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Molecular Characterization of the Major Maize Embryo Globulin Encoded by the Glb1 Gene

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Abstract and Figures

One of the most abundant proteins in maize (Zea mays L.) embryos is the molecular weight 63,000 globulin encoded by the Glb1 gene. To obtain DNA clones corresponding to Glb1, a cDNA library corresponding to RNA from developing maize embryos was constructed in a lambda expression vector and screened with antibodies specific for Glb1-encoded proteins. Here we report the complete nucleotide sequence, as determined from two overlapping clones, of pcGlb 1S, a 2009 base pair clone containing the entire translated region of Glb1. The deduced amino acid sequence of pcGlb 1S shows similarities to 7S-type seed storage proteins of wheat and legumes. Southern blot analysis of maize DNA confirms previous genetic studies which had indicated the presence of a single copy of Glb1 per haploid genome. Northern blot analysis indicates that Glb1 transcripts are present throughout most of embryo development and that expression of this gene is limited to seed tissues. Embryos homozygous for a Glb1 null allele, in which Glb1-encoded proteins are not detectable, contain low levels of Glb1 transcripts which are a different size from those encoded by functional alleles. This suggests that the defect in the null allele is at the level of gene transcription or RNA processing.
Content may be subject to copyright.
Plant
Physiol.
(1989)
91,
636-643
0032-0889/89/91
/0636/08/$01
.00/0
Received
for
publication
February
27,
1989
and
in
revised
form
May
19,
1989
Molecular
Characterization
of
the
Major
Maize
Embryo
Globulin
Encoded
by
the
Gibi
Gene1
Faith
C.
Belanger
and
Alan
L.
Kriz*
Department
of
Agronomy,
University
of
Illinois,
Urbana,
Illinois,
61801
ABSTRACT
One
of
the
most
abundant
proteins
in
maize
(Zea
mays
L.)
embryos
is
the
molecular
weight
63,000
globulin
encoded
by
the
Gibl
gene.
To
obtain
DNA
clones
corresponding
to
Gibl,
a
cDNA
library
corresponding
to
RNA
from
developing
maize
embryos
was
constructed
in
a
lambda
expression
vector
and
screened
with
antibodies
specific
for
GIbl-encoded
proteins.
Here
we
re-
port
the
complete
nucleotide
sequence,
as
determined
from
two
overlapping
clones,
of
pcGlb1S,
a
2009
base
pair
clone
containing
the
entire
translated
region
of
Gibl.
The
deduced
amino
acid
sequence
of
pcGlblS
shows
similarities
to
7S-type
seed
storage
proteins
of
wheat
and
legumes.
Southern
blot
analysis
of
maize
DNA
confirms
previous
genetic
studies
which
had
indicated
the
presence
of
a
single
copy
of
Gibl
per
haploid
genome.
Northem
blot
analysis
indicates
that
Gibl
transcripts
are
present
through-
out
most
of
embryo
development
and
that
expression
of
this
gene
is
limited
to
seed
tissues.
Embryos
homozygous
for
a
Gibl
null
allele,
in
which
GIbl-encoded
proteins
are
not
detectable,
contain
low
levels
of
Gibl
transcripts
which
are
a
different
size
from
those
encoded
by
functional
alleles.
This
suggests
that
the
defect
in
the
null
allele
is
at
the
level
of
gene
transcription
or
RNA
proc-
essing.
Maize
embryos
contain
large
amounts
of
saline-soluble,
water-insoluble
proteins
called
globulins.
The
major
globulin
component,
a
mol
wt
63,000
protein
designated
GLB1
(for-
merly
PROT)
(10),
is
one
of
the
most
abundant
proteins
in
mature
embryos.
GLB1
has
no
known
enzymatic
function
and
is
currently
believed
to
serve
as
a
storage
protein
(8).
Genetic
analysis
of
GLB
1
variants
indicated
that
this
protein
is
encoded
by
a
single
gene,
Glbl
(for
Globulin-i),
on
the
long
arm
of
chromosome
1
(22).
Several
alleles
of
this
gene,
including
a
CRM-
null,2
have
been
described
(17,
22).
Allelic
polymorphism
of
Glbl-encoded
proteins
is
observed
as
a
function
of
mobility
in
SDS-PAGE.
The
three
most
com-
monly
occuring
Glbl
alleles
have
the
designations
L,
I,
and
S,
for
Large,
Intermediate,
and
Small
proteins,
respectively.
By
convention,
the
protein
product
of
each
allele
is
indicated
in
the
upper
case
(e.g.
Glbl-S
encodes
GLB
1-S).
Pulse-chase
'This
contribution
from
the
University
of
Illinois
Agricultural
Experiment
Station
was
supported
by
grants
from
The
Standard
Oil
Company
of
Ohio,
a
wholly
owned
subsidiary
of
BP
America,
Inc.,
and
the
U.S.
Department
of
Agriculture
Competitive
Research
Grants
Office
(No.
88-37262-3427)
to
A.
L.
K.
2
Abbreviations:
CRM.
cross-reacting
material;
DAP.
days
after
pollination;
ds,
double-stranded.
radiolabeling
and
in
vitro
translation
experiments
have
indi-
cated
that
at
least
three
protein-processing
steps
occur
in
the
formation
of
the
mature
protein
from
the
primary
translation
product
(10).
The
final
processing
step
is
controlled
by
the
unlinked
gene
Mep
(chromosome
5,
near
Pr),
and
embryos
homozygous
for
the
recessive
mep
allele
accumulate
the
proc-
essing
intermediate
GLB1'
(10,
22).
We
have
recently
re-
ported
on
additional
characterization
of
maize
embryo
glob-
ulins
(8).
Here
we
report
the
isolation
and
characterization
of
a
full-
length3
cDNA
clone,
pcGlb
1S,4
corresponding
to
the
S
allele
of
the
Glbl
gene.
Using
this
clone
as
a
probe,
we
have
determined
that
expression
of
the
gene
appears
to
be
limited
to
seed
tissues
and
that
embryos
homozygous
for
the
null
allele
contain
small
amounts
of
transcript
corresponding
to
Glbl.
MATERIALS
AND
METHODS
Materials
Embryos
homozygous
for
the
GlbJ-L
and
Glbl-S
alleles
were
obtained
from
field-grown
plants
of
the
maize
(Zea
mays
L.)
inbred
lines
W64A
and
Va26,
respectively,
as
pre-
viously
described
(8).
The
GlbJ-O
null
allele
was
originally
identified
in
a
Black
Beauty
popcorn
line
(22).
Nitrocellulose
and
Magnagraph
nylon
membranes
were
obtained
from
Mi-
cron
Separations,
Inc.
(Westboro,
MA).
Random
priming
kits,
a
cDNA
synthesis
kit,
EcoRI,
T4
DNA
ligase,
SI
nu-
clease,
and
protein
and
RNA
mol
wt
standards
were
pur-
chased
from
Bethesda
Research
Laboratories
(Gaithersburg,
MD).
LambdaZAP
vector
arms,
Gigapack
packaging
extracts,
and
exonuclease
III/mung
bean
nuclease
deletion
kits
were
from
Stratagene
(La
Jolla,
CA).
[ac-32P]-labeled
dATP
(3000
Ci/mmol)
was
obtained
from
New
England
Nuclear
(Boston,
MA).
Oligo(dT)-cellulose
(type
7)
was
from
Pharmacia,
Inc.
(Piscataway,
NJ).
Preparation
of
GLB1-Specific
Antiserum
GLB
1-S
was
fractionated
from
whole
globulin
by
cryopre-
cipitation
(8)
and
stored
at
4°C
as
a
suspension
in
distilled
water
at
a
concentration
of
2
mg/mL.
A
volume
of
the
suspension
equivalent
to
600
ug
protein
was
emulsified
with
an
equal
volume
of
Freund's
complete
adjuvant
and
injected
'The
term
full-length
as
used
here
indicates
that
the
cDNA
clone
contains
the
entire
translated
region
of
the
gene.
4
The
GenBank
accession
number
for
pcGlblS
is
M24845.
636
CHARACTERIZATION
OF
THE
MAIZE
Glbl
GENE
PRODUCT
subcutaneously
into
multiple
sites
of
a
10-week
old
New
Zealand
White
rabbit.
A
second
inoculation
of
300
ug
GLB
1-
S
in
Freund's
incomplete
adjuvant
was
administered
2
weeks
later.
Blood
was
collected
from
the
marginal
ear
vein
3
weeks
after
the
secondary
inoculation
and
serum
was
prepared
from
the
blood
by
standard
methods.
Protein
Extraction
and
Immunoblot
Analysis
For
analysis
of
total
proteins
from
imbibed
mature
embryos
(8),
kernels
were
soaked
overnight
in
water
and
embryos
excised
from
the
softened
kernels
with
the
aid
of
a
sharpened
spatula.
For
developmental
analyses,
field-grown
ears
were
transported
to
the
laboratory
and
the
crowns
of
the
kernels
were
sliced
off
with
a
sharp
knife
or
scalpel
blade.
Embryos
were
removed
from
the
kernels
with
a
small
spatula,
imme-
diately
frozen
in
liquid
nitrogen,
and
stored
at
-70°C
until
needed.
Individual
mature
embryos
were
macerated
in
1
mL
SDS
sample
buffer
(2%
[w/v]
SDS,
50
mM
Tris
[pH
6.8]
5%
[v/v]
2-mercaptoethanol,
10%
[v/v]
glycerol)
at
a
ratio
of
50
mg/mL
(fresh
weight)
directly
in
1.5
mL
microcentrifuge
tubes
with
the
aid
of
a
fitted
pellet
pestle
(Kontes
Scientific
Glassware,
Morton
Grove,
IL).
Extracts
were
heated
to
100°C
for
2
min,
centrifuged
at
1
3,500g
for
5
min,
and
immediately
subjected
to
SDS-PAGE
as
previously
described
(8).
Five
,uL
of
each
sample
was
applied
to
a
12.5%
polyacrylamide
gel
and
subsequently
electroblotted
onto
nitrocellulose
by
using
a
semidry
blotting
apparatus
(PolyBlot;
American
Bionetics,
Hayward,
CA)
according
to
the
manufacturer's
specifications.
Immunoblot
analysis,
in
which
GLB
1-specific
antiserum
was
used
at
1:2000
dilution,
was
performed
as
previously
de-
scribed
for
analysis
of
maize
zeins
(13).
Nucleic
Acid
Isolation
and
Gel
Blot
Analysis
Total
RNA
was
isolated
from
frozen
tissue
by
using
the
guanidine-HCl
method
described
by
Cox
(3).
For
Northern
blot
analysis,
10
,ug
of
total
RNA
was
subjected
to
electropho-
resis
in
formaldehyde
agarose
gels
and
transferred
to
either
nitrocellulose
or
nylon
(Magnagraph)
membranes
as
de-
scribed
by
Selden
(23).
Hybridization
conditions
were
as
previously
described
(9).
Isolation
of
maize
nuclear
DNA
from
unfertilized
ears
and
Southern
blot
analysis
were
performed
as
previously
described
(9)
except
that
Magnagraph
membranes
were
used
as
the
transfer
medium.
Samples
for
gene
copy-number
reconstruc-
tions
contained
EcoRI-cleaved
DNA
from
a
genomic
clone
corresponding
to
GlbJ-L
(our
unpublished
data).
For
both
RNA
and
DNA
blots,
the
transferred
nucleic
acids
were
UV-
linked
to
the
membrane
by
using
a
Stratalinker
1800
appa-
ratus
(Stratagene).
For
use
in
hybridizations,
the
cDNA
insert
was
isolated
from
the
plasmid
clone
by
EcoRI
digestion,
separation
on
a
1%
agarose
gel,
and
binding
of
the
fragment
to
NA-45
paper
(Schleicher
and
Schuell,
Keene,
NH)
as
recommended
by
the
manufacturer.
The
isolated
insert
was
labeled
with
[a-
32p]
dATP
by
using
a
commercial
random
priming
kit
(BRL).
Construction
and
Screening
of
an
Embryo-Specific
cDNA
Library
To
obtain
cDNA
clones
corresponding
to
Glbl,
a
library
of
cDNA
sequences
was
prepared
in
a
bacteriophage
X-expres-
sion
vector.
Total
RNA
was
prepared
from
27
DAP
embryos
of
the
maize
inbred
line
Va26
(Glbi-SIS).
Polyadenylated
RNA
was
fractionated
from
total
RNA
by
oligo(dT)-cellulose
chromatography
(1).
First
and
second
strand
cDNA
synthesis
was
performed
by
using
a
commercial
cDNA
synthesis
kit
based
on
the
RNase
H
procedure
of
Gubler
and
Hoffman
(4).
The
ds
cDNA
was
protected
from
EcoRI
cleavage
by
treat-
ment
with
EcoRI
methylase,
and
EcoRI
oligonucleotide
link-
ers
(both
from
New
England
Biolabs,
Beverly,
MA)
were
added
to
the
ends
of
the
ds
cDNA
with
T4
DNA
ligase.
Subsequent
to
linker
cleavage
with
EcoRI,
excess
linkers
were
removed
by
passage
through
a
NACS
Prepac
column
(BRL)
as
described
by
the
manufacturer.
The
ds
cDNA
was
ligated
to
the
arms
of
the
expression
vector
LambdaZAP,
packaged
by
using
the
Gigapack
system,
and
the
resultant
recombinant
phage
was
used
to
infect
the
Escherichia
coli
host
BB4.
The
primary
library
consisted
of
approximately
500,000
clones
which
were
obtained
from
an
estimated
0.1
,ug
of
ds
cDNA.
The
initial
plating
of
the
library
was
screened
by
preparing
nitrocellulose
replicas
of
the
plates
by
the
plaque-lift
technique
as
described
by
Huynh
et
al.
(7).
Filters
were
agitated
in
a
solution
of
TTBS/
1%
(w/v)
nonfat
dry
milk
(TTBS:
50
mM
Tris
[pH
7.4]
0.15%
[v/v]
Tween-20,
140
mM
NaCl)
for
1
h,
then
transferred
to
a
solution
of
GLB1-specific
antibody
(1:500
dilution
in
TTBS)
and
agitated
overnight.
Immuno-
reactive
clones
were
detected
as
for
the
protein
immunoblots.
Plaques
were
picked,
plated,
and
subjected
to
three
additional
rounds
of
screening.
Of
10
initial
plaque
picks,
all
continued
to
yield
positive
reactions
after
the
final
screen.
The
cDNA
inserts
were
excised
from
LambdaZAP
as
recombinant
pBluescript
SK(-)
plasmids
according
to
the
manufacturer's
protocols.
Rescreening
of
the
library
with
a
radiolabeled
probe
was
essentially
as
described
by
Huynh
et
al.
(7).
DNA
Sequencing
For
nucleotide
sequence
analysis,
the
cDNA
inserts
in
pBluescript
were
subcloned
into
M13
mpl8
and
mpl9
(30)
to
obtain
inserts
in
opposite
orientations.
Overlapping
unidi-
rectional
deletions
corresponding
to
either
strand
were
pre-
pared
from
the
appropriate
M13
clone
RF
by
using
a
com-
mercial
exonuclease
III/mung
bean
nuclease
deletion
kit
(Stratagene).
The
deletions
were
sized
by
using
a
rapid
Si
nuclease
procedure
(16).
Dideoxynucleotide
sequencing
(21)
of
single-stranded
templates
with
a
modified
T7
DNA
polym-
erase
(Sequenase)
was
performed
by
using
a
commercial
sequencing
kit
(United
States
Biochemical
Corp.,
Cleveland,
OH).
The
deoxyguanine
triphosphate
(dGTP)
analog
7-deaza
dGTP
was
used
to
resolve
GC
compressions.
Analysis
of
DNA
sequences
was
performed
on
an
IBM
PC
AT
with
either
IBI/Pustell
Sequence
Analysis
software
(International
Bio-
technologies
Inc.,
New
Haven,
CT)
or
DNAStar
programs
(DNAStar,
Inc.,
Madison,
WI).
637
Plant
Physiol.
Vol.
91,
1989
RESULTS
Characterization
of
GLBI-Specific
Antibodies
The
specificity
of
GLB
1-specific
polyclonal
antibodies
was
determined
in
an
immunoblot
of
proteins
from
individual
embryos
of
varying
allelic
composition
(Fig.
1).
Lanes
1
and
2
of
Figure
1
are
from
embryos
of
the
inbred
lines
W64A
and
Va26
which
are
homozygous
for
the
GlbJ-L
and
Gibl-S
alleles,
respectively.
The
major
proteins
recognized
by
the
antiserum
are
GLB
1-L
and
GLB
1-S
and
their
respective
immediate
precursors
GLB
1'-L
and
GLB
l'-S
(10).
There
were,
however,
several
other
polypeptide
bands
of
lower
mol
wt
which
also
reacted
with
the
antiserum. Since
these
are
absent
in
embryos
homozygous
for
the
GlbJ-O
null
allele
(Fig.
1,
lane
3),
we
inferred
that
these
low
mol
wt
polypeptides
were
derived
from
Glb
1-encoded
proteins.
Such
polypeptides
were
present
in
GlbJ-L/O
and
Glbl-SIO
embryos
(Fig.
1,
lanes
4
and
5,
respectively),
but
at
levels
lower
than
those
observed
for
Gibi-LIL
or
S/S
embryos.
This
is
apparently
due
to
the
presence
of
only
a
single
functional
Glbi
allele
in
the
heterozygotes.
We
therefore
concluded
that
the
antiserum
was
specific
for
Glbl-encoded
proteins
and
was
appropriate
for
use
in
screening
a
cDNA
expression
library.
Isolation
and
Characterization
of
pcGIb1S
In
preliminary
experiments
we
observed
that
embryos
of
the
maize
inbred
line
Va26
contained
large
amounts
of
GLB
1-
S
protein.
We
therefore
chose
this
material
as
a
source
of
1
2
3 4
Figure
1.
Specificty
of
GLB1
antiserum.
Total
protein
extracts
from
embryos
of
varying
Glb1
genotypes
were
subjected
to
immunoblot
analysis
as
described
in
"Materials
and
Methods."
Lane
1,
Glb1-LIL
(W64A);
lane
2,
Glb1-S/S
(Va26);
lane
3,
Glb1-0/0;
lane
4,
Glb1-L/O
(W64A
x
Glb1-0/0);
lane
5,
Glb1-S/O
(Va
26
x
Glb1-0/0).
RNA
for
production
of
a
cDNA
library
as
described
above.
A
screen
of
250,000
clones
yielded
10
immunoreactive
clones
with
inserts
ranging
in
size
from
700
to
1800
bp.
Southern
blot
analysis
revealed
that
all
of
these
clones
hybridized
with
a
radiolabeled
probe
prepared
from
the
longest
insert.
The
1800
bp
clone,
designated
pcA
1,
was
chosen
for
further
characterization
and
subjected
to
nucleotide
sequence
analy-
sis.
A
300
bp
EcoRI/BstEII
restriction
fragment
from
the
5'
region
of
this
clone
was
used
as
a
radiolabeled
probe
to
rescreen
the
cDNA
library
for
a
full
length
cDNA
clone,
which
we
have
designated
pcGlb
lS.
The
5'
region
of
the
full
length
clone
was
sequenced
to
provide
55
nucleotides
of
overlap
with
the
original
1800
bp
clone
pcAl.
The
nucleotide
sequences
of
the
two
clones
were
identical
in
this
region
of
overlap.
Because
of
the
high
G
+
C
content
(66%)
of
these
clones,
it
was
necessary
to
use
the
dGTP
analog
deaza
dGTP
to
minimize
GC
compressions
and
obtain
unambiguous
se-
quence
data.
The
nucleotide
sequence
was
determined
from
both
strands
for
the
appropriate
regions
of
each
clone.
The
combined
sequence
of
the
two
cDNA
clones
and
the
deduced
amino
acid
sequence
are
shown
in
Figure
2.
The
sequence
surrounding
the
presumed
initiator
methionine
cor-
responds
to
a
relaxed
consensus
(27)
of
Kozak's
rule,
AN-
NATGG
(11,
14).
An
AT-rich
region
between
nucleotide
positions
+
1937
and
+
1949
is
considered
to
be
a
potential
polyadenylation
signal
and
contains
the
consensus
sequence
AATAAA
(18).
The
nucleotide
sequence
predicts
a
polypep-
tide
of
573
amino
acids
with
a
calculated
mol
wt
of
65,025
D.
From
SDS-PAGE
analysis
of
immunoprecipitated
in
vitro
translation
products,
the
mol
wt
for
the
primary
translation
product
of
Glbl-S
has
been
estimated
as
67,600
D
(10).
The
mol
wt
calculated
from
the
deduced
amino
acid
sequence
is
2,575
D
lower
than
the
experimentally
determined
estimate.
We
do
not
consider
this
discrepancy
to
be
significant
since
it
is
not
uncommon
for
proteins
to
exhibit
anomalous
migration
in
SDS-PAGE
(2).
The
presence
of
a
region
of
deduced
amino
acid
sequence
(underlined
in
Fig.
2)
identical
to
the
N-termi-
nal
region
of
experimentally
determined
GLB
1-S
protein
sequence
(8)
confirms
that
these
cDNA
clones
correspond
to
Gibi.
Previous
studies
demonstrated
that
GLBl'
and
GLB1
differ
at
only
one
terminus
(10).
The
present
sequence
analysis
indicates
that
the
final
processing
of
GLB
1'
to
GLB
1,
con-
trolled
by
the
Mep
gene
(22),
is
localized
to
the
N-terminal
portion
of
GLB
1'.
In
GLB1
'-S,
this
cleavage
probably
occurs
between
the
aspartate
and
glutamate
residues
at
positions
86
and
87
(Fig
2,
arrowhead).
The
single
internal
methionine
at
amino
acid
position
355
(Fig.
2,
boxed)
is
consistent
with
previous
cyanogen
bromide
cleavage
analysis
(10)
which
yielded
two
cleavage
fragments
corresponding
in
size
to
those
predicted
from
this
sequence
analysis.
The
amino
acid
composition
deduced
from
the
pcGlb
lS
sequence
in
the
region
corresponding
to
GLB
1-S
(Table
I)