Wessler, S.R. & Varagona, M.J. Molecular basis of mutations at the waxy locus of maize: correlation with the fine structure genetic map. Proc. Natl. Acad. Sci. USA 82, 4177-4181

Abstract

More than 40 mutant alleles of the waxy (Wx) locus of maize are available for molecular analysis. Previous studies have examined the nature of phenotypically unstable Wx mutant alleles caused by insertion of the maize transposable activator (Ac) and dissociation (Ds) elements. In this study we have used Southern blot analysis to characterize the locus in 22 strains harboring wx alleles with stable mutant phenotypes. Of these mutations, 17 are of spontaneous origin, 4 were induced by gamma rays, and 1 was induced by ethyl methanesulfonate. Of these 22 alleles, we find that 13 have either insertions or deletions within the Wx transcription unit. The insertions range in size from 150 base pairs to 6.1 kilobases. For 4 of the 6 deletions identified, the two breakpoints are within the Wx gene. For 9 other alleles we can detect no obvious lesions within or around the transcription unit. Evidence is presented that the insertions and deletions result in the mutant phenotype and are not polymorphisms. This conclusion is based on two findings: (i) a survey of inbred lines revealed only a single instance of polymorphism within the transcription unit, whereas all of the lesions described alter the transcription unit; and (ii) there is an excellent correlation between the position of these lesions on the physical map and their relative position on a fine structure genetic map of the locus.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
82,
pp.
4177-4181,
June
1985
Genetics
Molecular
basis
of
mutations
at
the
waxy
locus
of
maize:
Correlation
with
the
fine
structure
genetic
map
(spontaneous
mutation/DNA
insertions/polymorphism)
SUSAN
R.
WESSLER
AND
MARGUERITE
J.
VARAGONA
Department
of
Botany,
University
of
Georgia,
Athens,
GA
30602
Communicated
by
Oliver
E.
Nelson,
Jr.,
February
19,
1985
ABSTRACT
More
than
40
mutant
alleles
of
the
waxy
(Wx)
locus
of
maize
are
available
for
molecular
analysis.
Previous
studies
have
examined
the
nature
of
phenotypically
unstable
Wx
mutant
alleles
caused
by
insertion
of
the
maize
transposable
activator
(Ac)
and
dissociation
(Ds)
elements.
In
this
study
we
have
used
Southern
blot
analysis
to
characterize
the
locus
in
22
strains
harboring
wx
alleles
with
stable
mutant
phenotypes.
Of
these
mutations,
17
are
of
spontaneous
origin,
4
were
induced
by
y
rays,
and
1
was
induced
by
ethyl
methanesulfonate.
Of
these
22
alleles,
we
find
that
13
have
either
insertions
or
deletions
within
the
Wx
transcription
unit.
The
insertions
range
in
size
from
150
base
pairs
to
6.1
kilobases.
For
4
of
the
6
deletions
identified,
the
two
breakpoints
are
within
the
Wx
gene.
For
9
other
alleles
we
can
detect
no
obvious
lesions
within
or
around
the
transcription
unit.
Evidence
is
presented
that
the
insertions
and
deletions
result
in
the
mutant
phenotype
and
are
not
polymorphisms.
This
conclusion
is
based
on
two
findings:
(i)
a
survey
of
inbred
lines
revealed
only
a
single
instance
of
polymorphism
within
the
transcription
unit,
whereas
all
of
the
lesions
described
alter
the
transcription
unit;
and
(ii)
there
is
an
excellent
correlation
between
the
position
of
these
lesions
on
the
physical
map
and
their
relative
position
on
a
fine
structure
genetic
map
of
the
locus.
The
Wx
(waxy)
locus
of
Zea
mays
encodes
a
starch
granule-
bound
glucosyl
transferase
involved
in
starch
biosynthesis
(1).
It
is
expressed
in
the
endosperm
of
the
developing
kernel
and
in
the
pollen
grain
(2,
3).
The
locus
derives
its
name
from
the
waxy
appearance
of
mutant
kernels,
the
phenotype
reflecting
an
alteration
in
the
underlying
starch
composition.
Because
of
this
easily
identifiable
nonlethal
phenotype,
the
locus
has
been
the
subject
of
genetic
analysis
for
over
60
yr
(3,
4).
There
are
>40
Wx
mutants
of
both
spontaneous
and
induced
origins
available
for
analysis.
Some
are
stably
mutant
(i.e.,
the
mutant
phenotype
remains
unchanged)
both
somatically
and
germinally
in
all
genetic
backgrounds
tested.
Other
alleles
display
an
unstable
phenotype
because
of
the
insertion
of
transposable
elements
(5-8).
All
Wx
mutations
map
to
the
short
arm
of
chromosome
9.
To
understand
the
relationship
between
the
various
Wx
mutants,
Nelson
con-
structed
a
fine-structure
map
of
the
locus
(9,
10).
To
date
this
represents
the
most
rigorous
genetic
study
of
a
plant
gene.
This
ingenious
analysis
was
accomplished
by
determining
whether
various
wx
heteroalleles
exhibit
recombination.
The
restoration
of
Wx
expression
via
recombination
was
scored
by
staining
the
pollen
grains
of
these
heteroalleles
with
KI/12
and
counting
the
rare
black-staining
recombinants
that
con-
tain
amylose.
In
order
to
determine
the
molecular
basis
of
normal
and
mutant
Wx
expression,
the
locus
was
cloned
and
its
gene
product
was
characterized
(11).
It
was
found
that
the
Wx
locus
encodes
a
58-kDa
starch
granule-bound
protein
that
is
altered
in
some
strains
harboring
Wx
mutant
alleles
(wx)
and
missing
entirely
from
others
(11,
12).
Of
interest
to
us
was
the
finding
of
Echt
and
Schwartz
(12)
that
three
Wx
mutants
(strains
harboring
wx
alleles
R,
C31
and
90;
see
Table
1)
that
produced
altered
granule-bound
proteins
mapped
to
the
extremities
of
Nelson's
genetic
map.
This
result
suggested
that
all
or
most
of
the
Wx
mutations
could
be
found
within
the
structural
gene
of
the
Wx
protein.
Recent
studies
have
focused
on
a
molecular
description
of
the
transposable
elements
responsible
for
unstable
pheno-
types
in
plants
(11,
13-16).
However,
virtually
nothing
is
known
about
the
molecular
basis
of
stable
mutant
phenotypes
in
plants,
especially
those
of
spontaneous
origin.
The
Wx
locus
and
its
genetically
characterized
mutant
alleles
pro-
vides
a
unique
opportunity
to
address
this
question.
To
this
end
we
analyzed
the
lesions
responsible
for
22
mutant
Wx
alleles,
17
of
which
are
of
spontaneous
origin.
We
found
that
13
of
these
alleles
have
either
insertions
or
deletions
within
or
including
the
Wx
transcription
unit.
Furthermore,
we
found
that
there
is
an
excellent
correlation
between
the
position
of
an
insertion
or
deletion
on
the
physical
map
and
the
relative
position
of
these
mutations
on
Nelson's
genetic
map.
As
the
genetic
map
is
a
functional
map,
this
correlation
suggests
that
the
insertions
and
deletions
found
associated
with
mutant
alleles
are
in
fact
the
molecular
lesions
respon-
sible
for
the
mutant
phenotype.
MATERIALS
AND
METHODS
Maize
Strains.
The
following
inbred
lines
carrying
the
Wx
allele
were
obtained
for
this
study:
W23
x
K55
from
E.
Coe;
W23
from
G.
Neuffer;
W22
from
J.
Kermicle;
38-11
from
M.
Zuber;
HY,
Krug,
and
Oh45
from
B.
Bear;
and
Ga
209,
Ga
211,
and
Ga
219
from
A.
Flemming.
The
origins
of
the
Wx
mutant
alleles
are
described
in
Table
1.
Strains
carrying
the
wx
alleles
B5,
G,
K,
and
M
were
obtained
from
0.
Nelson,
strains
carrying
wx
alleles
I,
C2
and
BL2
were
obtained
from
C.
Echt,
and
the
remaining
wx
allele-carrying
strains
came
from
the
Maize
Genetics
Cooperation
Stock
Center
(Urbana,
IL).
The
strains
examined
in
this
study
were
all
homozygous
for
the
designated
Wx
or
wx
alleles.
Reagents.
Restriction
enzymes,
ligase,
and
DNA
polymer-
ase
I
were
from
either
Bethesda
Research
Laboratories
or
from
New
England
Biolabs.
Radioactive
dATP
and
dCTP
were
from
Amersham
(specific
activity,
400
Ci/mmol;
1
Ci
=
37
GBq).
Recombinant
Plasmids.
The
construction
of
plasmid
pWx5,
which
contains
the
Wx
transcription
unit
on
a
10.5-kilobase
(kb)
EcoRI
fragment,
has
been
described
(11).
Subclones
containing
probes
1,
2,
and
3
(see
Fig.
1D)
were
constructed
Abbreviations:
Wx,
waxy
gene;
wx,
mutant
Wx
allele;
bp,
base
pair(s);
kb,
kilobase(s);
RFLP,
restriction
fragment
length
polymor-
phism.
4177
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

4178
Genetics:
Wessler
and
Varagona
Table
1.
Strains
used
in
this
study
Molecular
wx
Nature
of
progenitor
allele
mutation
Lesion*
Wx
allelet
Origin*
90
Spontaneous
ND
HY
Brunson
R
Spontaneous
ND
HY
Richardson
B
Spontaneous
Deletion
HY
Bear
hybrid
G
Spontaneous
Insertion
W23
Bear
hybrid
I
Spontaneous
Insertion
HY
Bear
hybrid
K
Spontaneous
Insertion
HY
Bear
hybrid
M
Spontaneous
Insertion
HY
Bear
hybrid
Cl
Spontaneous
ND
HY
Blandy
farms
C2
y
rays
ND
HY
Blandy
farms
C3
y
rays
ND
HY
Blandy
farms
C4
Spontaneous
Deletion
HY
Blandy
farms
C31
y
rays
ND
HY
Blandy
farms
C34
y
rays
Deletion
Unknown
Blandy
farms
BJ
Spontaneous
Deletion
W23
Ashman
and
Brink
B2
Spontaneous
Insertion
W23
Ashman
and
Brink
B5
Spontaneous
Insertion
W23
Ashman
and
Brink
B6
Spontaneous
Deletion
HY
Ashman
and
Brink
B7
Spontaneous
Deletion
HY
Ashman
and
Brink
B8
Spontaneous
ND
W23
Ashman
and
Brink
c
Spontaneous
ND
HY
Collins
Stoner
Spontaneous
Insertion
HY
From
Assam
BL2
EMS
ND
HY
Briggs
EMS,
ethyl
methanesulfonate.
*ND
=
not
determined,
a
lesion
of
less
than
50
bp.
tThis
refers
to
one
of
the
four
Wx
alleles
described
in
Fig.
iD.
Few
of
the
direct
progenitors
are
actually
known.
tFrom
0.
Nelson
(9).
by
digestion
of
pWx5
with
Sal
I
and
subsequent
ligation
of
the
resulting
fragments
into
the
Sal
I
site
of
pUC9
(17).
Preparation
of
Maize
Genomic
DNA
and
Filter
Hybridiza-
tion.
DNA
was
purified
from
2-
to
4-week-old
plantlets
by
the
method
of
Shure
et
al.
(11).
To
rapidly
survey
many
maize
strains,
genomic
DNA
was
isolated
from
1
g
of
leaf
tissue
by
the
"miniprep"
protocol
of
Dellaporta
(18).
Restricted
DNA
was
electrophoresed
through
0.8-1.0%
agarose
and
blotted
by
the
method
of
Southern
(19)
as
modified
by
Fedoroffet
al.
(20).
Blots
were
hybridized
with
plasmids
containing
probes
1,
2,
or
3
(Fig.
1D)
that
were
labeled
by
nick-translation
(21)
to
a
specific
activity
of
1
x
108
to
5
x
108
cpm/j.g
by
using
radioactive
dATP
and
dCTP.
Autoradiography
was
for
16-48
hr
with
an
intensifying
screen.
RESULTS
Polymorphisms
Within
and
Around
the
Wx
Gene.
The
mutant
Wx
alleles
examined
in
this
study
are
described
in
Table
1.
Many
of
these
mutants
were
isolated
over
the
past
60
yr,
and
direct
progenitors
are
no
longer
available.
For
these
alleles
it
is
important
to
know
if
changes
in
the
size
of
restriction
fragments
reflect
a
mutation
or
a
polymorphism.
This
is
of
particular
importance
when
analyzing
plant
genes
which,
in
the
few
cases
examined,
exhibit
a
high
degree
of
restriction
fragment
length
polymorphism
(RFLP)
(22,
23).
To
determine
the
extent
of
RFLP
within
and
around
the
Wx
gene,
we
examined
several
inbred
lines
by
Southern
blot
analysis
with
Wx-specific
probes.
The
results
of
our
analysis
of
12
inbred
lines
are
summarized
in
Fig.
1.
The
cloned
Wx
gene
has
been
described
(11)
and
is
displayed
in
Fig.
iD
along
with
some
relevant
restriction
sites.
We
designated
this
allele
HY
after
an
inbred
line
we
have
examined
that
is
in-
distinguishable
from
the
cloned
locus.
By
hybridizing
labeled
restriction
fragments
to
dot
blots
(24)
containing
endosperm
poly(A),
we
and
others
have
delimited
the
Wx
transcription
unit
to
the
region
indicated
by
the
5'-to-3'
arrow
(unpublished
result;
ref.
15).
The
direction
of
transcription
is
inferred
by
the
position
of
homology
with
the
cDNA
clone
pcWx0.4
(ref.
11;
Fig.
ID).
Among
the
inbred
lines
examined
there
are
four
different
Wx
alleles.
The
names
given
these
alleles
(HY,
W22,
38-11,
and
W23)
represent
one
of
the
lines
that
harbors
that
particular
Wx
gene
and
flanking
sequences.
The
modified
restriction
maps
shown
in
Fig.
iD
were
generated
by
digest-
ing
genomic
DNA
with
either
Sal
I/Sst
I,
Sal
I,
or
Sal
I/Pst
I
and
hybridizing
with
a
combination
of
probes
1,
2,
or
3
(Fig.
iD)
or
subclones
of
these
probes.
Part
of
this
analysis
is
shown
in
Fig.
1
A,
B,
and
C.
We
found
that
the
DNA
corresponding
to
probe
1
is
highly
polymorphic.
When
genomic
DNA
was
digested
with
Sal
I/Sst
I
and
probed
sequentially
with
the
two
Sal
I-Sst
I
fragments
that
comprise
probe
1,
we
found
that
both
halves
reveal
RFLP.
The
3'
Sst
I-Sal
I
fragment
(with
respect
to
the
direction
of
Wx
tran-
scription)
is
conserved
in
alleles
HY,
W22
and
38-11
but
is
larger
in
W23
(Fig.
1A,
in
which
3'
fragments
are
noted
by
small
circles).
The
additional
250
base
pairs
(bp)
in
allele
W23
was
localized
to
the
Sst
I-EcoRV
fragment
(Fig.
ID).
The
5'
half
of
probe
1
hybridized
to
the
second
band
present
in
each
digest
of
Fig.
LA.
We
interpreted
these
RFLPs
as
insertions
or
deletions
(Fig.
1D)
within
this
fragment
because
the
results
of
additional
digests
with
HindIII/Sst
I
or
Sal
I
alone
were
consistent
with
this
interpretation
(data
not
shown).
It
should
be
noted
that
these
RFLPs
are
outside
of
the
region
believed
to
be
the
Wx
transcription
unit.
Among
the
inbred
lines,
only
one
RFLP
was
found
within
the
transcription
unit.
Digestion
with
Sal
I
revealed
that
the
Sal
I
fragments
represented
by
probes
2
and
3
are
essentially
conserved
in
all
strains
examined
(Fig.
1B).
However,
when
the
double
digest
of
Sal
I/Pst
I
was
probed
with
the
5'
and
3'
Sal
I-Pst
I
fragments
that
comprise
probe
2,
a
small
RFLP
was
detected
in
the
3'
half
in
allele
W23
(Fig.
1
C
and
D).
This
small
deletion
eliminated
the
Sst
I
site
in
this
strain
(Fig.
1D).
Additional
blots
indicated
that
the
sites
shown
at
the
3'
end
Proc.
Natl.
Acad.
Sci.
USA
82
(1985)

Proc.
Natl.
Acad.
Sci.
USA
82
(1985)
4179
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+
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I
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it
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IL.
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+
Pstl
Probe
2
FIG.
1.
Southern
blot
analysis
of
the
Wx
locus
in
various
inbred
lines.
(A-C)
Genomic
DNA
was
digested
with
the
enzyme
noted,
electrophoresed
through
agarose,
blotted
as
described,
and
hybridized
with
the
probe(s)
noted
and
displayed
in
D.
The
size
of
the
fragments
are
in
kb.
For
each
gel,
X
and
pBR322
restriction
fragments
were
transferred
and
probed
simultaneously
as
molecular
weight
markers.
(D)
Selected
restriction
sites
within
the
cloned
Wx
allele
in
strain
HY
(11),
the
position
of
probes
1,
2,
and
3,
and
a
summary
of
RFLPs
found
in
strains
W22,
38-11,
and
W23.
The
5'-to-3'
arrow
above
the
HY
map
represents
the
direction
and
approximate
limits
of
the
Wx
transcription
unit.
Dotted
lines
indicate
the
extent
of
uncertainty.
of
allele
HY
are
conserved
in
the
other inbred
lines.
How-
ever,
there
were
extensive
RFLP
outside
of
this
region
in
all
strains
examined.
These
data
demonstrate
that
the
Wx
transcription
unit
is
almost
perfectly
conserved
for
the
restriction
sites
examined.
Since
most,
if
not
all
of
the
mutations
fall
within
the
Wx
structural
gene
(ref.
12;
as
mentioned
in
the
Introduction),
observed
differences
between
the
restriction
maps
of
mutant
and
wild-type
alleles
should
represent
the
molecular
lesions.
Molecular
Lesions
Associated
with
Wx
Mutant
Alleles.
Genomic
DNA
was
isolated
from
strains
harboring
the
wx
alleles
described
in
Table
1.
All
were
digested
with
Sal
I,
blotted,
and
hybridized
with
probes
1,
2,
or
3.
Of
the
22
alleles
Insertions
A
Ic
0
in
>-
o
()
a
m
x
78
2
7.83L
e
n
6.1
.0-
qi
B
es
>
m
u
=e
x
5.30-
_*
examined
in
this
way,
9
showed
no
detectable
alterations.
That
is,
probes
2
and
3
hybridized
with
fragments
of
2.1
kb
and
0.8
kb,
respectively,
while
probe
1
hybridized
with
one
of
the
four
polymorphic
fragments
depicted
in
Fig.
ID.
In
Table
1
we
have
classified
these
lesions
as
"not
determined"
(ND).
It
should
be
noted
that
this
method
of
analysis
could
not
detect
lesions
that
altered
restriction
fragments
by
<50
bp.
We
found
that
seven
alleles
have
insertions
within
the
Wx
transcription
unit.
This
conclusion
is
based
on
the
following
evidence.
Four
alleles
(Stoner,
B5,
G
and
A{)
have
large
Sal
I
fragments
homologous
to
probe
2
(Fig.
2A).
In
addition,
probe
2
is
weakly
homologous
to
a
1.0-kb
Sal
I
fragment
of
Deletions
C
It
E
D
to
M
>I
M
10.8-
as
9.6r-
4
.
;'l
-9.4
5.2-
3.2-
**^
2.1-
*
4
1.
t
0.95-
0
0.80-
Probe
2
*
0.8-
*t
S
Probe
3
Probes
1,
2
+
3
FIG.
2.
Southern
blot
analysis
of
Wx
mutant
alleles.
DNA
from
13
homozygous
wx
alleles
were
digested
with
Sal
I,
electrophoresed
through
0.8%
agarose,
blotted,
and
hybridized
with
the
labeled
probe
shown
in
the
figure.
The
wild-type
control
included
on
each
blot
is
DNA
from
strain
HY
for
A
and
B
and
strains
W23
and
HY
for
C.
The
restriction
fragment
lengths
displayed
represent
average
values
of
many
gels.
In
C,
the
0.8-kb
fragment
of
the
BI
allele
did
not
transfer
well.
Similar
blots
of
BJ
DNA
clearly
reveal
this
fragment.
_1
0
fI
I
HY
I
lkb
VW23
Genetics:
Wessler
and
Varagona

4180
Genetics:
Wessler
and
Varagona
the
G
allele.
Three
other
mutant
alleles
(I,
K
and
B2)
possess
large
Sal
I
fragments
that
are
homologous
to
probe
3
(Fig.
2B).
For
each
allele,
multiple
restriction
digests
were
per-
formed
to
insure
that
the
altered
fragment
reflected
an
insertion
into
an
existing
fragment
rather
than
a
deletion
or
a
rearrangement
that
might
fuse
two
fragments
into
one
new
one.
For
the
probe
2
insertions
(Fig.
2A),
only
one
Sal
I
fragment
was
altered
[i.e.,
probe
3
hybridized
with
a
0.8-kb
fragment,
and
probe
1
was
homologous
in
each
case
to
a
fragment
that
corresponded
in
size
with
one
of
the
molecular
progenitors
(Table
1)].
Furthermore,
digestion
with
Sal
I/Pst
I
localized
each
insertion
to
either
the
5'
or
3'
side
of
probe
2
(Fig.
3).
The
probe
3
insertions
(Fig.
2B)
have
a
2.1-kb
Sal
I
fragment
homologous
to
probe
2,
and
the
size
of
DNA
fragments
hybridizing
with
probe
1
is
the
same
as
one
of
the
molecular
progenitors
(Table
1).
In
addition,
B2,
I
and
K
each
possess
a
Pst
I
fragment
homologous
with
probe
3
(Fig.
1D)
that
is
larger
than
its
Wx
counterpart
by
the
size
of
the
insertion
(data
not
shown).
These
data
are
summarized
in
Fig.
3.
Six
alleles
have
deletions
within
or
including
the
Wx
transcription
unit.
To
make
this
determination,
Sal
I
digests
of
genomic
DNA
isolated
from
strains
harboring
the
wx
alleles
B,
B1,
B6,
B7,
C4,
and
C34
were
blotted
and
hybrid-
ized
with
either
probes
1,
2,
or
3.
The
blot
in
Fig.
2C
was
hybridized
with
all
three
probes.
For
four
of
the
alleles,
we
can
identify
both
breakpoints
(B,
B1,
B6,
and
C4).
Both
probes
1
and
2
hybridized
to
the
5.2-kb
band
of
B
and
the
10.8-kb
band
of
BL.
When
a
Sal
I/Sst
I
digest
was
hybridized
with
probe
1,
we
found
that
the
5'
Sal
I-Sst
I
fragments
generated
were
intact
and
corresponded
with
a
particular
molecular
progenitor
(Table
1)
and
that
the
3'
Sal
I-Sst
I
fragment
was
fused
with
probe
2
sequences.
The
size
of
the
fused
fragment
permitted
an
estimate
of
the
deletion
size
for
both
B
and
B1
(Fig.
3).
The
B6
and
C4
deletions
have
both
breakpoints
in
the
2.1-kb
Sal
I
fragment.
A
Sal
I/Pst
I
digest
of
B,
B1,
B6,
and
C4
hybridized
with
probe
2
delimited
the
deletions
to
the
regions
shown
in
Fig.
3.
Only
the
5'
breakpoint
of
the
B7
allele
could
be
identified.
Both
Sal
I
(Fig.
2C)
and
Sal
I/Pst
I
digests
of
this
allele
indicated
that
the
Sal
Insertions
0
W<
to
5
rStoner
5.2
G
55
6.1
B2
0.15
I
and
K
'ml
4.5
'A
it
3.
a~
I~
it
C4
Ikb
B6
Deletions
....
B
BI
B7
C
34
FIG.
3.
Insertions
and
deletions
within
the
Wx
transcription
unit
in
13
mutant
Wx
alleles.
Except
for
the
B2
allele,
the
size
of
the
DNA
insertions
are
not
drawn
to
scale.
The
actual
length
of
these
inserts
are
shown
in
kb.
The
extent
of
deletions
are
depicted
by
stippled
regions.
Dotted
lines
indicate
uncertainty
in
the
precise
position
of
insertions
and
deletions
with
respect
to
the
restriction
sites
found
within
the
Wx
transcription
unit.
I
site
between
probes
1
and
2
is
intact.
Probe
2
had
weak
homology
with
a
Sal
I
fragment
of
-9.4
kb
(Fig.
2C),
which
is
probably
a
fusion
of
what
remains
of
probe
2
sequences
with
DNA
at
the
3'
end
of
the
deletion
(Fig.
3).
The
C34
allele
was
deleted
for
the
entire
Wx
gene
and
surrounding
DNA
(Figs.
2C
and
3).
The
cDNA
probe
pcWx0.4
[ref.
11;
-400
bp
in
length
and
homologous
with
the
3'
end
of
the
Wx
gene
(Fig.
1D)]
had
no
homology
with
alleles
B7
or
C34
(data
not
shown).
DISCUSSION
Our
analysis
of
stable
mutations
at
the
Wx
locus
was
complicated
by
two
factors:
(i)
the
lack
of
most
direct
progenitor
alleles
and
(ii)
the
presence
of
substantial
RFLP
in
the
maize
genome
(22,
23).
In
this
study
we
also
find
RFLP
when
the
Wx
locus
is
examined
in
many
inbred
lines.
However,
this
polymorphism
is
largely
confined
to
regions
outside
of
the
transcription
unit.
This
finding
was
essential
for
the
success
of
this
type
of
analysis
because
previous
genetic
and
biochemical
studies
(11,
12)
indicated
that
most
mutations
were
within
the
Wx
transcription
unit.
It
is
for
these
reasons
that
we
feel
confident
that
the
seven
insertions
and
six
deletions
found
associated
with
mutant
Wx
alleles
represent
molecular
lesions
rather
than
polymorphisms.
Is
it
possible
to
state
with
equal
confidence
that
the
lesions
detected
represent
the
primary
event
responsible
for
the
loss
of
Wx
expression?
Since
Wx
revertant
alleles
cannot
be
isolated
from
these
strains
[Nelson
(9)
estimates
that
the
rate
of
reversion
for
the
various
wx
alleles
ranges
from
0
to
2.7
x
l0-5],
it
is
conceivable
that
other,
less
obvious
alterations
are
also
present
at
the
locus.
To
address
this
question
we
have
compared
the
position
of
deletions
and
insertions
found
in
this
study
with
the
relative
position
of
these
alleles
on
the
fine-structure
genetic
map
of
the
locus
(9,
10).
Construction
of
the
genetic
map
was
primarily
based
on
a
determination
of
whether
the
Fls
of
wx
heteroalleles
exhibit
recombination,
thus
producing
a
functional
Wx
gene.
This
was
scored
by
determining
the
frequency
of
Wx
pollen
grains
produced
by
each
particular
wx
heteroallele.
The
relative
positions
of
the
Wx
mutations
as
deduced
in
this
manner
are
shown
in
Fig.
4.
Superimposed
upon
the
genetic
data
are
the
results
of
our
physical
analysis.
Under
each
allele
designation
is
the
nature
of
the
lesion
(as
determined
in
this
study)
and
the
ap-
proximate
position
within
the
transcription
unit
on
a
1
(5')
to
100
(3')
scale.
The
values
shown
for
insertions
represent
the
midpoints
of
the
target
restriction
fragments
(as
displayed
in
Fig.
3).
The
range
of
values
for
deletions
reflects
the
size
of
the
lesion
and
the
approximate
position
of
breakpoints.
We
also
have
included
in
this
analysis
three
transposable-element
insertion
mutations
that
have
been
characterized
at
the
molecular
level.
They
are
designated
m6
[wx-m6,
a
2.1-kb
Ds
element
insertion
(6,
11,
13)],
m8
[wx-m8,
a
2-kb
insertion
of
the
nonautonomous
element
of
the
suppressor-mutator
(Spm)-controlling
element
family
(7,
15)],
and
ml
[wx-ml,
a
392-bp
Ds
element
inserted
about
40
bp
5'
from
the
probe
2-3
junction
(Fig.
1D)
(ref.
5;
unpublished
data)].
It
was
possible
for
Nelson
to
map
these
elements
genetically
because,
as
nonautonomous
elements,
they
cannot
transpose
if
an
au-
tonomous
element
is
not
also
present
in
the
genome
(9).
It is
clear
from
Fig.
4
that
there
is
excellent
correlation
between
the
genetic
and
physical
maps.
Since
the
genetic
analysis
scores
the
restoration
of
Wx
gene
function,
these
data
strongly
suggest
that
the
lesions
we
have
characterized
are
responsible
for
the
mutant
phenotypes.
For
one
allele,
R,
the
results
of
our
study
are
inconsistent
with
the
extent
of
this
mutation
on
the
genetic
map.
Although
Nelson
found
that
R
failed
to
recombine
with
many
wx
alleles
(Fig.
4),
we
cannot
detect
any
obvious
lesion
for
this
allele.
Our
results
are
in
agreement
with
the
findings
of
Echt
and
Proc.
Natl.
Acad.
Sci.
USA
82
(1985)
3-.
we

Proc.
Natl.
Acad.
Sci.
USA
82
(1985)
4181
v
BZ
B
-(A)
7-12
o
90*
C
m6*
ml
(1)56
(D)71
88*
C4
BL2*
29-37
81
(A)6-14
M
(I)
33
C31+
Nk-
Stoner
(1)33
86
(A)23-43
m8*
MI79
82
(1)79
(1)(I
7
I
8
7
(A)
33
-100
C34
(A)
0-100
FIG.
4.
Correlation
between
the
genetic
map
and
the
physical
map
of
the
Wx
locus.
The
fine
structure
map
of
the
locus
is
that
of
Nelson
(9,
10).
It
is
constructed
as
a
complementation
map
such
that
any
two
mutants
do
not
recombine
if
their
horizontal
lines
overlap.
Lines
that
terminate
in
serations
indicate
that
there
is
no
mutant
more
distal
(or
proximal)
with
which
that
mutant
recombines.
Super-
imposed
on
these
horizontal
lines
are
the
molecular
nature
of
each
lesion
(I,
insertion;
A,
deletion)
and
the
relative
positions
of
the
lesions
found
associated
with
each
allele.
The
method
by
which
a
number
value
was
assigned
to
each
is
described
in
the
text.
V
(virescent)
and
Bz
(bronze)
are
genetic
loci
that
flank
the
Wx
locus.
+,
Alleles
that
are
indistinguishable
from
the
progenitor
alleles
by
Southern
analysis;
*,
mutations
caused
by
the
insertion
of
maize
transposable
elements.
Schwartz
(12),
who
analyzed
the
starch
granule-bound
pro-
teins
produced
by
this
strain.
This
allele
programs
the
synthesis
of
an
inactive
Wx
protein
of
correct
molecular
weight
(58
kDa)
but
altered
mobility
on
isoelectric-focusing
gels.
The
amount
of
protein
synthesized
is
comparable
with
nonmutant
strains.
Although
a
large
inversion
would
fail
to
recombine
with
many
wx
alleles
and
might
be
undetectable
by
our
Southern
analysis,
this
type
of
lesion
is
inconsistent
with
the
biochemical
data.
Two
of
the
wx
alleles
that
we
have
characterized
were
not
included
in
the
genetic
analysis
because
they
have
a
leaky
phenotype.
It
is
interesting
to
note
that
these
alleles,
G
and
B5,
have
large
insertions
within
the
Wx
gene
(Fig.
3).
We
have
determined
recently
that
strains
harboring
the
B5
allele
make
a
reduced
amount
of
a
wild-type-sized
transcript
(2.5
kb;
unpublished
data).
It
remains
to
be
determined
whether
this
element
is
in
intron
or
exon
sequences.
Spontaneous
mutations
in
Escherichia
coli
(25,
26),
yeast
(27)
and
Drosophila
melanogaster
(28,
29)
are
frequently
caused
by
DNA
insertions.
The
involvement
of
DNA
inser-
tions
in
unstable
plant
phenotypes
has
been
well
documented
(11,
13-15)
and
recently
reviewed
(16).
Far
less
is
known
about
the
molecular
lesions
responsible
for
stable
mutant
phenotypes
of
spontaneous
origin
in
plants.
To
our
knowl-
edge
in
only
one
instance
has
a
mutation
of
this
type
been
associated
with
DNA
insertions
or
rearrangements.
A
natu-
rally
occurring
mutant
allele
of
the
soybean
lectin
gene
contains
a
3.4-kb
insertion
that
has
the
structure
of
a
transposable
element
(30).
Of
the
17
mutant
alleles
of
spon-
taneous
origin
examined
in
this
study,
7
are
associated
with
insertions
and
5
with
deletions.
It
is
possible
that
2
of
the
insertions,
I
and
K,
are
the
same-both
have
4.5-kb
inser-
tions
into
the
same
region
(Fig.
3)
and
both
have
the
same
molecular
progenitor
(Table
1).
Only
5
alleles
have
lesions
that
must
be
smaller
than
our
limit
of
resolution
(50
bp).
These
results
suggest
that
gross
chromosomal
changes
make
up
a
large
proportion
of
spontaneous
mutations
in
maize.
The
existence
of
nonautonomous
transposable
elements
in
maize
poses
the
interesting
possibility
that
some
of
the
insertions
described
in
this
study
might
be
capable
of
trans-
position
when
crossed
with
a
strain
harboring
the
appropriate
autonomous
element.
The
Wx
insertion
mutations
have
been
crossed
with
strains
harboring
the
autonomous
elements
of
four
maize
controlling
element
families:
Ac-Ds,
Spm,
Bg,
and
Uq.
None
of
these
crosses
resulted
in
an
unstable
Wx
phenotype.
This
does
not,
of
course,
rule
out
the
possibility
that
these
elements
are
capable
of
transposition.
After
all,
they
did
at
one
time
transpose
into
the
Wx
locus.
They
may
represent
a
class
of
elements
activated
not
by
an
autonomous
element
but
possibly
by
stress
in
the
corn
field
(31,
32).
In
this
regard
they
may
have
a
similar
origin
to
insertions
we
find
associated
with
RFLP
outside
the
Wx
transcription
unit
in
alleles
38-11
and
W23
(Fig.
1D).
Finally,
these
insertions
may
be
defective
controlling
elements
no
longer
able
to
transpose.
The
answers
to
these
questions
await
the
cloning
and
detailed
characterization
of
these
mutations.
We
thank
Elizabeth
Oberthur
and
Dr.
Peter
Peterson
for
perform-
ing
crosses
with
our
insertion
mutations
and
the
Uq
controlling
element
system.
We
wish
to
express
our
gratitude
to
Dr.
Oliver
Nelson
for
valuable
discussions.
We
are
also
grateful
to
Ron
Okagaki
and
Drs.
George
Baran
and
Alan
Jaworski
for
discussions
and
comments
on
the
manuscript.
This
study
was
supported
by
National
Institutes
of
Health
Grant
GM32528
to
S.R.W.
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