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Genetic Uniformity in Two Populations of Drosophila melanogaster as Revealed by Filter Hybridization of Four-Nucleotide-Recognizing Restriction Enzyme Digests

Authors:

Abstract

A filter hybridization method is described for identifying restriction-site and insertion/deletion variation by using restriction enzymes that recognize four-nucleotide sequences and denaturing polyacrylamide gels for separating fragments. Eighty-seven lines of Drosophila melanogaster representing two natural populations were surveyed over a 2.7-kilobase region encompassing the alcohol dehydrogenase locus. Fifty distinct haplotypes were identified from 17 restriction-site and 11 insertion/deletion polymorphisms and from one allozyme polymorphism. There was no evidence for genetic differentiation between an East-Coast and a West-Coast (North American) sample. This technique has widespread applications in screening for DNA polymorphism.
Proc.
Natl.
Acad.
Sci.
USA
Vol.
83,
pp.
3562-3566,
May
1986
Population
Biology
Genetic
uniformity
in
two
populations
of
Drosophila
melanogaster
as
revealed
by
filter
hybridization
of four-nucleotide-recognizing
restriction
enzyme
digests
(polymorphism/restriction
polymorphism/insertion/deletion
variation)
MARTIN
KREITMAN*
AND
MONTSERRAT
AGUADOt
Museum
of
Comparative
Zoology
Laboratories,
Harvard
University,
Cambridge,
MA
02138
Communicated
by
C.
Clark
Cockerham,
December
19,
1985
ABSTRACT
A
filter
hybridization
method
is
described
for
identifying
restriction-site
and
insertion/deletion
variation
by
using
restriction
enzymes
that
recognize
four-nucleotide
se-
quences
and
denaturing
polyacrylamide
gels
for
separating
fragments.
Eighty-seven
lines
of
Drosophila
melanogaster
rep-
resenting
two
natural
populations
were
surveyed
over
a
2.7-
kilobase
region
encompassing
the
alcohol
dehydrogenase
locus.
Fifty
distinct
haplotypes
were
identified
from
17
restriction-site
and
11
insertion/deletion
polymorphisms
and
from
one
al-
lozyme
polymorphism.
There
was
no
evidence
for
genetic
differentiation
between
an
East-Coast
and
a
West-Coast
(North
American)
sample.
This
technique
has
widespread
applications
in
screening
for
DNA
polymorphism.
Two
techniques
dominate
attempts
to
identify
allelic
varia-
tion
at
the
DNA
level-Southern
blot
analysis
of
restriction
endonuclease
digestions
(1,
2)
and
direct
DNA
sequencing
(3).
However,
neither
technique
fully
satisfies
two
require-
ments
for
the
study
of
variation
in
natural
populations:
(i)
to
allow
sampling
of
a
large
number
of
alleles
and
(ii)
to
resolve
variable
sites
over
relatively
short
stretches
of
DNA.
The
problem
of
finding
nucleotide
polymorphisms
is
not
that
they
are
too
rare.
For
example,
in
his
sequence
survey
of
11
alleles
coding
for
alcohol
dehydrogenase
(Adh)
in
Drosophila
melanogaster,
Kreitman
(3)
identified
43
polymorphic
sites,
including
insertions/deletions
in
a
2.7-kilobase
(kb)
region.
However,
without
technical
innovation,
the
extension
of
this
work
to
population
surveys
has
remained
impractical.
One
such
innovation
that
allows
DNA
sequences
to
be
obtained
directly
from
genomic
DNA
(gDNA)
has
recently
been
reported
by
Church
and
Gilbert
(4).
In
this
paper,
a
modification
of
their
technique
is
described,
which
uses
enzymes
that
recognize
four-nucleotide
sequences
to
reveal
restriction-site
and
insertion/deletion
polymorphisms
within
small
regions
of
DNA.
We
have
applied
this
technique
to
estimate
the
extent
of
polymorphism
and
the
degree
of
genetic
divergence
at
the
Adh
structural
locus
in
two
popu-
lations
of
D.
melanogaster.
MATERIAL
AND
METHODS
Fly
Samples.
Isofemale
lines
were
established
from
wild-
caught
flies
collected
on
banana
bait.
One
sample
was
collected
at
Farmers
Market,
Raleigh,
NC
(October
1983)
and
one
at
Putah
Creek,
Davis,
CA
(October
1983).
Isochromo-
somal
lines
were
established
by
the
Curly
extraction
proce-
dure
using
SM5/B1L2
as
extractor
stock
(5).
Adh
allozyme
phenotype
was
determined
as
described
by
Kreitman
(6).
DNA
Preparation
and
Electrophoresis.
Total
nucleic
acid
was
extracted
from
approximately
0.5
g
of
frozen
(-70°C)
adult
flies
by
cell
lysis
with
potassium
acetate/NaDodSO4
and
extraction
with
phenol
(7).
Two
to
three
micrograms
of
gDNA
(estimated
from
ethidium
bromide-stained
agarose
gels
containing
total
nucleic
acid)
were
digested
first
for
4-6
hr
with
the
following
enzymes:
Alu
I,
Ban
I,
Dde
I/BamHI,
Hae
III,
Hha
I,
Msp
I,
Sau3A,
Sau96
I,
or
Taq
I;
then
the
samples
were
digested
for
0.5-1
hr
with
RNase
I
(100
pg/ml).
Samples
were
precipitated
with
ethanol,
washed,
dried
under
reduced
pressure,
and
resuspended
in
3
A.l
of
formamide
loading
buffer
(94%
formamide/0.05%
xylene
cyanol/0.05%
bromophenol
blue/10
mM
Na2EDTA,
pH
7.2).
After
incu-
bation
in
a
boiling
water
bath
or
90TC
dry
bath
for
5-10
min,
1.5-2.0
,ul
of
each
sample
was
loaded
with
a
Hamilton
syringe
onto
a
standard
30
cm
x
40
cm
x
0.4
mm
5%
polyacryla-
mide/7
M
urea
buffer
gradient
DNA
sequencing
gel
(8).
The
buffer
gradient
was
50-500
mM
in
Tris
borate/EDTA,
pH
8.3.
Fifty
to
sixty
samples
were
loaded
on
one
gel
in
0.35-mm
sharks'
teeth-comb-formed
slots.
Gels
were
run
at
1200-1300
V
until
the
bromophenol
blue
reached
35-40
cm
from
the
origin.
Electrophoretic
transfer
of
DNA
from
the
gel
to
New
England
Nuclear/DuPont
GeneScreen
(NEF
972)
and
sub-
sequent
UV
crosslinking
was
performed
as
described
in
(4).
Probe
Preparation.
A
2.7-kb
gel-purified
Sal
I-Cla
I
DNA
fragment
containing
the
Adh
structural
locus
(3)
was
nick-
translated
by
using
3000
or
5000
Ci
(1
Ci
=
37
GBq)
per
mmol
of
[a-32P]dATP
(DuPont/New
England
Nuclear)
to
a
specific
activity
of
8-10
x
108
cpm/Ag
of
DNA
(9).
The
probe
was
boiled
for
5-10
min
before
being
added
to
prewarmed
(65°C)
hybridization
buffer
(1%
crystalline
grade
bovine
serum
albumin
/1
mM
EDTA/0.5
M
sodium
phosphate,
pH
7.2/7%
NaDodSO4).
Hybridization/Wash.
Prehybridization/hybridization
was
at
55-65°C
either
using
polyethylene/polyester-laminated
bags
as
described
(4)
(5-10
x
107
cpm
in
12
ml
of
hybridiza-
tion
buffer)
or
more
recently
as
follows:
one
to
five
30
x
40
cm
wet
(50
mM
Tris
borate/10
mM
EDTA,
pH
8.3)
filters
placed
on
top
of
one
another
were
rolled
tightly
around
a
10-ml
sterile
disposable
pipette
and
placed
in
a
35
x
2.54
cm
(i.d.)
polycarbonate
(Lexan)
tube
sealed
at
one
end.
Approx-
imately
20
,ul
of
hybridization
buffer
(65°C)
per
cm
was
added,
and
the
open
end
of
the
tube
was
sealed
with
a
rubber
cork.
The
tube
was
placed
on
a
modified
Wheaton
tissue
culture
roller
set
at
5
rpm
in
an
incubator
at
55-600C
for
a
minimum
of
5
min.
This
solution
was
replaced
with
12-18
ml
of
hybridization
buffer
containing
5-50
x
107
cpm
of
probe
prewarmed
to
65°C.
It
is
important
to
orient
the
filters
so
they
"unwind"
when
rolling
against
the
inner
surface
of
the
tube
Abbreviations:
bp,
base
pair(s);
gDNA,
genomic
DNA;
Adh,
alcohol
dehydrogenase;
Adhs
and
AdhF,
isozymes
whose
relative
migration
is
slow
and
fast
in
electrophoresis.
*Current
address:
National
Institute
of
Environmental
Health
Sci-
ences,
P.O.
Box
12233,
Research
Triangle
Park,
NC
27709.
tPermanent
address:
Departament
de
Genetica, Universitat
de
Barcelona,
Av.
Diagonal,
645,
08028
Barcelona,
Spain.
3562
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.
Proc.
Natl.
Acad.
Sci.
USA
83
(1986)
3563
939
-
650
-
A/u
Hha
Dde
Hae
III
12345
123451234512345
aI
m0
0
284
-
280
-
168-_
164
-
_
69-
_
FIG.
1.
Autoradiograph
of
five
D.
melanogaster
DNA
samples
digested
with
four
restriction
enzymes
and
probed
with
a
2.7-kb
homologous
probe
encompassing
the
Adh
locus.
The
range
of
fragment
sizes
in
bp
is
shown
at
the
left.
For
some
fragments
(e.g.,
164/168
bp),
the
two
DNA
strands
have
slightly
different
electro-
phoretic
mobilities.
A
4-bp
insertion/deletion
difference
in
sample
3
can
be
easily
seen
in
the
Hha
I
(164-168
bp)
fragments.
A
1-bp
insertion/deletion
difference
between
samples
2
and
3
can
be
seen
in
the
Hae
III
(283-284
bp)
fragments.
and
remain
stationary
relative
to
the
tube.
Hybridization
was
for
12-18
hr.
The
filters
were
rinsed
several
times
with
prewarmed
(570C)
wash
solution
(1
mM
Na2EDTA/40
mM
sodium
phosphate/1%
NaDodSO4)
after
first
removing
the
probe
solution
from
the
tube.
Filters
were
then
transferred
to
a
large
tub
and
washed
on
a
rotary
shaker
either'eight
times
for
5
minii
(each
wash
at
room
temperature)
or'
three
times
for
30
min
(each
wash
at
570C;
1500-2000
ml
of
prewarmed
washsolu-
tion
was
used
for
each
wash
for
four
to
eight
filters).
After
the
last
wash,
filters
were
blotted
on
Whatman
3
MM
paper
to
remove
excess
liquid,
placed
in
SaranWrap
or
Seal-a-Meal
bags,
and
autoradiographed
for
2-4
days
on
Kodak
XAR
film
with
one
intensifying
screen.
RESULTS
Fig.
1
shows
an
autoradiograph
of
five
DNA
samples,
each
digested
with
Alu
I,
Dde
I,
Hae
III,
and
Hha
I;
a
2.7-kb
Adh
probe
was
used.
We
were
able
to
reliably
score
fragments
as
small
as
60-70
base
pairs
(bp)
after
3-day
exposures
and
insertion/deletion
differences
of only
one
base.
More
than
90%
of
the
four-nucleotide-recognizing
restriction
enzyme
sites
and
essentially
every
insertion/deletion
difference
could
be
detected.
In
a
random
sequence,
restriction
enzymes
that
recognize
four-nucleotide
sequences
are
expected
to
cut,
on
average,
every
256
bp,
a
16-fold
improvement
over
those
that
recog-
nize
six-nucleotide
sequences.
Knowledge
of
the
complete
sequence
of
the
2.7-kb
Adh
probe
(3)
allows
direct
calculation
of
the
fraction
of
all
possible
base
changes
'that
would
be
detected
in
this
sequence
given
any
constellation
of
enzymes.
Assuming
that
all
changes
are
equally
likely,
'we
estimate
that
l19%
of
all
possible
changes
would
be
detected
(discounting
sites
producing
fragments
that
would
be
too
small
to
score-
e.g.,
<
70
bp)
with
the
10
enzymes
used
in
this
study.
Thus,
all
changes
at
526
"site
equivalents"
(0.19
x
2723)
are
detectable
in
addition
to
all
insertion/deletion
differences
within
the
probed
region.
Fig.
2
shows
the
distribution
of
polymorphic
sites
in
a
sample
of
87
isochromosomal
lines:
27
from
Putah
Creek
and
60
from
Raleigh.
Not
including
the
allozyme
polymorphism,
a
total
of
28
polymorphisms
were
scored
representing
17
restriction
sites
and
11
insertions/deletions.
Two
restriction
sites
(Alu
I:
1068
and
Dde
I:
1551)
and
two
insertions/deletions
(655-685
and
-3320)
appear
only'once
in
the
87
lines.
The
remaining
polymorphisms
are
multiply
represented.
Fig.
3
shows
the
restriction,
insertion/deletion,
and
al-
lozyme
haplotypes
for
the
87
lines.
Three
insertion/deletion
sites
are
assigned
more
than
two
lengths.
Site
29
contains
insertions/deletions
located
3'
to
the
probed
region
and
were
detected
only
in
Dde
I
digestions.
It
is
likely
that
these
"alleles"
occur
at
more
than
one
position
within
the
approx-
imately
1000-bp
fragment,
and
some
may
be
combinations
of
two
or
more
other
insertions/deletions.
The
other
two
multiallelic
sites,
sites
23
and
26,
are'
likely
to
represent
variation
in
the
lengths
of
homonucleotide
sequences
(3).
The
sample
of
87
alleles
contains
a
total
of
50
haplotypes,
37
in
the
larger
Raleigh
sample
(n
=
60)
and
22
in
the
Putah
D
NONTRANSLATED
0
TRANSLATED
2
3
679
12
23
25
26
I
4 58
11
13
14
15
16
17-22
24
27
I0
-500
1
500
28
29
-A
AA
VV
3'
1000
1500
2000
2500
FIG.
2.
Distribution
of
29
polymorphic
sites.
Restriction
sites
are
shown
below
the
line
and
insertions/deletions
are
shown
above
the
line.
Insertions
are
numbered
3,
6,
7,
9,
12,
23,
25,
26;
deletions
are
numbered
2,
26,
28,
29.
Precise
locations
are
given
in
Table
2.
Population
Biology:
Kreitman
and
Aguadd
I
I
I
I
I
I
3564
Population
Biology:
Kreitman
and
Aguade
Ep.
1
SITE
NUMBER
10
1:
+
-
-
+
- -
+
-
-
+
+
-
++++
S
+
-
2:
+
----
+-+
-+
+
-
+-
+
+
+
F
+
_
3:
+
- - - -
-
+
-
+
+
-
+
-
+
+
S
-
+
4:
+----
-+-
+
+
-
+
-
+
+
F
+
_
5:
+
- -
+
-
-
+
+
-
+
+
-
++
++
S
+
-
6:
+
---+
+
+
-
+
+
-
+
-
+
+
F
+
_
7:
+
-
-
-
- -
+
+
-
+
+
-
+++
+
S
-
+
8:
+
----.
+ +
+
+
+
.
+ + +
+
F
+
-
9:
+-----+
+
-
-
+---
+
+
S
-
+
10:
+
----
+++
-
+
+
-
+
-
+
+
F
+
_
11:
+----+
+
-
-+
+-
-
+++
F
+-
12:
+--
--+
+ +
+
+
+-
+
+
+
F
+-
13:
+
- -
+
-
-
+
+
-
+
+
-
++ ++
S
+
-
14:
+
- -
+
-
-
+
+ _
+
+
_
+
-
+
+
F
+
-
15:
+
---
+ +
++
+
++++
+
+
++
F
+
-
16:
-
--
+
-
-
+ +
-
+
+
-
+
-
+
+
S
-
+
17:
+-.----+
+-+
-
-
-
--
F
+_
18:
+
----
+++
-
+
--
-
-
+
+
-
F
+
-
19:
+----
+
+
++++
-
+
+
+
F
+
_
20:
+
.
+
+
-
-
-+
-
--
-+
+
S
-
+
21:
+---
+
+
+-
+
+
-
++++
F
+
-
22:
+
--
-
-
-+
-
+
+
-
+
-
+
+
F
+
-
23:
+
----+
+
-
-+
+
-
+
-
+
+
F
+
-
24:
+
-
-
+
-
--
-
+
-
+
+
-
+-+
+
+
S
+
-
25:
+
-
- -
-
-
+
-
- -
+
-
-
--
-+
S
-
+
26:
+
-
-
+
+
+ +
+
S
+
27:
+
+
-
+
--
-
+
-
+
+
-
+
+.+
+
S
+
-
28:
+
-
-
+ -
-
+ +
-
+ +
-
+
-
+
-
S
-
+
29:
+
-
-
+
-
-+
+
-
+
+
-
++
+ +
F
+
-
30:
+
.
+
+
-
-
-+
-
--
-+
+
S
+
-
31:
+
-
-
+
-
--
-+
-
+
+
-
+-+
+
+
S
+
-
32:
+
----+
+
+
+
-
+
_
+
+
F
+
-
33:
+
-
-
+ -
-
+ +
-
+
+
-
+
-
+
+
S
-
+
34:
+
-
-
-
- -
- -
-
-
-
-
-
-
- -
S
+
35:
- -
+
---
+ +
-
+
+
-
+
-
+
+
S
+
-
36:
+
-
-
+
-
-
+
+
-
+
+
-
+
-
+
+
S
+
-
37:
+
-
-
+
-
-
+ +
-
+
+
- +
-
+
+
S
+
-
38:
+
----
+-+
-
+
+
-
+
+-+
+
F
+
-
39:
+
-
-
+
-
-
+ +
-
+
+
-
+
-
+ +
S
+
-
40:
+
--+--+
+
-+
+
_
+
_
+
+
F
+
-
41:
-
-
+
---
+
+
-
+
+
-
+
-
+
+
S
+
-
42:
+
-
- -
-
-
+
-
+
+
-
+
-
+
+
S
-
+
43:
+-
-
+
- -
+ +
-
+
+
-
+
-
+
+
S
-
+
44:
+
+
-
+
---
+
-
+
+
-+
++
+
S
+
-
45:
+
-++
+++
-
+
+
_
+
_
+
+
F
+
_
16:
+
-
-
+ +
-
+ +
-
+
+
-
+
-
+
+
S
+
-
47:
+
-
-
- -
-
+
-
+
+
-
+
+-+
+
S
+
-
48:
+
-
+
-
-
-
+
+
- -
+
-
--
-+
+
S
-
+
49:
+
-
-
+
-
-
+
+
-
-
+
++
+++
S
+
-
50:
+
+
+
+
+
+
_+
_
+
+
F
+
-
Proc.
Natl.
Acad.
Sci.
USA
83
(1986)
OE.
20
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+4+
-+-+
-+-+
-4--
-4_4+
-4_4+
4+-
4+-
4+-
4+-
4+-
4+-
3+-
4+-
4+-
4+-
44-
4+-
44-
4+-
5+-
-1
+
-
4+-
44-
4+-
_3+_
4-
4+-
5
+-
5+-
4+-
4+-
-4--_
5--
44+--
-4+4-
-4--_
34+--
4+
-
4+
-
5+
-
5+
-
3+
-
4+
-
-4--_
3--
4--
44+--
44+--
4+
-
-4+--
4+
-
4+
-
29
_+
_
-+
4
_+
4
-+
-
_+
4
_+
2
_+
3
-+4--
4+---
-1
+
-
2
_
+
_ _
-1
+_ _
-1
+
-
3
+1
+_ _
+1
+
-
_
+1
+
-
_
-1
+
_ _
+
+
2
+-4--
-4---
-4---
-4---
-4---_
Ra
0
0
0
0
8
1
3
1
0
0
0
1
0
0
0
2
1
1
0
0
0
5
1
2
1
2
1
1
2
1
1
1
1
1
6
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
Pu
1
1
1
1
2
2
2
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
FIG.
3.
Fifty
haplotypes
(Hap.)
identified
from
29
polymorphisms
in
87
lines.
Frequencies
(Freq.)
in
the
two
samples
(Ra,
Raleigh;
Pu,
Putah
Creek)
are
shown
to
the
right
of
the
haplotypes.
Creek
sample
(n
=
27).
There
is
an
average
of
5.75
differences
between
alleles
for
the
combined
sample
of
87,
including
restriction-site
and
insertion/deletion
polymorphisms
(Table
1).
The
two
populations
are
strikingly
similar
in
this
respect,
with
the
Raleigh
sample
having
5.70
and
the
Putah
Creek
sample
having
5.45
average
differences
between
alleles.
Table
1
also
provides
a
summary
for
the
two
Adh
al-
lozymes.
Consistent
with
previous
evidence
suggesting
a
more
recent
ancestry
for
the
AdhF
allele
(3,
10),
the
Adhs
allele
(average
no.
of
differences
=
3.74)
has
more
than
twice
the
average
number
of
restriction-site
differences
compared
to
AdhF
(average
no.
of
differences
=
1.50).
Surprisingly
though,
the
average
number
of
insertions/deletions
are
essentially
identical
(1.71
and
1.69
for
Adhs
and
AdhF,
respectively).
Table
2
gives
the
individual
frequencies
for
each
of
the
29
polymorphic
sites
in
the
two
populations.
By
using
Fisher's
exact
probability
test
to
compare
frequencies,
only
two
sites,
6
and
17,
are
significant
at
the
5%
level,
and
one
site,
29,
is
significant
at
the
1%
level.
As
described
above,
this
latter
site
may
conflate
insertions/deletions
at
several
different
sites.
Site
17
is
the
AdhF/Adhs
allozyme
polymorphism.
The
frequency
of
AdhS
is
higher
in
the
Raleigh
sample
(70%)
than
in
the
Putah
Creek
sample
(40%).
Other
than
these
two
sites,
only
one
additional
site
shows
a
significantly
different
fre-
quency
in
the
two
populations.
Since,
at
the
5%
significance
level,
1
in
20
sites
are
expected
to
be
significant
under
the
null
hypothesis
of
no
difference,
the
similarity
of
frequencies
at
26
of
29
sites
offers
clear
evidence
for
genetic
homogeneity
of
the
two
populations.
Comparison
of
haplotype
frequencies
in
the
two
population
samples
is
a
potentially
more
powerful
method
for
identifying
genetic
differences.
The
distribution
of
87
alleles
among
the
50
distinct
haplotypes
in
the
two
populations
is
shown
in Fig.
Proc.
Natl.
Acad.
Sci.
USA
83
(1986)
3565
Table
1.
Adh
locus
restriction-site
and
insertion/deletion
(Ins/Del)
haplotype
variation
in
87
lines
Type
of
polymorphism
Restriction
Restriction
Ins/del
Sample
+
ins/del
only
only
Ra
+
Pu
(n
=
87)
No.
of
haplotypes
50
30
30
Mean
differences
5.75
3.65
2.1
Haplotype
diversity
0.96
0.93
0.89
Ra
(n
=
60)
No.
of
haplotypes
37
24
26
Mean
differences
5.70
3.60
2.10
Haplotype
diversity
0.95 0.92
0.90
Pu
(n
=
27)
No.
of
haplotypes
22
15
12
Mean
differences
5.54
3.61
1.93
Haplotype
diversity
0.95
0.90
0.87
Adh5
(n
=
53)
No.
of
haplotypes
29
20
19
Mean
differences
5.45
3.74
1.71
Haplotype
diversity
0.92
0.90
0.86
AdhF
(n
=
34)
No.
of
haplotypes
21
10
13
Mean
differences
3.19
1.50
1.69
Haplotype
diversity
0.93
0.80
0.84
The
average
number
of
differences
(Mean
differences)
is
the
number
of
sites
segregating
between
two
alleles.
Haplotype
diversity
i5
calculated
as
1
-
S(p1)2
where
pi
is
the
frequency
of
the
ith
haplotype.
Ra,
Raleigh;
Pu,
Putah
Creek;
AdAF,
allele
for
alcohol
dehydrogenase-fast,
isozymes
that
migrate
faster
in
electrophoresis
than
isozymes
specified
by
Adhs,
the
allele
for
alcohol
dehydrogen-
ase-slow.
3.
The
distribution
is
strongly
skewed-35
of
the
50
haplo-
types
are
represented
only
once;
14
haplotypes
are
multiply
represented,
9
of
which
are
present
in
both
population
samples.
There
is
no
significant
difference
between
the
distribution
of
haplotypes
in
the
two
samples
(P
>
0.1).
The
third
most
abundant
haplotype,
35,
is
found
only
in
the
Raleigh
sample.
However,
because
this
haplotype
is
an
Adhs
allele,
and
the
number
of
slow
alleles
is
so
much
greater
in
the
Raleigh
sample
(42
vs.
11),
it
is
not
unexpected
that
some
Adhs
haplotypes
would
have
no
Putah
Creek
representatives.
Therefore,
taking
the
Adh
allozyme
frequency
difference
into
account,
there
is
a
striking
similarity
in
the
pattern
of
haplotype
representation
in
the
two
samples.
This
offers
additional
support
for
genetic
homogeneity
of
the
two
pop-
ulations.
DISCUSSION
Technical
Considerations.
In
preparing
the
gels,
we
used
a
steep
buffer
gradient
to
allow
greater
migration
of
large
fragments
without
losing
small
ones.
Five
percent
polyacryl-
amide
gels
provide
adequate
resolution
of
small
fragments
and
reasonable
separation
of
fragments
up
to
1000
bp
in
length.
We
noticed
a
tendency
for
enzyme
digestions
pro-
ducing
an
excess
of
large
fragments
(e.g.,
digestions
with
enzymes
that
recognize
a
six-nucleotide
sequence)
to
resolve
poorly,
possibly
a
result
of
the
poor
entry
of
large
fragments
into
the
gel
matrix.
We
detected
no
significant
increase
in
32P-labeled
filter
background
when
probing
from
one
to
five
filters
in
as
high
as
5
x
108
cpm/18
ml
of
hybridization
solution.
The
use
of
Lexan
tubes
for
hybridization
makes
the
technique
simpler,
safer,
and
less
expensive
(requires
less
probe
per
filter).
We
noticed
no
difference
in
signal
for
single
filters
hybridized
in
bags
or
from
one
to
five
filters
simulta-
neously
hybridized
in
one
tube.
This
allows
hybridization
of
up
to
20
filters
at
a
time
in
four
tubes.
Applicability.
The
technique
with
restriction
enzymes
that
recognize
a
four-nucleotide
sequence
should
be
useful
not
only
in
evolutionary
studies
but
also
in
identifying
specific
haplotypes
associated
with
genetic
disease.
In
our
study,
most
of
the
variable
restriction
sites
and
all
of
the
inser-
tion/deletion
sites
were
detected
by
using
only
three
or
four
enzymes.
The
ability
to
identify
small
insertions/deletions
using
only
a
few
enzymes
may
be
of
particular
value
for
genetic
screening,
since
this
type
of
mutation
is
abundant
and
in
some
cases
is
"multiallelic"
(e.g.,
site
23).
Population
Survey.
Comparison
of
the
87
lines
reported
in
this
study
with
11
sequenced
Adh
alleles
from
a
world-wide
collection
(3)
provides
some
evidence
for
genetic
differenti-
ation
of
populations.
None
of
the
10
restriction-site
and
insertion/deletion
haplotypes
predicted
from
the
sequences
of
the
11
alleles
are
exactly
represented
in
the
sample
of