Vol. 83, No. 3, 1978 August 14,1978
BIOCHEMICAL
RESTRICTION
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1125-1131
ENDONUCLEASE ANALYSIS OF
HUMAN GLOBIN GENES IN CELLULAR DNA Saul
Surrey,
Jill
S. Chambers, Elias
and
of Hematology Hospital
Philadelphia,
Received
Muni
Schwartz
Division The Children's
Diane
of Philadelphia
Pennsylvania
19104
June 22,1978
Summary: Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hint II, Barn HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were tra sferred to nitro-cellulose filters and identified by hybridization to [ 3Y P]cDNA for total adult globin mRNA. The a-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous a-thalassemia, a genetic disorder due to deletion of a-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated a-globin gene loci. The inherited are
characterized
globin
chains
radioactive that
group
by absent of hemoglobin
globin
with
globin
and
globin
genes which [2-61.
of the
chain,
The development restriction provides
[l].
studies
Recent
for
lack
a new approach
techniques
have been
and rabbit
globin
digestion
used for
DNA sequences
using
of DNA sequences
genes localizing
and subsequent
there
8-chain
sizing,
[g-11].
Using
of
have
indicated
of fetal for
hemo-
specific
gel
is
structural prosynthesis [6,7].
DNA sequences
by
electrophoresis diseases.
mapping similar
absent
are present
specific
of human genetic
detection,
of the
of the corresponding
where for
syndromes
hybridization
persistence
of production
to the study
thalassemia
of one or more
hereditary
the structural for
the
DNA in solution
in Do- thalassemia,
of methods
endonuclease
cellular
have deletions
account
In contrast,
8-globin
synthesis
a-thalassemia,
8-thalassemia
called
or decreased
cDNA to sheared
some patients
teins
of human disorders
[8] These
and cloning methods,
of mouse
we have
com-
0006-291X/78/0833-1125$01.00/0
1125
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 83, No. 3, 1978
pared
restriction
BIOCHEMICAL
endonuclease
infant
with
hydrops
using
several
globin
genes
zygous
a-thalassemia.
fetalis
AND BIOPHYSICAL
digests
of normal
due to homozygous
restriction
endonucleases
and demonstrate
MATERIALS
human DNA to DNA from an
a-thalassemia.
identify
specific
RESEARCH COMMUNICATIONS
deletion
Our studies
the fragments of a-gene
containing
fragments
in homo-
AND METHODS
Globin mRNA was prepared using peripheral blood containing 60 per cent reticulocytes from a 23 year old man with pyruvate kinase deficiency. Globin synthesis studies [12] of his reticulocytes showed equal a- and B-chain production. The globin mRNA was prepared by a sequence of acid precipitation, phenol-chloroform extraction, double passage through oligo-dT cellulose and sucrose density gradient centrifugation, using modifications of methods previously described [13]. The resulting poly[A] containing 9-11s RNA was used as a template for oligo(dT) -primed synthesis of radioactive cDNA by reverse transcriptase from av&'iyeloblastosis virus [14], with [32P]GTP(23000 Ci/mmole; Amersham Corp., Illinois) as the source of radioactivity. High molecular weight DNA was isolated [15] from the spleen of a fetus with hydrops fetalis due to homozygous a-thalassemia and from spleens removed from children with hemolytic anemia. The DNA was cleaved with an excess of a specific restriction endonuclease in each reaction at 37'C for 24 hours, using buffer conditions described by the enzyme suppliers (New England Biolabs, Solutions containing lo-20 ug of Inc. and Bethesda Research Labs, Inc.). DNA fragments in 20 ul aliquots were separated by electrophoresis in a 1% neutral, horizontal agarose gel employing a tris-acetate buffer system [16]. Electrophoresis was performed at 20 milliamps constant current for 16-18 The DNA samples were placed so that normal hours at room temperature. and a-thalassemia samples treated with the same enzyme were in adjacent wells. Restriction endonuclease fragments from A and 9x174 phage DNA were used as molecular weight markers. Following electrophoresis the DNA fragments were denatured with alkali, neutralized and transferred to a cellulose nitrate filter as described bx Southern [8]. The DNA on the filters was annealed for 24-48 hours at 65 C with 1 to 5ng/ml of high specific activity globin cDNA, and the filters were subsequently washed [8]. Radioactive bands were located by radioautography with Kodak XRP-1 film and DuPont Cronex Hi-Speed intensifying screens. RESULTS AND DISCUSSION Since
analysis
of a-globin Specific but
genes. after
DNA sequences fragments
absent
Figure digestion
I,
found
1 shows with
in restriction
the patterns
various
not with
solution
has demonstrated
a-thalassemia
[2,3],
endonuclease
digests
DNA may be identified
and controls but
in
in homozygous
in a-thalassemia
a-thalassemia and Alu
of hybridization
endonucleases. were
Hae III.
found
with
A single
1126
any globin of normal
as containing
of globin
specific Distinct Eco RI, 20.5
differences
kb a-globin
III,
DNA
a-globin
fragments
Hind
deletion
Hint
obtained between II,
DNA fragment
Barn HI
BIOCHEMICAL
Vol. 83, No. 3, 1978
Figure
1.
in
pattern.
the Eco RI digest
This
finding
two a-chain In that
DNA [17]. to their
differs
fragments, report
radioactive
on neutral
resolved
problem.
this
system.
of normal from
gels
state close
and that
We observed study
absent
described
8.7 and 7.8 kb,
was found
In the previous
DNA but
that
the authors
probe
was performed
gel
RESEARCH COMMUNICATIONS
Autoradiographic detection of globin genes in restriction endonuclease digests of human DNA. DNA from a control (C) and a fetus with homozygous e-thalassemia (H) was cleaved with various restriction endonucleases (Eco RI, Hind III, Barn HI, Alu I and Hae III). The resultant DNA fragments were separated by electrophoresis on a 1% agarose gel [16] and transferred to cellulose nitrate filters as described by Southern cDNA against globin mRNA from an adult was used 181. Radioactive for detection of DNA fragments containing globin gene sequences as described in Materials and Methods. The DNA specimens on the electrophoretic strip on the right are from undigested samples.
is present
noted
AND BIOPHYSICAL
in the a-thalassemia
in a recent
in Eco RI digests that
a large
to the origin
[17]
1127
a large
amount
which
of normal
amount
of DNA hybridizing
when electrophoresis
the use of an alkaline
no such difficulties
report
gel
system
in employing of low molecular
a neutral weight
Vol. 83, No. 3, 1978
material
BIOCHEMICAL
containing
the presence present
globin
of contaminant
study
only
the high
localization
degradation
genes
lanes
a-chain
possibility
an RNA linker
DNA with the
that
an RNA linker
RNA
would
RNA sites
been previously
has
(Fig.
thalassemia 3.2,
1),
1.7 and 0.9 a clear
normal
the
to more u-globin
results
of cDNA-driven
studied
have
our finding
Since
Hind
III
of the
hybridization
the two a-globin
loci
to
the pres-
[17].
Another
kb Eco RI fragment. gel
The existence
from
mammalian
cells
III
than
the patterns (4.9,
4.1
I (1.6,
two Hind
of a-globin in
kb),
Hint
a-globin
indicating
1203 indicate likely
fragments
is
the 4.1 kb band
other and a(3.7, There
DNA bands
of genetic
The most
II
1.3 kb).
kb band,
A variety
with of normal
1.4,
III
the 4.9
loci.
sequences
digestion
hybrid-
data
[19]
that
most humans
explanation that
than
in
in
there
and
for are
of double-stranded
a-globin
cDNA [21,22],
are
therefore
approximately
4.1 kb apart,
measured
The enzyme
while
we have
only
identified
off
the gel
(less
should
produce
two. than
about
at least
The third 500 bp),
1128
three
may have or it
a-globin been
small
may overlap
twice
the 4.9 band.
center
centers.
If
fractionation,
at the
their
migrate
samples
for
to agarose
after
experiments
of a-globin intensity
in
and Alu
cDNA molecules.
a-globin cuts
Hind
in intensity
of unequal
as many separate
found
kb)
duplication
prior
in
the
relate
study
two DNA fragments.
by differences
4.1 much darker
ization
which
may account
DNA isolated
Barn HI (14.5
difference
from
the undigested
the 20.5
alkali
gene fragments
the following:
kb),
DNA, with
with
apparent
recent
seen
the present
in methods
in
were
the
[18].
identified
DNA, are
present
in mitochondrial
a-globin
in
in this
to generate
reported
The specific enzymes
treated
be hydrolyzed
of alkali-labile
is
were
is
procedure
found
is
in
digestion
variations
fragments
in
gene sequences
In addition,
experimental
suggesting
fragmentation;
to the origin
These
ence of two smaller
the Eco RI digests,
globin
of DNA before adjacent
the
RESEARCH COMMUNICATIONS
or alkali-induced
containing
on right).
of DNA during
is
in
1, Eco RI lanes). weight
of globin
1, "DNA"
(Fig.
bands
(Fig.
molecular
was observed
nucleases
discrete
the Eco RI digests study
genes
AND BIOPHYSICAL
from
DNA bands, enough other
to
globin
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DNA bands. The identification hybridization
between
y-globin.
This
amounts,
with
sequence
bridization
is
amino
less
studies
present contain fragments
using
a partially
less
intense
bands
(10.2,
of 1.8,
1.3,
different semia.
4.9,
in
studies
facilitate
Hind
1.9 kb)
III I (2.3,
sequences of a-, partial
1.0,
0.9
5.9,
3.9
and 2.4 kb DNA preB- and reported
3.0 kb)[17].
The [Fig.11 genes,
to multiple
represent Hint
kb).
in both
of splenic to globin
corresponding too short
S- and 6- gene purification
Because
recently
4.5,
[23-241
II
Since
copy
rDNA
B- and g-globin (5.7,
1.5 kb),
Hae III
fragments
DNA preparations,
of a-globin
hybridizing
are
y-chains,
B-cDNA to y-globin
which
hy-
they
must
DNA sequences.
deletion
of material
to those
8.2 kb),
present
enzyme digests
new fragments
$- and
The putative
(6.6,
amino
to S- and g-genes
in the Eco RI DNA digests
digests (17.2,
B- or g-chain
demonstrate
the deletion
identification
in size
cDNA probe
the other
kb) and Alu
at least
or the remaining
similar
cDNA contaminants
bands
extensive
and a-thalassemia
or both.
of ribosomal
2.1
Since
genes,
2.8,
is
equal
and cDNA-driven
DNA, each of the
of radioactive
restriction
digests,
will
(7.6,
1.0 and 0.9 kb were
each contain These
are
there
between
normal
6- and
mRNA from
cross-hybridization[13].
of both
B-globin
the following:
Barn HI
slight
cross-hybridization
The major are
here
purified
or hybridization
genes
hybridize
homology
for
globin
However,
B-cDNA will
S- or 6-globin
detected
may represent
genes.
show only
by cross-
in approximately
chains,
sequence
the Eco RI digests either
g-gene
acid
total
&-globin
B- and that
complicated
molecules
mRNA.
of B- and &globin
in
using
a- and B-chain
indicate
is
cDNA and DNA sequences
was prepared
between
of cross-hybridization
sumably
study
reactions
and hybridization
bands
B-globin
or no y- or A-chain
homology
There
k51.
this
mRNA contains little
DNA fragments
radioactive
The cDNA in
an adult.
acid
of 8-globin
gene sequences
DNA from
to be detected
fragments
in these
of the fragments
1129
homozygous
cDNA did to a-chain
in several
not
a-thalas-
appear
mRNA is by these different
in preparation
in
either
methods.
these complete, The
enzyme digests for
mo-
Vol. 83, No. 3, 1978
lecular
cloning
intervening
and further
sequences
of thalassemia. ping
BIOCHEMICAL
of the
prenatal
disorders
by restriction obtained
for
genes,
duplication
be possible and these
diagnosis
of all using studies
of homozygous
endonuclease
globin
cloned are
RESEARCH COMMUNICATIONS
of loci,
and mutations
identification
[22],
may allow
blasts
in globin
should
B- and Y-globin
a-,
analysis
Precise loci
AND BIOPHYSICAL
in
double-stranded in progress.
of DNA from
of genes,
the many genetic
gene fragments
a-thalassemia
mapping
ordering
forms
and map-
cDNA for These
and other amniotic
methods thalassemic fluid
fibro-
by amniocentesis.
ACKNOWLEGEMENTS We are indebted to Drs. Frances M. Gill and Thomas Kinney for providing some of the tissues used in these studies. We thank Drs. John M. Taylor, Ueli Schibler and Susan Astrin of the Institute for Cancer Research in Philadelphia for advice regarding their modification of the Southern "blot" method, Dr. Robert Perry for encouragement, advice and much helpful criticism, and Dr. J.W. Beard, through the Office of Program Resources and Logistics, Viral Oncology Program, National Cancer Institute, for providing reverse transcriptase for these studies. We are grateful to Carol Way for expert help in photography and in preparation of the manuscript. This work was supported in part by grants from the National Institutes of Health (AM 16691) and the Cooley's Anemia Fund. REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13.
Weatherall, D.J. and Clegg, J.B. (1972) The Thalassemia Syndromes, 2nd edn., Blackwell, Oxford. Ottolenghi, S., Lanyon, W.G., Paul, J., Williamson, R., Weatherall, D.J., Clegg, J.B., Pritchard, .I., Pootrakal, S. and Boon, W.H. (1974) Nature 251, 389-392. Taylor, J.M., Dozy, A., Kan, Y.W., Varmus, H.E., Lie-Injo, L.E., Ganesan, J. and Todd, D. (1974) Nature 258, 392-394. Kan, Y.W., Hallard, J.P., Dozy, A.M., Charache, S. and Kazazian, H.H. (1975) Nature 258, 162-163. Ottolenghi, S., Comi, P., Giglioni, B., Tolstoshev, P., Lanyon, W.G., Mitchell, G.J., Williamson, R., Russo, G., Musumeci, S., Achiliro, G., Tsistrakis, G.A., Charache, S., Wood, W.G., Clegg, J.B. and Weatherall, D.J. (1976) Cell 9, 71-80. Ramirez, F., O'Donnell, J.V., Marks, P.A., Bank, A., Musumeci, S., Schiliro, Pizzarelli, G., Russo, G., Luppis, B. and Gambino, R. (1976) Nature 263, 471-475. * Tolstoshev, P., Mitchell, J., Lanyon, G., Williamson, R., Ottolenghi, S., Comi, P., Giglioni, B., Masera, G., Modell, B., Weatherall, D.J. and Clegg, J.B. (1976) Nature 259, 95-98. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. Tilghman, S.M., Tiemeier, D.C., Polsky, F., Edgell, M.H., Seidman, J.G., Leder, A., Enquist, L.W., Norman, B. and Leder, P. (1977) Proc. Natl. Acad. Sci. USA 4406-4410. Jeffreys, A.J. and Flavell, R.A. (1977) Cell 12, 429-439. Jeffreys, A.J. and Flavell, R.A. (1977) Cell 12, 1097-1108. Schwartz, E. (1974) Semin. Hemat. 11, 549-567. Kan, Y.W., Holland, J.P., Dozy, A.M. and Varmus, H.E. (1975) Proc. Natl. Acad, Sci. USA 72, 5140-5149.
1130
G.,
Vol. 83, No. 3, 1978
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Efstratiadis, A., Maniatis, T., Kafatos, F.C., Jeffrey, A. and Vournakis, J.N. (1975) Cell 4, 367-378. Blin, N. and Stafford, D.W. (1976) Nucl. Acids Res. 3, 2303-2308. Sugden, B., DeTroy, B., Roberts, R.J. and Sambrook, J. (1975) Anal. Biochem. 68, 36-46. Mears, J.G., Ramirez, F., Leibowitz, D., Nakamura, F., Bloom, A., Kanotey-Ahulu, F. and Bank, A. (1978) Proc. Natl. Acad. Sci. USA 75, 1222-1226. Chang, A.C.Y., Lansman, R.A., Clayton, D.A. and Cohen, S.N. (1975) Cell 6, 231-244. Schwartz, E. (1976) Amer. J. Hum. Genet. 28, 423-426. Tolstoshev, P., Williamson, R., Eskdale, J., Verdier, G., Godet, J., Nogon, V., Trabuchet, G. and Benabadji, M. (1977) Eur. J. Biochem. 78, 161-165. Orkin, S.H. (1978) J. Biol. Chem. 253, 12-15. Wilson, J.T., Wilson, L.B., deRie1, J.K., Villa-Komaroff, L., Efstratiadis, A., Forget, B.A. and Weissman, S.M. (1978) Nucl. Acids Res. 5, 563-581. Loeb, L.A., Tartof, K.D. and Travaglini, E.C. (1973) Nature New Biol. 242, 66-69. Travaglini, E.C., Dube, D.K., Surrey, S. and Loeb, L.A. (1976) J. Mol. Biol. 106, 605-621.
1131
BIOCHEMICAL
RESTRICTION
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1125-1131
ENDONUCLEASE ANALYSIS OF
HUMAN GLOBIN GENES IN CELLULAR DNA Saul
Surrey,
Jill
S. Chambers, Elias
and
of Hematology Hospital
Philadelphia,
Received
Muni
Schwartz
Division The Children's
Diane
of Philadelphia
Pennsylvania
19104
June 22,1978
Summary: Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hint II, Barn HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were tra sferred to nitro-cellulose filters and identified by hybridization to [ 3Y P]cDNA for total adult globin mRNA. The a-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous a-thalassemia, a genetic disorder due to deletion of a-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated a-globin gene loci. The inherited are
characterized
globin
chains
radioactive that
group
by absent of hemoglobin
globin
with
globin
and
globin
genes which [2-61.
of the
chain,
The development restriction provides
[l].
studies
Recent
for
lack
a new approach
techniques
have been
and rabbit
globin
digestion
used for
DNA sequences
using
of DNA sequences
genes localizing
and subsequent
there
8-chain
sizing,
[g-11].
Using
of
have
indicated
of fetal for
hemo-
specific
gel
is
structural prosynthesis [6,7].
DNA sequences
by
electrophoresis diseases.
mapping similar
absent
are present
specific
of human genetic
detection,
of the
of the corresponding
where for
syndromes
hybridization
persistence
of production
to the study
thalassemia
of one or more
hereditary
the structural for
the
DNA in solution
in Do- thalassemia,
of methods
endonuclease
cellular
have deletions
account
In contrast,
8-globin
synthesis
a-thalassemia,
8-thalassemia
called
or decreased
cDNA to sheared
some patients
teins
of human disorders
[8] These
and cloning methods,
of mouse
we have
com-
0006-291X/78/0833-1125$01.00/0
1125
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 83, No. 3, 1978
pared
restriction
BIOCHEMICAL
endonuclease
infant
with
hydrops
using
several
globin
genes
zygous
a-thalassemia.
fetalis
AND BIOPHYSICAL
digests
of normal
due to homozygous
restriction
endonucleases
and demonstrate
MATERIALS
human DNA to DNA from an
a-thalassemia.
identify
specific
RESEARCH COMMUNICATIONS
deletion
Our studies
the fragments of a-gene
containing
fragments
in homo-
AND METHODS
Globin mRNA was prepared using peripheral blood containing 60 per cent reticulocytes from a 23 year old man with pyruvate kinase deficiency. Globin synthesis studies [12] of his reticulocytes showed equal a- and B-chain production. The globin mRNA was prepared by a sequence of acid precipitation, phenol-chloroform extraction, double passage through oligo-dT cellulose and sucrose density gradient centrifugation, using modifications of methods previously described [13]. The resulting poly[A] containing 9-11s RNA was used as a template for oligo(dT) -primed synthesis of radioactive cDNA by reverse transcriptase from av&'iyeloblastosis virus [14], with [32P]GTP(23000 Ci/mmole; Amersham Corp., Illinois) as the source of radioactivity. High molecular weight DNA was isolated [15] from the spleen of a fetus with hydrops fetalis due to homozygous a-thalassemia and from spleens removed from children with hemolytic anemia. The DNA was cleaved with an excess of a specific restriction endonuclease in each reaction at 37'C for 24 hours, using buffer conditions described by the enzyme suppliers (New England Biolabs, Solutions containing lo-20 ug of Inc. and Bethesda Research Labs, Inc.). DNA fragments in 20 ul aliquots were separated by electrophoresis in a 1% neutral, horizontal agarose gel employing a tris-acetate buffer system [16]. Electrophoresis was performed at 20 milliamps constant current for 16-18 The DNA samples were placed so that normal hours at room temperature. and a-thalassemia samples treated with the same enzyme were in adjacent wells. Restriction endonuclease fragments from A and 9x174 phage DNA were used as molecular weight markers. Following electrophoresis the DNA fragments were denatured with alkali, neutralized and transferred to a cellulose nitrate filter as described bx Southern [8]. The DNA on the filters was annealed for 24-48 hours at 65 C with 1 to 5ng/ml of high specific activity globin cDNA, and the filters were subsequently washed [8]. Radioactive bands were located by radioautography with Kodak XRP-1 film and DuPont Cronex Hi-Speed intensifying screens. RESULTS AND DISCUSSION Since
analysis
of a-globin Specific but
genes. after
DNA sequences fragments
absent
Figure digestion
I,
found
1 shows with
in restriction
the patterns
various
not with
solution
has demonstrated
a-thalassemia
[2,3],
endonuclease
digests
DNA may be identified
and controls but
in
in homozygous
in a-thalassemia
a-thalassemia and Alu
of hybridization
endonucleases. were
Hae III.
found
with
A single
1126
any globin of normal
as containing
of globin
specific Distinct Eco RI, 20.5
differences
kb a-globin
III,
DNA
a-globin
fragments
Hind
deletion
Hint
obtained between II,
DNA fragment
Barn HI
BIOCHEMICAL
Vol. 83, No. 3, 1978
Figure
1.
in
pattern.
the Eco RI digest
This
finding
two a-chain In that
DNA [17]. to their
differs
fragments, report
radioactive
on neutral
resolved
problem.
this
system.
of normal from
gels
state close
and that
We observed study
absent
described
8.7 and 7.8 kb,
was found
In the previous
DNA but
that
the authors
probe
was performed
gel
RESEARCH COMMUNICATIONS
Autoradiographic detection of globin genes in restriction endonuclease digests of human DNA. DNA from a control (C) and a fetus with homozygous e-thalassemia (H) was cleaved with various restriction endonucleases (Eco RI, Hind III, Barn HI, Alu I and Hae III). The resultant DNA fragments were separated by electrophoresis on a 1% agarose gel [16] and transferred to cellulose nitrate filters as described by Southern cDNA against globin mRNA from an adult was used 181. Radioactive for detection of DNA fragments containing globin gene sequences as described in Materials and Methods. The DNA specimens on the electrophoretic strip on the right are from undigested samples.
is present
noted
AND BIOPHYSICAL
in the a-thalassemia
in a recent
in Eco RI digests that
a large
to the origin
[17]
1127
a large
amount
which
of normal
amount
of DNA hybridizing
when electrophoresis
the use of an alkaline
no such difficulties
report
gel
system
in employing of low molecular
a neutral weight
Vol. 83, No. 3, 1978
material
BIOCHEMICAL
containing
the presence present
globin
of contaminant
study
only
the high
localization
degradation
genes
lanes
a-chain
possibility
an RNA linker
DNA with the
that
an RNA linker
RNA
would
RNA sites
been previously
has
(Fig.
thalassemia 3.2,
1),
1.7 and 0.9 a clear
normal
the
to more u-globin
results
of cDNA-driven
studied
have
our finding
Since
Hind
III
of the
hybridization
the two a-globin
loci
to
the pres-
[17].
Another
kb Eco RI fragment. gel
The existence
from
mammalian
cells
III
than
the patterns (4.9,
4.1
I (1.6,
two Hind
of a-globin in
kb),
Hint
a-globin
indicating
1203 indicate likely
fragments
is
the 4.1 kb band
other and a(3.7, There
DNA bands
of genetic
The most
II
1.3 kb).
kb band,
A variety
with of normal
1.4,
III
the 4.9
loci.
sequences
digestion
hybrid-
data
[19]
that
most humans
explanation that
than
in
in
there
and
for are
of double-stranded
a-globin
cDNA [21,22],
are
therefore
approximately
4.1 kb apart,
measured
The enzyme
while
we have
only
identified
off
the gel
(less
should
produce
two. than
about
at least
The third 500 bp),
1128
three
may have or it
a-globin been
small
may overlap
twice
the 4.9 band.
center
centers.
If
fractionation,
at the
their
migrate
samples
for
to agarose
after
experiments
of a-globin intensity
in
and Alu
cDNA molecules.
a-globin cuts
Hind
in intensity
of unequal
as many separate
found
kb)
duplication
prior
in
the
relate
study
two DNA fragments.
by differences
4.1 much darker
ization
which
may account
DNA isolated
Barn HI (14.5
difference
from
the undigested
the 20.5
alkali
gene fragments
the following:
kb),
DNA, with
with
apparent
recent
seen
the present
in methods
in
were
the
[18].
identified
DNA, are
present
in mitochondrial
a-globin
in
in this
to generate
reported
The specific enzymes
treated
be hydrolyzed
of alkali-labile
is
were
is
procedure
found
is
in
digestion
variations
fragments
in
gene sequences
In addition,
experimental
suggesting
fragmentation;
to the origin
These
ence of two smaller
the Eco RI digests,
globin
of DNA before adjacent
the
RESEARCH COMMUNICATIONS
or alkali-induced
containing
on right).
of DNA during
is
in
1, Eco RI lanes). weight
of globin
1, "DNA"
(Fig.
bands
(Fig.
molecular
was observed
nucleases
discrete
the Eco RI digests study
genes
AND BIOPHYSICAL
from
DNA bands, enough other
to
globin
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DNA bands. The identification hybridization
between
y-globin.
This
amounts,
with
sequence
bridization
is
amino
less
studies
present contain fragments
using
a partially
less
intense
bands
(10.2,
of 1.8,
1.3,
different semia.
4.9,
in
studies
facilitate
Hind
1.9 kb)
III I (2.3,
sequences of a-, partial
1.0,
0.9
5.9,
3.9
and 2.4 kb DNA preB- and reported
3.0 kb)[17].
The [Fig.11 genes,
to multiple
represent Hint
kb).
in both
of splenic to globin
corresponding too short
S- and 6- gene purification
Because
recently
4.5,
[23-241
II
Since
copy
rDNA
B- and g-globin (5.7,
1.5 kb),
Hae III
fragments
DNA preparations,
of a-globin
hybridizing
are
y-chains,
B-cDNA to y-globin
which
hy-
they
must
DNA sequences.
deletion
of material
to those
8.2 kb),
present
enzyme digests
new fragments
$- and
The putative
(6.6,
amino
to S- and g-genes
in the Eco RI DNA digests
digests (17.2,
B- or g-chain
demonstrate
the deletion
identification
in size
cDNA probe
the other
kb) and Alu
at least
or the remaining
similar
cDNA contaminants
bands
extensive
and a-thalassemia
or both.
of ribosomal
2.1
Since
genes,
2.8,
is
equal
and cDNA-driven
DNA, each of the
of radioactive
restriction
digests,
will
(7.6,
1.0 and 0.9 kb were
each contain These
are
there
between
normal
6- and
mRNA from
cross-hybridization[13].
of both
B-globin
the following:
Barn HI
slight
cross-hybridization
The major are
here
purified
or hybridization
genes
hybridize
homology
for
globin
However,
B-cDNA will
S- or 6-globin
detected
may represent
genes.
show only
by cross-
in approximately
chains,
sequence
the Eco RI digests either
g-gene
acid
total
&-globin
B- and that
complicated
molecules
mRNA.
of B- and &globin
in
using
a- and B-chain
indicate
is
cDNA and DNA sequences
was prepared
between
of cross-hybridization
sumably
study
reactions
and hybridization
bands
B-globin
or no y- or A-chain
homology
There
k51.
this
mRNA contains little
DNA fragments
radioactive
The cDNA in
an adult.
acid
of 8-globin
gene sequences
DNA from
to be detected
fragments
in these
of the fragments
1129
homozygous
cDNA did to a-chain
in several
not
a-thalas-
appear
mRNA is by these different
in preparation
in
either
methods.
these complete, The
enzyme digests for
mo-
Vol. 83, No. 3, 1978
lecular
cloning
intervening
and further
sequences
of thalassemia. ping
BIOCHEMICAL
of the
prenatal
disorders
by restriction obtained
for
genes,
duplication
be possible and these
diagnosis
of all using studies
of homozygous
endonuclease
globin
cloned are
RESEARCH COMMUNICATIONS
of loci,
and mutations
identification
[22],
may allow
blasts
in globin
should
B- and Y-globin
a-,
analysis
Precise loci
AND BIOPHYSICAL
in
double-stranded in progress.
of DNA from
of genes,
the many genetic
gene fragments
a-thalassemia
mapping
ordering
forms
and map-
cDNA for These
and other amniotic
methods thalassemic fluid
fibro-
by amniocentesis.
ACKNOWLEGEMENTS We are indebted to Drs. Frances M. Gill and Thomas Kinney for providing some of the tissues used in these studies. We thank Drs. John M. Taylor, Ueli Schibler and Susan Astrin of the Institute for Cancer Research in Philadelphia for advice regarding their modification of the Southern "blot" method, Dr. Robert Perry for encouragement, advice and much helpful criticism, and Dr. J.W. Beard, through the Office of Program Resources and Logistics, Viral Oncology Program, National Cancer Institute, for providing reverse transcriptase for these studies. We are grateful to Carol Way for expert help in photography and in preparation of the manuscript. This work was supported in part by grants from the National Institutes of Health (AM 16691) and the Cooley's Anemia Fund. REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13.
Weatherall, D.J. and Clegg, J.B. (1972) The Thalassemia Syndromes, 2nd edn., Blackwell, Oxford. Ottolenghi, S., Lanyon, W.G., Paul, J., Williamson, R., Weatherall, D.J., Clegg, J.B., Pritchard, .I., Pootrakal, S. and Boon, W.H. (1974) Nature 251, 389-392. Taylor, J.M., Dozy, A., Kan, Y.W., Varmus, H.E., Lie-Injo, L.E., Ganesan, J. and Todd, D. (1974) Nature 258, 392-394. Kan, Y.W., Hallard, J.P., Dozy, A.M., Charache, S. and Kazazian, H.H. (1975) Nature 258, 162-163. Ottolenghi, S., Comi, P., Giglioni, B., Tolstoshev, P., Lanyon, W.G., Mitchell, G.J., Williamson, R., Russo, G., Musumeci, S., Achiliro, G., Tsistrakis, G.A., Charache, S., Wood, W.G., Clegg, J.B. and Weatherall, D.J. (1976) Cell 9, 71-80. Ramirez, F., O'Donnell, J.V., Marks, P.A., Bank, A., Musumeci, S., Schiliro, Pizzarelli, G., Russo, G., Luppis, B. and Gambino, R. (1976) Nature 263, 471-475. * Tolstoshev, P., Mitchell, J., Lanyon, G., Williamson, R., Ottolenghi, S., Comi, P., Giglioni, B., Masera, G., Modell, B., Weatherall, D.J. and Clegg, J.B. (1976) Nature 259, 95-98. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. Tilghman, S.M., Tiemeier, D.C., Polsky, F., Edgell, M.H., Seidman, J.G., Leder, A., Enquist, L.W., Norman, B. and Leder, P. (1977) Proc. Natl. Acad. Sci. USA 4406-4410. Jeffreys, A.J. and Flavell, R.A. (1977) Cell 12, 429-439. Jeffreys, A.J. and Flavell, R.A. (1977) Cell 12, 1097-1108. Schwartz, E. (1974) Semin. Hemat. 11, 549-567. Kan, Y.W., Holland, J.P., Dozy, A.M. and Varmus, H.E. (1975) Proc. Natl. Acad, Sci. USA 72, 5140-5149.
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Efstratiadis, A., Maniatis, T., Kafatos, F.C., Jeffrey, A. and Vournakis, J.N. (1975) Cell 4, 367-378. Blin, N. and Stafford, D.W. (1976) Nucl. Acids Res. 3, 2303-2308. Sugden, B., DeTroy, B., Roberts, R.J. and Sambrook, J. (1975) Anal. Biochem. 68, 36-46. Mears, J.G., Ramirez, F., Leibowitz, D., Nakamura, F., Bloom, A., Kanotey-Ahulu, F. and Bank, A. (1978) Proc. Natl. Acad. Sci. USA 75, 1222-1226. Chang, A.C.Y., Lansman, R.A., Clayton, D.A. and Cohen, S.N. (1975) Cell 6, 231-244. Schwartz, E. (1976) Amer. J. Hum. Genet. 28, 423-426. Tolstoshev, P., Williamson, R., Eskdale, J., Verdier, G., Godet, J., Nogon, V., Trabuchet, G. and Benabadji, M. (1977) Eur. J. Biochem. 78, 161-165. Orkin, S.H. (1978) J. Biol. Chem. 253, 12-15. Wilson, J.T., Wilson, L.B., deRie1, J.K., Villa-Komaroff, L., Efstratiadis, A., Forget, B.A. and Weissman, S.M. (1978) Nucl. Acids Res. 5, 563-581. Loeb, L.A., Tartof, K.D. and Travaglini, E.C. (1973) Nature New Biol. 242, 66-69. Travaglini, E.C., Dube, D.K., Surrey, S. and Loeb, L.A. (1976) J. Mol. Biol. 106, 605-621.
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