419
Vol.
6.
No.
7
0270-7306/86/072409-11$02.00/0
Copyright
©3
1986,
American
Society
for
Microbiology
Six
Mouse
oL-Tubulin
mRNAs
Encode
Five
Distinct
Isotypes:
Testis-
Specific
Expression
of
Two
Sister
Genes
ALFREDO
VILLASANTE,'
DASHOU
WANG,'
PAUL
DOBNER,2
PATRICK
DOLPH,'
SALLY
A.
LEWIS,'
AND
NICHOLAS
J.
COWAN'*
Department
of
Biochemistry,
New
York
University
Medical
Center,
New}
York,
New
York
10016,1
and
Department
of
Molecular
Genetics
and
Microbiology,
University
of
Massachusetts
Medical
Center,
Worcester,
Massachusetts
016052
Received
19
February
1986/Accepted
4
April
1986
Five
mouse
a-tubulin
isotypes
are
described,
each
distinguished
by
the
presence
of
unique
amino
acid
substitutions
within
the
coding
region.
Most,
though
not
all
of
these
isotype-specific
amino
acids,
are
clustered
at
the
carboxy
terminus.
One
of
the
a-tubulin
isotypes
described
is
expressed
exclusively
in
testis
and
is
encoded
by
two
closely
related
genes
(Ma3
and
Ma7)
which
have
homologous
3'
untranslated
regions
but
which
differ
at
multiple
third
codon
positions
and
in
their
5'
untranslated
regions.
We
show
that
a
subfamily
of
a-tubulin
genes
encoding
the
same
testis-specific
isotype
also
exists
in
humans.
Thus,
we
conclude
that
(i)
the
duplication
event
leading
to
a
pair of
genes
encoding
a
testis-specific
ca-tubulin
isotype
predated
the
mammalian
radiation,
and
(ii)
both
members
of
the
duplicated
sequence
have
been
maintained
since
species
divergence.
A
second
ot-tubulin
gene,
Mca6,
is
expressed
ubiquitously
at
a
low
level,
whereas
a
third
gene,
MaL4,
is
unique
in
that
it
does
not
encode
a
carboxy-terminal
tyrosine
residue.
This
gene
yields
two
transcripts:
a
1.8-kilobase
(kb)
mRNA
that
is
abundant
in
muscle
and
a
2.4-kb
mRNA
that
is
abundant
in
testis.
Whereas
the
1.8-kb
mRNA
encodes
a
distinct
a-tubulin
isotype,
the
2.4-kb
mRNA
is
defective
in
that
the
methionine
residue
required
for
translational
initiation
is
missing.
Patterns
of
developmental
expression
of
the
various
at-tubulin
isotypes
are
presented.
Our
data
support
the
view
that
individual
tubulin
isotypes
are
capable
of
conferring
functional
specificity
on
different
kinds
of
microtubules.
Microtubules
are
assembled
from
heterodimers
of
a-
and
,B-tubulin
together
with
microtubule-associated
proteins.
They
function
in
a
wide
variety
of
ways
in
eucaryotic
cells;
for
example,
they
have
specific
functions
in
the
mitotic
and
meiotic
spindle,
in
the
centriole,
in
the
manchette
and
flagellar
axoneme
of
spermatozoa,
in
axonal
transport
in
neurons,
and
in
the
marginal
bands
of
platelets.
Clearly,
associated
proteins
such
as
kinesin
(28)
play
a
crucial
role
in
conferring
these
and
other
specific
functions
on
micro-
tubules.
Additionally,
a-
and
f-tubulin
proteins
themselves
show
significant
heterogeneity,
and
the
tubulin
isotypes
described
to
date
vary
in
their
patterns
of
expression
in
an
evolutionarily
conserved
manner
(16).
This
heterogeneity
in
a-
and
,-tubulins
offers
the
potential
of
contributing
to
diversity
of
microtubule
function
in
eucaryotic
cells,
either
through
differential
polymerization
of
the
various
tubulin
subunits,
or
by
virtue
of unique
interaction(s)
with
associ-
ated
proteins.
In
mammals,
a-
and
,B-tubulins
are
encoded
by
large
multigene
families
(5).
A
significant
fraction
of
the
genes
in
these
families
are
pseudogenes
(5,
13),
a
fact
that
makes
it
difficult
to
distinguish
functional
from
nonfunctional
genomic
tubulin
sequences.
To
circumvent
this
problem
and
to
study
the
important
question
of
the
tubulin
repertoire
of
mammals,
we
decided
to
study
expressed
mouse
tubulin
genes
at
the
mRNA
level
by
exhaustive
screening
of
cDNA
libraries.
We
recently
reported
the
sequence
and
regulated
expression
of
three
mouse
1-tubulin
and
two
mouse
a-
tubulin
isotypes
(16).
Here
we
present
the
complete
se-
quence
of
four
additional
mouse
a-tubulin
mRNAs
and
compare
them
with
the
completed
sequences
of
the
two
*
Corresponding
author.
previously
isolated
a-tubulins.
Gene-specific
probes
were
generated
to
study
the
developmental
expression
of
these
a-tubulins.
We
find
that
two
sister
genes
are
expressed
exclusively
in
testis
and
that
a
third
gene
gives
rise
both
to
an
a-tubulin
protein
that
is
abundant
in
muscle
and
a
mysteri-
ous
larger
transcript
abundant
in
testis
that
lacks
the
N-
terminally
encoded
methionine.
Finally,
we
describe
a
gene
that
is
expressed
ubiquitously at
a
low
level.
We
discuss
the
implications
of
these
results
for
the
functional
diversity
of
mammalian
microtubules.
MATERIALS
AND
METHODS
cDNA
cloning
and
sequencing.
RNA
was
prepared
from
the
testes
and
bone
marrow
of
adult
Swiss
Webster
mice
by
the
method
of
Berk
and
Sharp
(2)
and
fractionated
on
oligo(dT)
cellulose.
The
poly(A)+
mRNA
was
used
as
a
template
for
the
synthesis
of
cDNA
as
previously
described
(14),
and
the
cDNA
was
used
to
construct
libraries
in
Xgtll
(32).
These
libraries
were
not
amplified,
to
avoid
a
differential
amplifi-
cation
and
consequent
skewed
representation
of
the
cloned
cDNAs.
The
libraries
were
replicated
onto
nitrocellulose
(1),
and
the
filters
were
probed
with
a
gel-purified
insert
from
the
chicken
a-tubulin
cDNA
pTl
(4),
32p
labeled
by
nick
trans-
_lation.
Duplicate
nitrocellulose
filters
were
hybridized
with
the
mixed
nick-translated
inserts
of
the
3'
untranslated
region
subclones
of
Mal
and
Ma2
(16)
to
eliminate
from
further
analysis
cDNAs
identifiable
as
those
we
had
already
characterized.
Hybridizations
were
carried
out
at
42°C
in
a
mixture
of
50%
formamide,
5
x
SSC
(1
x
SSC
is
0.15
M
NaCl
plus
0.015
M
sodium
citrate),
20
mM
phosphate
buffer
(pH
6.5)
and
1
x
Denhardt
solution.
The
library
filters
were
washed
to
a
final
stringency
of
2x
SSC
at
50°C.
Positive
hybridizing
plaques
that
did
not
hybridize
to
the
3'
probes
2409

2410
VILLASANTE
ET
AL.
10
Met
Arg
Glu
Cys
Ile
Ser
Ile
His
Val
Gly
TTTTTCACTTCCTCAGTTTTCGCGGACCACTTCAGGACTAAAT
ATG
CGT
GAG
TGC
ATC
TCC
ATC
CAC
GTT
GGC
GTTGAGGACCAGTGGTGAGGMCGGCCGAGGCGGGTCTGAGCGGGTCTCCGGAGTTCAGC
T
T
G
G
CTCGTAGGATCGGCTGAGGTACTCTGCTAGGAGTTCAGC
T
TG G
CTACGTGAGACGTACAGCCCAAACTCATC
C
T
AIL
T G
G
CCTCCTCCTCGCCTCCGCCATCCACCCGGCTGCCGCGAACGAGCAACC
CTCTTAGTTGTCGGGAACGGTAACCGAGACCCGGTGTCTGCTTCTATCTCTCACCCTCGCCTTCTAACCCGTTGCTATC
20
Gln
Ala
Gly
Val
Gln
Ile
Gly
Asn
Ala
Cys Trp
Glu
Leu
CAG
GCT
GGT GTC
CAG
ATC
GGC
MT
GCC
TGC
TGG GAG
CTC
A
A
G
A
T
G
Tyr
Cys Leu
Glu
His
GI
TAC
TGC
CTG
GAA
CAT
GG
T
T
30
ly
Ile
Gln
Pro
Asp
Gly
iC
ATC
CAG
CCT
GAT
GGC
T
C
T
T
T
C
T
G
T
G
40
Gln
Met
Pro
Ser
Asp
Lys
CAG
ATG CCA
AGT
GAC
AAG
C
A
C
A
C
C
T
Thr
Ile
Gly
Gly
ACC
ATT
GGG
GGA
C
C
C C
T
Gly
Asp
Asp
Ser
Phe
GGA
GAT
GAC
TCC
TTC
G
C
A
G
C
G
C
A
50
60
70
,
Asn
Thr
Phe
Phe
Ser
Glu
Thr
Gly
Ala
Gly
Lys
His
Val
Pro
Arg
Ala
Val
Phe
Val
Asp
Leu
M
AAC
ACC
TTC
TTC
AGT
GAG
ACA
GGA
GCT
GGC
AAG
CAT
GTG
CCC
CGG
GCA
GTG
TTC
GTA
GAC
CTG
A
T
C
C
A
T
G
A
T
C
C
A
T
G
ED
CJ
A T
A
A
T
C
T
G
80
Val
Ile
Asp
Glu
Val
Arg
Thr
Gly
Thr Tyr
Arg
Gln
Leu
Phe
His
Pro
GTC
ATC
GAT
GAA
GTT
CGC
ACC
GGC
ACC
TAC
CGC
CAG
CTC
TTC
CAT
CCT
Gn
G
C
G
A
G
T
T
C
A
G=
A
A
G
T
C
A
A
GEr
A
El
T
C
A
C
Asn
Asn
Tyr
Ala
Arg
Gly
AAT
AAC
TAT
GCC
CGT
GGC
T
A
A
C
T
A
A
T
TT
Gly
Leu
Gln
Gly
Phe Leu
Val
Phe
GGT
CTC
CAG
GGC
TTC
TTG
GTT
TTC
A
G
CCem
A
G
C
C
ez
A
T
C
A
A
Tyr
Gly
Lys
Lys
Ser
Lys
Leu
Glu
TAC
GGA
MG
AAG
TCC
AAG
CTG
GAG
T
C
T
C
T
C
A
A
90
Gl
u
GAG
100
Gln
Leu
Ile
Thr
Gly
Lys
Glu
Asp
Ala
Al
a
CAG
CTC ATC
ACA
GGC
MG
GAG GAT
GCT
GCC
G
T
AA
A
T
G
A
A
TT
G
A
AA
110
120
130
His
Tyr
Thr
Ile
Gly
Lys
Glu
Ile
Ile
Asp
Leu
Val
Leu
Asp
Arg
Ile
Arg
Lys
Leu
Ala
Asp
Gln
Cys
Thr
CAC
TAC ACC
ATT
GGC
MG
GAG
ATC
ATT
GAC CTT
GTC
CTG
GAC
AGG
ATT
CGC
MG
CTG
GCT
GAC
CAG
TGC
ACA
C
A
Tj
G
TC
A
C
A
C
Tm
G
T
T
Tj
G
C
C
A
A
C
Tm
G
T
C
r
G
C
C
_
T
140
Hi
s
Ser
CAC
AGC
170
Phe
Ser
Ile
TTC
TCC
ATT
T
Ez
C
C
Phe
Gly
Gly
Gly
TTT
GGT
GGG
GGA
A
A
C
A
T
C
A
C
C
C
Tyr
Pro
Al
a
Pro
TAC
CCA
GCC
CCC
G
G
150
160
Thr
Gly
Ser
Gly
Phe
Thr
Ser
Leu
Leu
Met
Glu
Arg
Leu
Ser
Val
Asp
ACT
GGC
TCT
GGC
TTC
ACC
TCC
CTG
CTG
ATG
GAG
CGG
CTC
TCT
GTG
GAT
A
G
G
TED
G
T
A
C
A
G
G
T
Ej
G
T
A
C
C
T
T
T
C
180
Gln
Val
Ser
Thr
Ala
Val
Val
Glu
Pro
Tyr
Asn
Ser
Ile
Leu
CAG
GTT
TCC
ACT
GCT
GTG
GTT
GAG
CCC
TAC
AAT
TCC
ATC
CTC
T
A
G
C
G
T
C
G
T
A
G
C
G
T
C
G
A
G
G
A
G
C
G
190
Thr
ACC
Thr
His
Thr
Thr
Leu
ACC
CAC
ACC ACC
CTG
G
G
T
Glu
His
Ser
Asp
GAG
CAC
TCT
GAT
T
C C
T
C
C
A
A
C
200
Cys
TGT
Ala Phe
Met
Val
Asp
Asn
Glu
GCC
TTC
ATG
GTA
GAC
MT
GAG
T
G
T
C
A
T
G
T
C
A
G
C
A
210
Ala
Ile
Tyr
GCC
ATC
TAT
C
C
Asp
Ile
C)
GAC
ATC
TC
T
T
T
es
Arg
Arg
Asn
Leu
Asp
Ile
3T
CGT
AGA
MC
CTC
GAC
ATT
C
G
C
C
G
T
C
G
C
C
G
T
C
C
C
T
A
T
Arg
Pro
Thr
Tyr
Thr
Asn
Leu
Asn
Arg
CGC
CCA
ACC
TAC ACC
AAC
CTT
AAC
CGC
T
C
A
C T
T
T
C
A
C
T
T
T
C
T
A
A
G
230
Leu
Ile
Ser
Gln
Ile
Val
Ser
CTT
ATT
AGC
CAG
ATT
GTG
TCT
G
gml
G
G
EM
A
C
C
A
C
C
T
G
A
E
A
Ser
Ile
Thr
TCC ATC
ACT
T
A
A
A
240
Al
a
GCT
C
C
Ser
Leu
Arg
Phe
TCC
CTC
AGA
TTT
G
G
A
G
C
T
G
C
C
250
Asp
Gly
Ala
Leu
Asn
Val
GAT
GGG
GCC
CTG
MT
GTT
C
G
C
G
C
G
Ma6
Ma3
Ma7
MQ4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Glu
Pro
Thr
GAA
CCC
ACG
G
T
G
T
G
T
T
C
Ma6
Ma3
Ma7
Ma4
Mla1
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
maaa
220
GI
u
GAG
A
A
MOL.
CELL.
BIOL.

FIVE
MOUSE
a-TUBULIN
ISOTYPES
2411
Asp
Leu
Thr
Glu
Phe
Gln
Thr
Asn
Leu
GAT
CTG
ACA
GAA
TTC
CAG
ACC
AAC
CTG
C
T
A
C
T
A
C
G
260
270
280
Val
Pro
Tyr
Pro
Arg
Ile
His
Phe Pro Leu
Ala
Thr
Tyr
Ala
Pro
Val
Ile
Ser
Ala Glu
Lys
GTA
CCC
TAC
CCT
CGC
ATC
CAC TTC
CCT
CTG
GCC
ACT
TAT
GCC
CCT
GTC
ATC TCT
GCT
GAG
AAA
G
A
A
A
C
C
G
A
G
G
A
A
G
C
AG
G
C
C
T
T
C
A
A
T
A
G
T
Ala
Tyr
His
Glu
Gln Leu
Thr
Val
Ala
GCC
TAC
CAT
GAG
CAG
CTT
ACA
GTA
GCA
A
G
G
A
G|
G
G
C
290
300
GIu
Ile
Thr
Asn
Ala
Cys Phe
Glu
Pro Ala
Asn
Gln
Met
GAG
ATC
ACC
MT
GCC TGC
TTT
GAG
CCA
GCC
AAC
CAG
ATG
T
C
T
T
C
T
T
T
310
Val
Lys
Cys Asp
Pro
Arg
His
Gly
GTG
AAA
TGT
GAC
CCT
CGC
CAT
GGT
C
G
C
C
C
G
C
G
C C
Lys
Tyr
Met
Ala
Cys
Cys
Leu
Leu
Tyr
AAA
TAC
ATG
GCT
TGC
TGC
CTG
CTG
TAC
C
IA
T
C
T
G
C
A
320
330
340
Arg
Gly
Asp
Val
Val
Pro
Lys
Asp
Val
Asn
Ala
Ala
Ile
Ala
Thr
Ile
Lys
Thr
Lys
Arg
Thr
CGT
GGT
GAT
GTG
GTT
CCC
AAA
GAT
GTC
MT
GCT
GCC
ATT
GCC ACC ATC
MG
ACC
AAG
CGT
ACC
G
G
C
G
T
A
A
A
G
G
G T
C
C
A
G
G
A
T
&J
G
C13
C
G
Ilie
Gin
Phe
Val
Asp
Trp
Cys
Pro
Thr
ATC
CAG
TTT
GTG
GAC
TGG
TGC
CCC
ACT
A
T
T
G
A
T
G
A
A
350
360
Gly
Phe
Lys
Val
Gly
Ile
Asn
Tyr
Gln
Pro
Pro
Thr
Val
Val
Pro
GGC
TTC
MG
GTT
GGC
ATT
MC
TAC
CAG
CCT
CCC
ACT
GTG
GTA
CCC
A
T
G
T
C
T
A
T
G
T
T
C
T
C
A
A
G
T
T
370
Gly
Gly
Asp
Leu
Ala
Lys
GGT
GGT
GAC
CTG
GCC
MG
GA
A
GA
G
Val
Gl
n
Arg
Ala
Val
Cys
Met
Leu
Ser
GTG
CAG
AGA
GCT
GTG
TGC
ATG
CTG
AGC
C
G
C
C
G
C
T
380
Asn
Thr
Thr
Ala
AAC
ACC
ACA
GCC
T
G
T
G
A
C
390
Ile
Ala Glu
Ala
Trp
Ala
Arg
Leu
Asp
ATT
GCT
GAG
GCC
TGG
GCT
CGC
CTA GAT
C
A
C
G
C
C
A
C
G
C
A
C
T
G
C
His
Lys
Phe
Asp
Leu
Met
Tyr
CAC
AAG
TTT
GAT
CTG
ATG
TAT
A
C C
C
A
C
C
C
C
T
Lys
Arg
Ala
Phe
Val
His
Trp
Tyr
AAG
CGT
GCC
TTT
GTG
CAC
TGG
TAT
A
T
C
G
T
C
A
G
T
A
410
420
Val
Gly
Glu
Gly
Met
Glu
Glu
Gly
Glu
Phe
Ser
Glu
Ala
Arg
Glu
GTG
GGT
GAG
GGC
ATG
GAG
GAG
GGT
GAG
TTC
TCT
GAG
GCC
CGT
GAG
A
A
A
G
C
G
A
A
A
G
C
G
A
G
T A
Asp
Met
Ala
Ala
Leu
Glu
GAC
ATG
GCT
GCC
CTA GAG
AA
G
G
IC0A
G
G
T
A
440
450
Asp
Tyr
Glu Glu
Val
Gly
Al
a
Asp
Ser
Ala
Glu
Gly
Asp
Asp
Glu
Gly
Glu
Glu
---
---
Tyr
Ma6
GAT
TAT
GAG
GAG
GTT
GGG
GCA GAT
AGT
GCT
GAA
GGA
A
GAT
GAG
GGT
GAG
GAA
***
***
TAT
TM
CTCATGTGCTGCCATTCTATACTTCTGTGGT
Mca3
C
A
G
C
TCC
LJr--
A
***
A
O
C
GC
GG
GTW
GTC
CATT
Ma7
A
G
C
TCC
_I
Lt
D
A
***
A
LE2U
C
G
GCGCATGGGTCTGGCTGGCGGCCGTCCATTT
Ma4
A
A
C
C
TCC
G
A
A
G
***
***
***
G
ACCGCTACTTGGAGCCTGTTCACTGTGTTTA
Mal
T
TC
QM
C
Im
r
]
A
2
GA
C
ATTfATGTCACAAGGTGCTGCTTCCACAGG
Ma2
T
TC
cm1
C
I=
A
L3iJ[9i
C
GTCCATTCCTTGAGCCCCCTGTGTCGTCAAA
Ma6
'la3
Ma7
Ma4
Mal
Ma2
CTCATCTTTGTCTTTGTGTGGCTCTAACTGTCCTAAACTGTCAATAAAGGTGTTTCCGTTGTACGGTGGCAn
ATGTCTTCCCCACCATTGGAMIAAGGATATATTATTAn
A
TTGCAAAATCCTTTCGAAATAAACAGTCTCCTTGCAn
GXTGTTTATTGTGTTCCAACACAGAAAGTTGTGGTCTGATCAGTTAATTTCTATGTGGCAATGTGTGCTTTCATACAGTTACTGIACTTATGAATGATTGATTTTGACAGAGACCCCAAG
TGC
TCCAGTATTAGTTGCAGGCACCTGATGCTTCTGTGCTGTTTCCATTCTGTGATCATGTCTTCTCCATGTTGTACCTCTTAAGTTTTCCATGATGTCTCAAAC
TAA
Ma
1
CTGCCCATTTCACTTATGGGTTTTM&IAMAATACTCCC
FIG.
1.
Complete
sequences
of
six
a-tubulin
cDNA
clones,
Ma6,
Ma3,
Ma7,
Ma4,
Mal,
and
Ma2.
Only
the
amino
acid
sequence
of
Ma6
is
shown.
Open
boxes
in
the
other
sequences
indicate
amino
acid
changes
with
respect
to
Ma6.
Asterisks
denote
amino
acid
deletions
introduced
into
the
carboxy-terminal
region
so
as
to
maximize
homology;
spaces
indicate
sequence
identity.
Polyadenylation
signals
are
underlined.
Regions
selected
for
the
synthesis
of
antisense
oligodeoxyribonucleotides
for
use
as
gene-specific
probes
are
indicated
by
dashed
lines.
The
sequence
shown
for
Ma4
is
that
of
the
short
(1.8
kb)
transcript
(see
the
text).
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
Ma6
Ma3
Ma7
Ma4
Mal
Ma2
400
Al
a
GCC
430
Lys
AAG
A
VOL.
6,
1986

2412
VILLASANTE
ET
AL.
7
16
50
54
75788082
115117
126128
137
150170
232
287
317
334340
425
437
Ma6
-
I-I-i
Si-T-i
i-L---u-T-T-i
iT-M-
ADSAEGD**DEGEEY
MCa3/7
-,ii
v
,--V
V-I
uAA-G4'S-I/M-
-
'-L
VDSVEAEA*EEGEEY
Ma4
McaI
MCa2
-V-Mw'
,T-C-i-I-N-Pii
P-S
-
i
I
i-S-l
----A-S-'
i-
IDSYE*DSEDEGEE
-
i
i'-I
--
i-w
/-G-
/-S-i-i
-
{-
VDSVEGEGEEEGEEY
______-
i
-o
i
i-S-il-
J'-
i
VDSVEGEGEEEGEEY
FIG.
2.
Summary
of
isotype-specific
amino
acid
substitutions.
The
reference
sequence
is
Mot6.
At
positions
where
another
isotype
encodes
different
amino
acids
than
those
encoded
by
Ma6,
those
amino
acids
are
indicated
by
the
one-letter
code.
Only
amino
acid
differences
with
Mcx6
are
indicated,
with
the
exception
of
the
carboxy-terminal
region,
where
the
complete
sequence
of
each
isotype
is
included.
from
Mal
or
Ma2
were
picked
and
purified.
DNA
was
prepared
from
a
1-ml
culture
of
each
of
these
phages
(S.
A.
Lewis
and
N.
J.
Cowan,
Mol.
Cell
Biol.,
in
press),
cut
with
EcoRI,
and
resolved
on
5%
polyacrylamide
gels.
Each
excised
insert
was
subcloned
into
M13
and
sequenced
by
the
Sanger
dideoxy
chain
termination
method
(22).
Approxi-
mately
75
a-tubulin
cDNA
clones
from
each
library
were
examined
in
this
manner.
Sequences
of
representative
cDNAs
were
completed
by
subcloning
Bal
31
exonuclease-
deleted
fragments
into
M13
as
described
elsewhere
(15).
The
3'
untranslated
probe
for
Ma3/MO7
and
the
5'
probe
for
MA4L
(where
L
is
the
long
transcript)
were
generated
by
this
method.
Antisense
oligodeoxyribonucleotides
were
synthe-
sized
for
use
as
gene-specific
probes
for
M(X6
and
Ma4;
the
sequences
they
hybridize
to
are
indicated
in
Fig.
1.
RNA
blot
transfer
experiments.
RNA
was
prepared
(2)
from
10
different
tissues
dissected
from
mice
of
various
ages
(see
figure
legends).
RNA
concentrations
were
determined
at
A260.
Samples
(10
or
20
p.g)
were
electrophoresed
on
1%
agarose
gels
containing
2.2
M
formaldehyde
(3).
The
RNA
was
transferred
to
nitrocellulose
(24),
and
the
blots
were
probed
by
the
following
methods:
(i)
with
gel-purified
insert
from
the
subcloned
3'
untranslated
region
of
Ma
,
32p
labeled
by
nick
translation
(21)
or
(ii)
with
one
of
the
various
oligodeoxyribonucleotides
shown
in
Fig.
1,
32p
labeled
with
polynucleotide
kinase.
Hybridization
and
wash
conditions
for
the
nick-translated
insert
were
as
described
above
for
the
library
screening.
For
synthetic
oligonucleotides,
hybridiza-
tion
was
performed
for
24
h
at
42°C
in
a
mixture
of
20%
formamide,
5x
SSC,
5x
Denhardt
solution,
and
20
mM
phosphate
buffer
(pH
6.5).
The
blots
were
washed
to
a
final
stringency
of
2
x
SSC
at
60°C.
RESULTS
Complete
sequence
of
six
a-tubulin
cDNAs.
To
maximize
the
chance
of
identifying
novel
cx-tubulin
isotypes,
two
cDNA
libraries
[constructed
with
poly(A)+
mRNA
from
mouse
bone
marrow
and
testis]
were
screened.
The
choice
of
these
tissues
was
based
on
the
observation
that
testis
and
lymphatic
tissues
(e.g.,
spleen
and
thymus)
all
express
a-tubulin
at
relatively
high
abundance,
as
measured
in
RNA
blot
transfer
experiments
with
a
coding
region
probe.
How-
ever,
because
this
high
abundance
could
not
be
accounted
for
by
the
expression
of
previously
described
cx-tubulin
isotypes
alone
(16),
it
seemed
reasonable
to
expect
the
expression
of
other,
novel
ox-tubulin
isotypes
in
these
tis-
sues.
In
addition,
the
existence
of
unique
kinds
of
microtubule
structures
(i.e.,
the
manchette
in
spermatozoa
and
the
marginal
band
in
platelets)
made
testis
and
bone
marrow
particularly
favorable
sources
in
our
search
for
novel
tubulin
isotypes.
This
search
was
facilitated
by
screen-
ing
each
library
both
with
a
chicken
a-tubulin
probe
(to
identify
cloned
a-tubulin
coding
sequences)
(4)
and
with
a
A
B
28S-
Z
o
=
E
-
0
o
U,
C
CY
-r-
NM
Co
18S-
28S-
FIG.
3.
Expression
of
Ma6
in
tissues
of
the
adult
mouse.
Total
RNA
was
prepared
from
brain
(br),
heart
(he),
kidney
(ki),
liver
(li),
lung
(lu),
muscle
(mu),
spleen
(sp),
stomach
(st),
and
testis
(te)
of
adult
mice.
Portions
(20
,ug)
of
each
sample
were
resolved
on
1%
agarose
gels
containing
2.2
M
formaldehyde
(3)
and
transferred
to
nitrocellulose
(24).
The
blot
was
probed
with
the
oligonucleotide
complementary
to
the
3'
untranslated
region
of
Ma6
(Fig.
1),
32p
labeled
with
polynucleotide
kinase.
The
positions
of
18S
and
28S
rRNAs
are
indicated
as
size
markers.
Weak
but
detectable
expres-
sion
of
Ma6
in
brain
and
muscle
was
evident
upon
longer
exposure
(data
not
shown).
18S-
S
e.g
FIG.
4.
Testis-specific
expression
of
Ma3/Ma7.
(A)
An
RNA
blot
identical
to
that
described
in
the
legend
of
Fig.
3
was
probed
with
the
excised
insert
of
a
subclone
of
Ma3
containing
the
entire
3'
untranslated
region,
32P
labeled
by
nick
translation
(21).
(B)
Samples
(10
,ug)
of
RNA
from
mouse
testis
at
days
10,
15,
22,
and
32
were
resolved
on
a
formaldehyde-agarose
gel
(3),
blotted
to
nitrocellulose
(24),
and
probed
with
the
same
probe
used
in
panel
A.
MOL.
CELL.
BIOL.
Sjik-W_
q.
.,

FIVE
MOUSE
a-TUBULIN
ISOTYPES
2413
mixed
probe
consisting
of
the
subcloned
3'
untranslated
regions
of
two
previously
described
mouse
o-tubulin
isotypes,
Mal
and
Ma2
(16).
This
latter
probe
served
to
identify
(and
thereby
eliminate)
many
of
those
clones
encod-
ing
previously
characterized
isotypes.
Four
novel
a-tubulin
cDNAs
were
identified
with
this
procedure.
In
each
case,
the
complete
sequence
was
deter-
mined
from
a
series
of
extensively
overlapping
cloned
frag-
ments
with
identical
sequences
within
the
region
of
overlap.
The
sequence
of
each
cDNA
(Ma3,
Ma4,
Ma6,
and
MO7)
and
the
oa-tubulin
isotype
that
it
encodes
is
shown
in
Fig.
1,
together
with
the
completed
sequence
of
Mal
and
Ma2.
Each
cDNA
represents
a
cloned
copy
of
a
distinct
gene
product,
because
each
possesses
unique
5'
and
(with
the
exception
of
Ma3
and
MO7)
unique
3'
untranslated
regions.
Also,
each
differs
from
the
others
in
the
coding
region
with
regard
to
silent
substitutions;
more
important
(again
with
the
Ha44
AGTTCTGGGTCCTCGGCCGCCCACAGGCGTCGGCGAAAG
Ma4L
C
A
T
T
T
G
Ha44
GCTGCCGCCCCGGCCGGGGACCAGGAAGCGTCAGGCAGC
Ma4L
C
A
M
T
C
G
G
G
T
T
Ha44
TGGCAAGGGCTCCCCGGGGACGCGCCACAGCCTCACAGC
Ma4L
*T
A
G
T
G
C
TTA
T
C
TG
C
A
G
Ha44
CGGCCCGAGTCTCCTGGGAGGCAGGGCTGGAAGGGCAGG
Ma4
C
***
C
GAC
G
A
C
A
28S
-
18S
-
B
28S-
18
--S
W
Oa
t
*
U
C
28S-
Ha44
GGTGAAGGCCAGCTGTGGCCGCTTGGGAAGGACCGCCTC
Ma4L
A
G
GGACA
TT
TT
T
G
18S-'
Ha44
GCCTGCTCCCGACCTAAGTCAGAACACCTGGATGACCGG
MaL4L
AG
T
AT
C
A
G
T
C
CC
TGA
Ha44
TGCCTCCAGGACGCAGGTGCAGGTGAGACTCGCCCTGCC
!4a4L
C
T
A
TG
L
A
C
*
T
*
TT
Ha44
ACAGCACCCTGCATCTCCGCGGAGGCCCTCGGGAGCCCA
MAL4
C
G
T
TTC
A
TC
AT
G
Ha44
GCGTGTCTGCTCAAAACGAGGAAAGMTGGTTAAAGCCC
Ma4L
GACA
G
T
C
AT*********
FIG.
6.
Expression
of
Ma4
in
adult
mouse
tissues.
Blots
of
RNA
from
nine
adult
mouse
tissues
identical
to
those
shown
in
Fig.
3
were
used
with
the
following
probes
from
Ma4.
(A)
An
antisense
oligo-
nucleotide
complementary
to
the
3'
untranslated
region
of
Ma4,
32P-labeled
with
polynucleotide
kinase.
The
extent
of
this
probe
is
indicated
in
Fig.
1.
(B)
An
antisense
oligonucleotide
complementary
to
24
nucleotides
of
the
a-tubulin
coding
region
that
uniquely
recognizes
Ma4
(also
indicated
in
Fig.
1),
32P-labeled
with
polynu-
cleotide
kinase.
(C)
The
excised
insert
from
a
subclone
of
Ma4
encompassing
sequences
from
5'
to
amino
acid
2
(see
Fig.
5
and
the
text),
32P
labeled
by
nick
translation.
Ha44
GAATCGCGACTCTTAATCCCAGCGGGACAG
ALc4i
GGC
T
CT
*
G
A
A
CC
FIG.
5.
Homology
between
the
5'
end
of
the 2.4-kb
transcript
of
Mci4
(i.e.,
Mcx4L,
as
discussed
in
the
text)
and
the
corresponding
region
of
a
human
a-tubulin
gene,
Ha44.
The
sequence
immediately
upstream
to
the
triplet-encoding
amino
acid
2
of
the
human
gene
Ha44
(Dobner
et
al.,
submitted)
is
shown
together
with
the
corre-
sponding
sequence
at
the
5'
end
of
Ma4L.
The
sequences
have
been
aligned
for
maximum
homology,
and
only
differences
in
MA4L
compared
with
Ha44
are
indicated.
Asterisks
denote
deletions.
Termination
codons
in
the
sequence
of
Mo4L
are
underlined.
exception
of
Ma3
and
MO7),
each
differs
by
virtue
of
a
limited
number
of
amino
acid
differences.
Comparison
of
the
amino
acid
sequences
of
the
five
ot-tubulin
isotypes
shows
that
the
majority
of
these
differences
lie
within
the
15
carboxy-terminal
amino
acids.
Within
this
region,
Mal
and
Ma2
are
identical,
as
are
Ma3
and
MO7.
However,
multiple
differences
exist
between
the
carboxy-terminal
regions
of
Mcl/Mct2,
Ma3/Ma7,
Ma4,
and
Moa6.
Four
of
the
five
isotypes
defined
by
the
six
cDNA
clones
contain
an
encoded
carboxy-terminal
tyrosine;
the
isotype
encoded
by
Ma4
is
VOL.
6,
1986
"
0
.-
3
CL
0
m
=
-.e
=
3
E
cn
o
*.
#a
-fto
0
A,
1.
-s
--i
It
"VI
I

2414
VILLASANTE
ET
AL.
br
he
ki
li
lu
Ma6
OD
*
e
-
mu
Sp
00
0
st
a
S
.
e
S
FIG.
7.
Developmental
expression
of
Ma4
and
Ma6.
Total
RNA
from
brain
(br),
heart
(he),
kidney
(ki),
liver
(ii),
lung
(lu),
spleen
(sp),
stomach
(st),
and
thymus
(th)
was
prepared
from
mice
of
ages
3,
6,
10,
15,
22,
and
32
days
(left
to
right).
RNA
from
muscle
(mu)
and
testis
(te)
was
also
prepared
from
mice
of
ages
10,
15,
22,
and
32
days
(left
to
right).
Portions
(10
p.g)
of
each
RNA
sample
were
electrophoresed
on
1%
agarose
gels
containing
2.2
M
formaldehyde
(3)
and
transferred
to
nitrocellulose.
Duplicate
blots
were
probed
with
the
oligonucleotide
complementary
to
the
3'
untranslated
regions
of
Ma4
and
Ma6
(see
Fig.
1),
32P
labeled
with
polynucleotide
kinase.
A
duplicate
set
of
blots
was
also
probed
with
the
3'
untranslated
region
probe
for
Ma3/Ma7,
but
the
probe
failed
to
detect
bands
in
any
of
the
RNA
samples
even
after
very
long
exposure
times
(data
not
shown)
except
those
from
testis
(see
Fig.
4B).
Weak
but
detectable
expression
of
Ma6
in
developing
brain
and
muscle
is
evident
upon
longer
exposure
(data
not
shown).
unusual
in
that
it
lacks
a
carboxy-terminal
tyrosine.
The
amino
acid
substitutions
characteristic
of
each
isotype
are
summarized
in
Fig.
2.
Low-level
expression
of
a
ubiquitously
expressed
a-tubulin
isotype
(Ma6).
To
define
the
developmental
and
tissue-
specific
patterns
of
isotype-specific
a-tubulin
gene
expres-
sion,
probes
were
constructed
for
the
unique
3'
untranslated
regions
of
each
isotype.
In
the
case
of
the
isotype
encoded
by
Ma3
and
MO7,
a
probe
was
generated
as
a
3'
untranslated
region
subclone;
in
the
case
of
Ma4
and
Ma6,
where
the
3'
untranslated
regions
are
relatively
short,
a
synthetic
antisense
oligonucleotide
was
prepared.
The
regions
encom-
passed
by
the
gene-specific
oligonucleotide
probes
are
shown
in
Fig.
1.
Their
specificity
was
determined
in
control
experiments
in
which
each
oligonucleotide
was
tested
(i)
for
its
ability
to
uniquely
detect
a
cloned
complementary
se-
quence,
and
(ii)
for
its
lack
of
cross-hybridization
with
noncomplementary
cDNA
clones
(data
not
shown).
The
32P-labeled
Ma6-specific
oligonucleotide
was
used
in
a
blot
transfer
experiment
to
examine
the
relative
abundance
of
Ma6
sequences
in
RNAs
prepared
from
adult
mouse
brain,
heart,
kidney,
liver,
lung,
muscle,
spleen,
stomach,
and
testis.
The
data
(Fig.
3)
show
that,
with
the
exception
of
brain
and
muscle,
where
Ma6
expression
is
barely
detect-
able,
the
abundance
of
this
isotype
varies
somewhat
among
the
tissues
examined.
The
size
of
the
Ma6
transcript
is
1.8
kilobases
(kb);
however,
in
testis,
a
second
transcript
of
2.4
kb
is
evident.
The
nature
and
significance
of
the
large
Ma6-specific
transcript
in
testis
is
unknown.
Two
genes
encode
an
identical
testis-specific
isotype.
Two
of
the
cDNAs
(Ma3
and
Ma7)
encode
an
identical
a-tubulin
isotype
and
indeed
share
an
identical
3'
untranslated
region,
Ma4
..e*.S0
*...ea
@
0a
Ma6
te
th
Ma4
*,
*
*
.
MOL.
CELL.
BIOL.

FIVE
MOUSE
a-TUBULIN
ISOTYPES
2415
TT
CTC
TGG
CAG
Val
Gly
GTG
GGT
C
Ile
Asn
Tyr
Gln
ATT
MC
TAC
CAG
Thr
Val
Val
Pro
Ma3
ACT
GTG
GTC
CCT
H2a
A
C
Ma3
H2a
Ala
Val
Cys
Met
Leu
GCC
GTG
TGC
ATG
CTG
Gly
Gly
Asp
Leu
Ala
GGG
GGA
GAC
CTG GCC
380
Ser
Asn
AGC
MT
C
370
Lys
AM
G
Thr
Thr
Ala
ACC
ACG
GCC
Val
Gl
n
Arg
GTG
CAG
CGG
Ile
Ala
Glu
ATC
GCA
GAG
T
G
Ala
Trp
Ala
GCC
TGG
GCC
400
Al
a
GCC
Ly
s
Arg
MG
CGA
T
390
Arg
CGC
Leu
Asp
His
Lys
Phe
Asp
Leu
Met
Tyr
CTG
GAC
CAC
MA.ATTT
GAC
CTG
ATG
TAC
T
G
C
T
C
T
Al
a
Phe
Val
GCC
TTT
GTG
His
CAT
C
420
Met
Glu
Glu
Gly
Glu
Phe
Ser
Glu
Ma3
ATG
GAG
GM
GGG
GAG
TTC
TCC
GAG
H2a
A
G
A
T
410
Trp
Tyr
Val
Gly
Gl
u
Gly
TGG
TAC
GTG
GGA
GM
GGC
C
Al
a
Arg
GCC
CGG
C
GI
u
Asp
Leu
GAG
GAC
U1'
430
Ala
Ala
Leu
Glu
Lys
Ma3
GCA
GCG
CTG
GAG
MG
H2a
T
A
440
Ma3
Ser
Val
H2a
TCC
GTG
Asp
GAC
T
Tyr
Glu
Glu
Val
Gly
Val
Asp
TAT
GM
GAG
GTG
GGC
GTG
GAT
Glu
Ala
Glu
Ala
Glu
Glu
Gly
Glu
Glu
GM
GU
GAG
-GC
GM
GM
GGG
GAG
GAG
T
T
C
A
A
450
Tyr
TAC
TGA
Ma3
GCGCATGGGTCTGGCTGGCGGCCGTCCATTTATGTCTTC
H2a
G
G*
G
G
.TC
CA
bGG
G
GA
GTT
G
CCCCC
T
Ma3
CCCACCATTGGMATAAAGGATATATTATTAM
H2a
T
T
C
TT
C
G*
MGGCCMG
TT
FIG.
8.
A
human
a-tubulin
gene,
H2a,
encodes
the
same
isotype
as
Ma3/Ma7.
The
3'
exon
of
the
human
a-tubulin
gene,
H2a,
was
sequenced
and
compared
with
the
sequence
of
Ma3.
Only
sequence
differences
are
indicated.
Amino
acids
characteristic
of
this
isotype
are
underlined.
The
3'
untranslated
regions
are
aligned
for
maximum
homology;
deletions
are
indicated
by
asterisks;
polyadenylation
signals
are
indicated
with
a
thick
underline.
at
least
within
the
extent
of
the
overlapping
clones
examined
(Fig.
1).
However,
multiple
third
codon
position
differences
exist
throughout
the
coding
region,
and
the
5'
untranslated
regions
are
somewhat
dissimilar.
Use
of
a
32P-labeled
subcloned
3'
untranslated
region
probe
to
determine
the
pattern
of
tissue-specific
expression
of
Ma3
and
Ma7
showed
that
these
cDNAs
are
expressed
exclusively
in
testis
(Fig.
4A).
Even
after
lengthy
exposure,
no
Ma3/Ma7
tran-
script
could
be
detected
in
somatic
tissues
at
any
stage
of
development,
nor
could
one
be
detected
in
four
stages
of
whole
mouse
embryo
(data
not
shown).
We
conclude
that
expression
of
the
genes
encoding
Ma3
and
Ma7
is
testis
Ma3
H2a
360
Pro
CCC
Pro
CCT
C
Ma3
H2a
Ma3
H2a
VOL.
6,
1986

2416
VILLASANTE
ET
AL.
TABLE
1.
Summary
of
as-tubulin
isotype-specific
expression
in
mice
Approx
relative
levels"
of
expression
in:
Gene
Brain
Heart
Kidney
Liver
Lung
Muscle
Spleen
Stomach
Testis
Thymus
Malb
++
(-)
++
-
- -
+
Ma22b
+++
++
+
++
-
++
+
+
+++
Ma3/Ma7
+
+
+
+
Ma6
(-)
-
-
+
-
(-)
+
+
M4'
+
++
+
+
+ +
+
+
+
Ma4Ld
+
-
+
+
+
+ +
+,
Highest
expression;
-,
lowest
expression;
(-),
trace.
b
Data
from
Lewis
et
al.
(16).
Ma4
=
1.8-kb
mRNA.
d
Ma4L
=
2.4-kb
mRNA.
specific.
The
developmental
regulation
of
these
genes
was
examined
in
a
blot
transfer
experiment
by
using
RNA
from
testes
of
male
mice
of
different
ages.
The
data
show
barely
detectable
expression
at
day
15
and
abundant
expression
at
day
22,
increasing
thereafter
until
sexual
maturity
(Fig.
4B).
Therefore,
the
expression
of
the
genes
encoding
Ma3
and
Ma7
appears
linked
to
the
process
of
spermatogenesis.
The
3'
untranslated
region
probes
used
in
these
experiments
recognize
both
Ma3
and
Ma7
mRNAs.
However,
since
Ma3
and
Ma7
cDNAs
were
isolated
in
similar
numbers
from
the
testis
cDNA
library,
we
assume
that
the
relative
contribu-
tions
of
the
genes
to
the
total
mRNA
level
are
approximately
equal.
A
single
gene
yields
two
transcripts,
one
encoding
an
o-tubulin
and
one
of
unknown
function.
Multiple
overlapping
clones
were
isolated
from
the
bone
marrow
and
testis
cDNA
libraries
that
encode
a
novel
a-tubulin
isotype,
Ma4
(Fig.
1
and
2).
Curiously,
sequence
analysis
of
the
cDNA
clones
encoding
this
isotype
from
testis
(Mca4L)
showed
that
they
lacked
the
initiator
ATG
codon.
Instead,
these
clones
in-
cluded
what
appeared
to
be
an
amino-terminal
extension.
However,
shortly
upstream
from
the
position
expected
for
the
initiator
methionine,
both
the
a-tubulin
reading
frame
and
all
other
reading
frames
were
closed
(Fig.
5).
In
contrast,
clones
encoding
the
identical
isotype
isolated
from
the
bone
marrow
cDNA
library
did
contain
the
initiator
ATG
codon
in
the
expected
position
(Fig.
1).
Because
the
Ma4-encoding
cDNA
clones
from
testis
and
bone
marrow
are
absolutely
identical
in
sequence
3'
to
the
position
expected
for
the
initiator
methionine
(including
the
3'
untranslated
regions),
both
transcripts
almost
certainly
derive
from
the
same
gene.
To
investigate
the
tissue
distribution
of
these
transcripts,
an
RNA
blot
transfer
experiment
was
performed
with
a
32p
labeled
antisense
oligonucleotide
derived
from
3'
untrans-
lated
region
sequences.
This
probe
detected
a
1.8-kb
tran-
script
that
was
particularly
abundant
in
muscle
and
was
present
in
all
other
somatic
tissues
examined;
however,
the
1.8-kb
transcript
was
absent
in
testis,
which
exclusively
expressed
a
2.4-kb
transcript,
Mot4L
(Fig.
6A).
A
2.4-kb
transcript
was
also
detectable,
albeit
at
lower
abundance,
in
brain,
heart,
and
striated
muscle.
To
verify
that
the
mRNAs
recognized
by
the
Ma4
3'
untranslated
region
probe
encode
a-tubulin,
an
oligonucleotide
complementary
to
a
heterolo-
gous
region
within
the
Ma4
coding
sequence
(Fig.
1)
was
synthesized
and
used
as
a
gene-specific
probe
on
a
duplicate
blot
(Fig.
6B).
Control
experiments
in
which
cDNA
clones
encoded
all
a-tubulin
isotypes
showed
this
oligonucleotide
to
be
Ma4-specific
(data
not
shown).
The
results
are
indeed
identical
to
those
obtained
with
the
3'
probe.
Finally,
a
probe
corresponding
to
the
region
of
the
testis
Ma4
transcript
(i.e.,
Ma4L)
5'
to
amino
acid
2
was
used
on
a
duplicate
blot
(Fig.
6C).
This
probe
hybridized
only
to
the
2.4-kb
transcript.
The
nature
and
significance
of
the
bizarre
2.4-kb
Ma4
testis
transcript
that
lacks
an
initiator
methionine
is
discussed
below.
Developmental
regulation
of
Ma4
and
Mot6.
The
genes
encoding
Ma6
and
Max4
are
expressed
in
a
wide
variety
of
adult
mouse
tissues
(Fig.
3
and
6).
To
examine
the
pattern
of
developmental
expression
of
these
two
isotypes,
32P-labeled
gene-specific
antisense
oligonucleotides
were
used
as
probes
in
blot
transfer
experiments,
using
equal
samples
of
RNA
from
tissue
dissected
from
mice
aged
3,
6,
10,
15,
22,
and
32
days.
In
brain
and
in
muscle,
where
adult
levels
of
Ma6
expression
are
barely
detectable
(Fig.
3),
there
are
equally
low
levels
of
Ma6-specific
mRNA
during
early
development
(Fig.
7).
In
other
tissues
showing
more
significant
levels
of
adult
Ma6
expression,
the
relative
abundance
of
Ma6-
specific
mRNA
does
not
change
dramatically
as
a
function
of
postnatal
age.
However,
the
2.4-kb
Ma6
transcript
ex-
pressed
in
adult
testis
(Fig.
3)
does
not
appear
in
testis
RNA
from
mice
aged
32
days
or
less.
In
the
case
of
Ma4,
where
two
transcripts
are
derived
from
a
single
gene
(see
above),
some
distinct
developmental
changes
are
evident.
Whereas
the
level
of
Ma4
expression
stays
relatively
unchanged
in
developing
kidney,
liver,
lung,
spleen,
and
immature
thy-
mus,
there
is
a
marked
developmental
increase
in
the
level
of
expression
of
both
the
1.8-kb
and
the
2.4-kb
transcripts
in
ATG
p
P;
EXON
2
p
~
~~~~~~~~~~~~~~~~~~
Ma4
LIz
iii
0
0.5
1.0
kb
Ma4L
E//////
-
FIG.
9.
Three
classes
of
Ma4
and
their
relationship
to
the
human
a-tubulin
gene,
Ha4
(Dobner
et
al.,
submitted).
Homologies
be-
tween
Ha44
and
three
Ma4
cDNA
classes
(Ma4,
Ma4L,
and
C;
see
the
text)
are
shown
as
hatched,
solid,
and
open
boxes
and
the
dashed
line.
The
figure
shows
the
probable
mechanism
for
the
generation
of
mRNAs
represented
by
the
cloned
cDNAs.
PL
and
P
are
two
promoters
that
yield
transcripts
spliced
as
shown.
Alterna-
tively,
it
is
conceivable
that
Ma4
and
Ma4L
are
derived
from
a
transcript
from
PL
alone,
and
that
this
transcript
is
differentially
spliced
(as
shown
by
the
dotted
line).
MOL.
CELL.
BIOL.

FIVE
MOUSE
a-TUBULIN
ISOTYPES
2417
brain
(Fig.
7).
In
testis,
where
the
2.4-kb
transcript
is
exclusively
expressed
in
the
adult
(Fig.
6),
this
transcript
is
undetectable
before
32
days;
instead,
a
very
low
level
of
the
1.8-kb
transcript
is
evident
at
early
developmental
stages
(Fig.
7).
A
human
a-tubulin
gene
subfamily
encodes
the
same
isotype-specific
carboxy-te
rminal
amino
acids
as
do
mouse
cDNAs
Me3
and
Ma7
and
shares
3'
untranslated
region
homology.
In
an
earlier
report,
we
described
the
isolation
of
a
subfamily
of
closely
related
human
a-tubulin
genes
(31).
The
identification
of
these
genes
as
representing
of
a
related
subfamily
rested
on
restriction
mapping
data:
whereas
the
majority
of
restriction
sites
were
common
to
each
gene,
there
were
a
large
number
of
differences,
primarily
outside
the
coding
regions,
that
could
not
be
explained
solely
in
terms
of
allelic
differences.
The
isolation
of
the
two
closely
related
mouse
a-tubulin
cDNAs
Ma3
and
Ma7
prompted
us
to
examine
the
sequence
of
the
human
a-tubulin
gene
subfamily
described
above.
The
sequence
of
the
last
exon
of
one
of
the
members
of
this
family,
H2a,
is
presented
in
Fig.
8
and
is
compared
with
the
homologous
region
in
the
mouse
cDNA
clone
Ma3.
The
amino
acid
sequence
encoded
by
H2a,
including
the
characteristic
carboxy-terminal
region,
is
identical
to
that
encoded
by
Ma3
and
Ma7,
and
there
is
significant
(58%)
conservation
of
the
3'
untranslated
regions
as
well.
We
therefore
surmise
that
this
human
a-tubulin
subfamily
encodes
the
testis-specific
a-tubulin
isotype,
and
that
the
existence
of
multiple
genes
encoding
a
testis-
specific
a-tubulin
is
a
feature
common
to
all
mammalian
species.
DISCUSSION
In
this
paper,
we
describe
the
characteristics
and
differ-
ential
expression
of
five
a-tubulin
isotypes
in
the
developing
mouse.
The
amino
acid
differences
between
these
isotypes
are
summarized
in
Fig.
2,
and
their
approximate
relative
levels
of
expression
in
10
different
tissues
are
summarized
in
Table
1.
One
isotype,
encoded
by
two
genes,
Ma3
and
Ma7,
is
absolutely
testis
specific.
A
second,
Ma6,
is
a
minor
a-tubulin
in
all
tissues.
A
third
gene,
Ma4,
gives
rise
to
two
transcripts,
the
shorter
of
which
encodes
an
a-tubulin
abun-
dant
in
muscle,
and
the
longer
of
which
lacks
an
initiation
codon
in
the
expected
position.
The
other
two
a-tubulins,
Mal
and
Ma2,
are
ubiquitously
expressed,
although
the
former
is
expressed
primarily
in
lung
and
brain
(16).
Like
the
,B-tubulins
in
mouse
and
other
vertebrate
species
(10,
16,
26),
the
a-tubulins
described
here
differ
most
from
each
other
in
the
sequence
of
the
carboxy-terminal
15
amino
acids
(Fig.
2).
As
in
,-tubulins
(16,
25),
other
amino
acid
differences
occur
throughout
the
length
of
the
protein,
although
not
noticeably
as
clusters.
All
of
these
amino
acid
differences
must
have
real
significance
(and
not
result
from
neutral
sequence
drift),
because
they
are
completely
con-
served
in
mammals
in
each case
in
which
a
cross-species
comparison
of
the
primary
structure
of
the
same
isotype
can
be
made.
For
example,
two
human
a-tubulins,
bal
and
kal,
have
amino
acid
sequences
(and
expression
patterns)
iden-
tical
to
those
of
Mal
and
Ma2,
respectively
(6).
Similarly,
an
a-tubulin
cDNA
encoding
the
same
carboxy
terminus
and
bearing
3'
untranslated
region
homology
to
Ma6
has
been
isolated
from
Chinese
hamster
ovary
cells
(9).
Ma4
also
has
a
structurally
identical
counterpart
in
monkeys
and
humans
(P.
R.
Dobner,
E.
Kislanskis,
B.
M.
Wentworth,
and
L.
Villa-Komaroff,
submitted
for
publication).
Finally,
we
show
here
that
a
human
a-tubulin
gene
encodes
the
same
isotype
as
Ma3/Ma7.
The
carboxy
terminus
of
both
a-
and
1-tubulins
is
thought
to
be
on
the
outer
surface
of
the
microtubule
(29),
and
there
is
evidence
that
it
is
this
do-
main
that
modulates
microtubule
assembly
and
binds
microtubule-associated
proteins
(23).
It
seems
likely,
there-
fore,
that
these
a-tubulin
isotypes
contribute
to
the
diversity
of
microtubule
function
by
differentially
binding
micro-
tubule-associated
proteins.
Other,
non-carboxy-terminal
isotype-specific
amino
acid
differences
may
also
have
subtle
effects
on
the
interaction
between
a-
and
p-subunits
or
on
the
dynamics
of
microtubule
assembly.
Microtubules
form
the
basis
of
many
specialized
struc-
tures
in
testis,
such
as
the
mitotic
and
meiotic
spindles
and
the
flagellar
axoneme
of
the
spermatozoan.
In
Drosophila
melanogaster,
a
mutation
in
a
3-tubulin
gene
has
been
described
that
disrupts
all
these
functions
(11,
12).
(Re-
cently,
however,
it
has
been
reported
that
the
expression
of
this
gene
is
not
testis
specific;
it
is
apparently
expressed
in
the
early
stages
of
embryogenesis
as
well
[19].)
It
does
not
follow
that
the
mammalian
testis-specific
a-tubulin
described
here
(encoded
by
Ma3
and
Ma7)
is
an
a-tubulin
equivalent
of
the
D.
melanogaster
1-tubulin.
Indeed,
the
time
course
of
appearance
of
the
isotype
encoded
by
Ma3/Ma7
would
be
consistent
with
postmeiotic
expression,
suggesting
that
this
mammalian
a-tubulin
might
be
especially
tailored
for
either
the
flagellar
axoneme
or
the
manchette.
In
1984,
the
isolation
of
a
cDNA
clone
for
a
testis-specific
mouse
a-tubulin
was
reported
(7).
The
sizes
of
the
tran-
scripts
and
3'
untranslated
region
corresponding
to
this
cDNA
are
completely
inconsistent
with
all
of
the
a-tubulins
described
here,
including
those
encoded
by
the
testis-
specific
cDNAs
Ma3
and
Ma7.
Although
it
is
possible
that
we
failed
to
find
an
example
of
the
previously
reported
cDNA
in
our
screening
experiments,
we
feel
that
this
is
highly
unlikely,
given
the
exhaustive
nature
of
our
search
in
which
about
75
a-tubulin
cDNAs
from
testis
were
se-
quenced.
A
more
likely
explanation
(given
that
this
previ-
ously
reported
cDNA
was
a
single
isolate)
is
that
it
repre-
sents
a
cloning
artifact.
We
present
data
here
that
demonstrate
the
existence
of
a
multigene
subfamily
encoding
the
testis-specific
a-tubulin
isotype
in
both
humans
and
mice
(Fig.
8).
Thus,
the
dupli-
cation
of
an
ancestral,
testis-specific
a-tubulin
gene
must
have
occurred
at
some
time
before
the
mammalian
radiation,
and
the
function
of
(minimally)
two
of
the
resulting
copies
has
been
maintained
since
then,
at
least
in
mice.
An
inter-
esting
feature
of
this
subfamily
is
the
occurrence
of
a
fourth
intervening
sequence
at
amino
acid
352,
in
addition
to
the
three
introns
common
to
all
other
vertebrate
a-tubulin
genes
described
to
date
(5,
30).
A
similar
situation
exists
in
the
vertebrate
a-actins,
for
example,
where
the
smooth-muscle-
specific
a-actin
has
one
intron
in
addition
to
those
it
shares
with
all
other
a-actins
(27).
It
is
possible
that
this
extra
intron
plays
some
functional
role:
for
example,
it
could
contain
a
tissue-specific
transcriptional
enhancer
sequence,
or
it
could
act
to
prevent
gene
conversion
between
these
genes
and
the
non-testis-specific
a-tubulins.
Ma4
was
represented
in
our
libraries
by
three
classes
of
cDNA,
all
of
which
were
identical
downstream
from
the
codon
encoding
amino
acid
2.
One
transcript,
Ma4
(Fig.
1),
represents
the
1.8-kb
mRNA
and
encodes
a
divergent
a-
tubulin,
abundant
in
muscle
and
heart
but
absent
in
testis.
A
second
transcript,
Ma4L
(the
extended
5'
end
of
which
is
shown
in
Fig.
5),
represents
the
2.4-kb
mRNA
expressed
in
mature
testis
and
more
weakly
in
brain,
heart,
and
muscle
(Fig.
6).
A
third
transcript
contains
approximately
1
kb
of
VOL.
6,
1986

2418
VILLASANTE
ET
AL.
semirepetitive
DNA
5'
to
amino
acid
2
(data
not
shown).
Because
all
these
transcripts
are
identical
3'
to
amino
acid
2,
we
conclude
that
they
each
derive
from
the
same
gene.
A
comparison
with
the
corresponding
human
ot-tubulin
gene,
Hcx44
(Dobner
et
al.,
submitted),
clarifies
the
relationship
of
these
three
transcripts
to
one
another
(Fig.
5
and
9).
M(X4
and
MQ4L
appear
to
be
transcripts
from
two
different
pro-
moters,
both
of
which
splice
to
the
triplet
encoding
amino
acid
2
of
the
gene,
which
is
the
start
of
the
second
exon
of
this
and
all
other
hiterto
described
vertebrate
a-tubulin
genes
(5),
whereas
the
third
cDNA
(designated
C)
(Fig.
9)
is
a
copy
of
an
unspliced
transcript.
Because
the
long
mRNA
(Mot4L)
has
(i)
no
initiation
codon
in
frame
with
the
a-tubulin
coding
region,
(ii)
many
stop
codons
in
the
region
upstream
from
amino
acid
2,
and
(iii)
a
seemingly
random
set
of
mutations
relative
to
the
human
gene
H(X44
in
this
upstream
region
(Fig.
5)
which
preserve
neither
reading
frame
nor
amino
acids,
we
conclude
that,
at
least
in
mice,
this
tran-
script
is
unlikely
to
be
translated.
The
intron-exon
structure
of
vertebrate
a-tubulin
genes
may
facilitate
the
occurrence
of
misspliced
or
unspliced
transcripts
such
as
those
de-
scribed
above.
The
first
exon
of
such
a
gene
has
as
its
3'
end
the
triplet
encoding
the
initiator
methionine
ATG.
This
sequence
is
imperfect
with
respect
to
the
consensus
donor
splice
signal
sequence
(17).
Such
a
gene
structure
thus
lends
itself
to
the
use
of
alternative
promoters,
although
in
this
case,
such
use
may
be
adventitious.
Ma6
also
gives
rise
to
two
transcripts
in
testis
(Fig.
3),
perhaps
by
a
similar
mechanism.
The
a-tubulin
encoded
by
the
short
(1.8
kb)
transcript
of
Ma4
is
the
most
divergent
of
the
five
we
describe
and
is
unique
in
having
no
encoded
carboxy-terminal
tyrosine
residue.
In
this
regard,
it
is
interesting
to
note
that
an
enzyme
has
been
previously
characterized
that
is
particu-
larly
abundant
in
brain
and
muscle
and
that
specifically
tyrosinylates
the
carboxy
terminus
of
some
but
not
all
ot-tubulins
(20).
Ma4
was
expressed
in
all
somatic
tissues
examined,
at
the
highest
level
in
striated,
smooth,
and
cardiac
muscle,
where
it
appears
to
be
the
dominant
a-
tubulin.
Microtubules,
while
not
abundant
in
muscle,
act
as
a
part
of
the
cytoskeleton
and
in
the
organization
and
movement
of
organelles
(8).
Perhaps
Ma4
has
evolved
to
play
a
specialized
role
in
one
of
these
nonmitotic
functions,
in
organelle
transport,
for
example.
Although
tubulin
is
a
heterodimer
of
a-
and
P-tubulin
subunits,
a-
and
,-tubulin
genes
do
not
appear
to
be
ex-
pressed
in
pairs.
For
example,
we
found
no
brain-specific
a-tubulin
corresponding
to
the
brain-specific
,B-tubulin,
M,B4
(16).
Similarly,
in
an
extensive
seach
of
our
testis
cDNA
library,
we
found
no
testis-specific
3-tubulin
sequences
analogous
to
Ma3
or
Ma7
(although
we
did
isolate
a
testis-
abundant
3-tubulin
[unpublished
data].
Furthermore,
de-
spite
evidence
for
a
hematopoetic
tissue-specific
1-tubulin
(18;
unpublished
data),
we
found
no
such
a-tubulin
cDNA
in
an
exhaustive
search
of
our
bone
marrow
cDNA
library.
Thus,
although
ubiquitous
ox-
and
,-tubulins
may
be
coordi-
nately
expressed
(16),
the
same
does
not
seem
to
be
true
for
these
highly
specialized
tubulins
in
testis,
brain,
and
bone
marrow.
We
conclude
that
the
incorporation
of
one
of
these
unique
isotypes,
either
at
or
P,
is
sufficient
to
confer
special-
ization
of
function
on
a
microtubule.
ACKNOWLEDGMENTS
This
work
was
supported
by
grants
from
the
Muscular
Dystrophy
Association
and
by
a
Public
Health
Service
grant
from
the
National
Institutes
of
Health.
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