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Identical
Nucleotide
Sequences
From
By l.S.
We
have
determined
bp
long
EcoRI
fragment
the
ered
to
and
Icy.2
cell
anemia
contain
enhancer
patient
No 31
and
F and
from
low
third
chromosome
carried
tional
hereditary
persistence
cell
have
hereditary
cause
of this
within
the
been
found
5’
G
in
nd-#{176}’y-HPFH
Bodine
and
that is located
and
identified
from
the
or the
an
for
in
of
5’
DNA
from
F)
with
promoters
subjects
with
have
either
the
enhancer
about
is located
element
400
bp 3’ to its
within
a 750-bp
EcoRI/HindIII
fragment.
This fragment,
added to a plasthe ‘y-globin
gene promoter
fused to the
chloramphenicol
the CAT activity
this
sequence,
acetyltransferase
(CAT)
in K562 cells some sixfold
which
of transcripts
initiated
gene transcription-initiation
tive role in enhancer
sequence
appears
might
affect
the
sites
function,
that
function.
normal
G.y
the number
and
Ay
globin
in vitro,
may play a definiand mutations
within
its
We have
determined
the
sequence
of this -750-bp
EcoRI/Hind
III fragment
(and the
i,550-bp
segment
located
3’ to it) that was isolated
from
chromosomes
of four
different
subjects
who had either
distinct
patient
increases
in their Hb F level, such as an Arabian
and a Chinese
nd-HPFH
heterozygote,
or who
low levels
55
had
of Hb F.
presented
globin
AND
METHODS
Samples.
DNA
samples were isolated from peripheral
WBCs
during earlier investigations
and were stored in the cold.
Cloning and sequence
analysis.
For three samples the 15.3-kb
Bam HI fragment
(3A,5#{246})
and for the fourth (the Yugoslavian
control)
the 13-kb BglII
fragment
(5’#{176}’y-5’fl) were cloned
into
EMBL3
bacteriophage
vector.7 Identification
of positive clones was
with appropriate
probes (‘yIVS-II;
3’”y;
5’b)
Differentiation
of the
two types of clones from the Yugoslavian
subject was after digestion
with Xmn I and probing with ‘yIVS-II
(normal chromosome,
8 kb;
triplication
chromosome,
7 and 5 kb9), and that of the clones from
the Chinese HPFH heterozygote
was after digestion with Xmn I and
probing
with
the 4’ probe (normal
chromosome,
7.6 kb; HPFH
chromosome,
3 kb3). The 2.3-kb
EcoRI
fragment
(3fry)
was
subcloned into the Bluescript
plasmid (Stratagene)
as described.’0”
DNA
sequence
analysis
was by the dideoxy
chain
termination
method of Sanger
et al’2 with a modification
of the sequenase
protocol (Stratagene).
Several primers and the reverse primers were
used that were prepared
with an ABI synthesizer.
Data
are
Blood,
with
mutations
seen
within
were
published
between
the
except
for
data
make
These
in 0y and
& Stratton.
the
samples
Chinese.
increases
as nucleotide
gene (=No
this
an
it
sequence
are
production.
A
Inc.
numbers
relative
to the Cap
site of the
Ay
1).
RESULTS
are
data
for the
in Fig
as published
sequence
by the
sequence.
tions; four
of the
listing
observed
G
Vol 73, No 3 (February 15), 1989: pp 845-848
EcoRI
fragment
nucleotide
all four
for
T substitution
-+
for the Chinese
(2,360
bp)
.
below
There
were 2 1 differences
insertions
of one nucleotide,
nucleotide)
tional
entire
The data
listed are for the normal
by Shen et al.’3 Deviations
are mdi-
I
presented
the
published
(1 6 simple
substituone deletion
of one
samples
was
nd-HPFH
analyzed;
observed
an addi-
+ 3412
at position
chromosome.
DISCUSSION
from four
55 adult
samples
was an Arabian
DNA
levels
for
(20%
to 30%)
haplotype
No
of Hb
31
subjects
have been analyzed.
who is mildly
affected,
has
F in his blood,
second
The
.
was
severely
affected
with
low levels of
homozygous
for haplotype
No l9. The
with
a nd-HPFH
who
a normal
had
chromosome
haplotype
that
was
tion6’9;
the
with
the HPFH
is uncommon
a Yugoslavian
2.3-kb
Submitted
Supported
41544 and
Blood
Children’s
fragment
a ‘y-globin
that
was
Hospital,
Shanghai.
PR
gene
triplica-
analyzed
came
Biology,
ofMedical
Medical
Genet-
China.
October 11, 1988; accepted November
10, 1988.
by USPHS
Research
Grants HLB-05168
and HLBby HLB-29623
from
the National
Heart,
Lung and
Institute.
Address
of Cell
reprint
requests
and
Molecular
to Dr Titus
H.J.
Biology,
Medical
Augusta,
GA 30912-2100.
The publication
costs ofthis
charge payment.
This article
“advertisement”
in accordance
indicate
S
a normal
of Hb F (nearly
20%),
and 40% of a ‘y2.3 The
From the Department
of Cell and Molecular
College ofGeorgia,
Augusta
and the Laboratory
ics, Shanghai
55 patient,
Hb F (<10%)
and
third was a Chinese
determinant
has a specific
among
Chinese.3
The fourth
with
EcoRI
One
high
and is homozygous
a black
hematology,
level of Hb A2, but an elevated
level
which consisted
of about
60% of a’y2
subject
MATERIALS
the
chromosome
differences
made
different
gene,
increased
to 23-fold.2
Thus
to act by increasing
from
with
Sequence
signal,
containing
specific
a normal
were
four
in
by Grune
cated
which
was
the
polyadenylation
mid
associated
was
Twenty-one
differences
the
mutation
that
fourth
a comparison
of
additional
mutations
ndAyHPFH.l
and
the
subject.
no
sequences
Huisman
in a
Often
specific
an
Elements
a nondeletional
gene
Ley2 recently
identified
3’ to the Ay globin gene,
when
a 1989
(Hb
patients
with
globin
1 9. A
seen
F (HPFH).
although
A,.,
No
and
a Yugoslavian
unlikely
a nondele-
hemoglobin
in certain
Hb
is unclear,
patient
haplotype
fetal
for
Enhancer
and T.H.J.
subject.
from
sequence;
hemoglobin
observed
Chinese
sickle
anemia
for
Lanclos,
by Bodine
Arabian
determinant
K.D.
observed
cell
of fetal
2.360-
Gene
Chromosomes
is consid-
sickle
and in subjects
the
and
a black
the
Zeng,
this
a homozygosity
persistence
increase
of the
F and
been
(55)
anemia
(nd)
Y.T.
Hb
increases
production
sickle
element
a homozygosity
IGNIFICANT
Huang,
gene
Globin
3’’y
Different
chromosomes;
globin
were
high
with
S
Hb
with
H.J.
four
A
chromosomes
Four
sequence
from
3’ to the
the
The
haplotype
nucleotide
fragment
is located
Han,
of the
Huisman,
College
Department
of Georgia,
article were defrayed
in part by page
must therefore
be hereby
marked
with 18 U.S.C. section 1734 solely to
this fact.
I 989 by Grune
& Stratton,
Inc.
0006-4971/89/7303-0037$3.00/0
845
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846
HAN
GAATTCAAGG
TTTAGTCAGG
TGTAGCAATT
CTATTTTATT
AGGAGGAATA
CTATTTCTAA
2027
TGGCACTTAG
CTTTTCACAG
CCCTTGTGGA
TGCCTAAGAA
AGTGAAATTA
ATCCCATGCC
2087
CTCAAGTGTG
CAGATTGGTC
ACAGCATTTC
AAGGGAGAGA
CCTCATTGTA
AGACTCTGGG
2147
GGAGGTGGGG
ACTTAGGTGT
AAGAAATGAA
TCAGCAGAGG
CTCACAAGTC
AGCATGAGCA
2207
TGTTATGTCT
GAGAAACAGA
CCAGCACTGT
GAGATCAAAA
TGTAGTGGGA
AGAATTTGTA
2267
CAACATTAAT
TGGAAGGTTT
ACTTAATGGA
ATTTTTGTAT
AGTTGGATGT
TAGTGCATCT
2327
TGATGGTGTT
ACGGACCTGG
TGTTTGTGTC
TCCTCAAAAT
2387
ET
AL
C
CTATAAGTAA
GAGTTTAATA
AA
TCACATGCTG
AATCCCCAAC
TCCCAACTGA
CCTTATCTGT
GGGGGAGGCT
TTTGAAAAGT
2447
AATTAGGTTT
AGCTGAGCTC
ATAAGAGCAG
ATCCCCATCA
TAAAATTATT
TTCCTTATCA
2507
A
GAAGCAGAGA
GACAAGCCAT
TTCTCTTTCC
TCCCGGTGAG
GACACAGTGA
GAAGTCCGCC
2567
ATCTGCAATC
CAGGAAGAGA
ACCCTGACCA
CGAGTCAGCC
TTCAGAAATG
TGAGAAAAAA
2627
CTCTGTTGTT
GAAGCCACCC
AGTCTTTTGT
ATTTTGTTAT
AGCACCTTAC
ACTGAGTAAG
2687
G
GCAGATGAAG
TCTCAGGTTT
AAGGAGAAAA
GCAAAGCTCC
AAATAAGCTT
CCTCGTCCCC
GGGTTTTGAG
TATGTTTCAG
TGAACTACAG
G
ACCATGTT*A
2747
CATAAAATAC
CTACTCTACT
2807
T-
I
ACTCTCATCT
ATAAGACCCA
AATAATAAGC
CTGCGCCCTT
CTCTCTAACT
TTGATTTCTC
2867
CTATTTTTAC
TTCAACATGC
TTTACTCTAG
CCTTGTAATG
TCTTTACATA
CAGTGAAATG
2927
TAAAGTTCTT
TATTCTTTTT
TTCTTTCTTT
CTTTTTTCTC
CTCAGCCTCA
GAATTTGGCA
2987
CATGCCCTTC
CTTCTTTCAG
GAACTTCTCC
AACATCTCTG
CCTGGCTCCA
TCATATCATA
3047
AAGGTCCCAC
TTCAAATGCA
GTCACTACCG
TTTCAGGATA
TGCACTTTCT
TTCTTTTTTG
3107
GTAAAGAAAT
AAACGAATGA
3167
A
TTTTTTGTTT
Fig 1 .
nucleotide
Sequence
present.
TTTTTAAGTC
data for the
The fragments
AAAGCAAATT
TCTTGAGAGA
2.360-bp
EcoRl fragment.
located
3’ to the
globin
from the four chromosomes
had identical
differences
gene.’3
with
(-).
the
the same nucleotide
published
data.
observed;
().
no
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
IDENTICAL
NUCLEOTIDE
SEQUENCES
847
CTAC1’GCATA
GGCAGAGCAG
CCCCGAGGGC
CGCTGGTTGT
TCCTTTTATG
GTTATTTCTT
3227
GATGATATGT
TAAACAAGTT
TTGGATTATT
TATGCCTTCT
CTTTTTAGGC
CATATAGGGT
3287
AACTTTCTGA
CATTGCCATG
GCATGTTTCT
TTTAATTTAA
TTTACTGTTA
CCTTAAATTC
3347
T
AGGGGTACAC
GTACAGGATA
TGCAGGTTTG
TTTTATAGGT
AAAAGTGTGC
CATGGTTTTA
3407
ATGGGTTTTT
----T
TTTTTCTTGT
AAAGTTGTTT
AAGTTTCTTG
TTTACTCTGG
ATATT*GGCC
A----
3467
TTTGTCAGAA
GAATAGATTG
GAAAATCTTT
TTCCCATTCT
GTAGATTGTC
TTTCGCTCTG
3527
ATGGTAGTTT
CTTTTGCTGA
GCAGGAGCTC
TTTAGTTTAA
TTAGATTCCA
TTGGTCAATT
3587
TTTGCTTTTG
CTGCAATTGC
TTTTCACGCT
TTCATCATGA
AATCTGTGCC
CGTGTTTATA
3647
TCATGAATAG
TATTGCCTTG
ATTTTTTTCT
AGGCTTTTTA
TAGTTTGGGG
TTTTTCATTT
3707
AAGTCTCTAA
TCCATCCGGA
GTTAATTTTG
GATAAGGTAT
AAGGAAGGAG
TCCAGTTTCA
3767
I
TTTTTCAGCA
TATGGCTAGC
CAGTTCTCCC
CCATCATTTA
TTAAATTGAA
AATCCTTTCC
3827
CCATTGCTTG
CTTTTGTCAG
GTTTCTAAAA
GA*CAGATGG
C
TTGTAGGTAC
AATATGCAGT
3887
TTCTTCAAGT
CATATAATAC
CATCTGAAAT
CTCTTATTAA
TTCATTTCTT
TTAGTATGTA
3947
TGCTGGTCTC
CTCTGCTCAC
TATAGTGAGG
GCACCATTAG
CCAGAGAATC
TGTCTGTCTA
4007
GTTCATGTAA
GATTCTCAGA
ATTAAGAAAA
ATGGATGGCA
TATGAATGAA
ACTTCATGGA
4067
TGACATATGG
AATCTAATGT
GTATTTGTTG
AATTAATGCA
TAAGATGCAA
CAAGGGAAAG
4127
A
GTTGACAACT
GCAGTGATAA
A
CCTGGTATTG
ATGATATAAG
AGTCTATAGA
TCACAGTAGA
4187
AGCAATAATC
ATGGAAAACA
ATTGGAAATG
GGGAACAGCC
ACAAACAAGA
AAGAATCAAT
4247
ACTACCAGGA
---T
AAGTGACTGC
AGGTCACTTT
TCCTGGAGCG
GGTGAGAGAA
AAGTGGAAGT
4307
T*GCAGTAAC
-A
TGCCGAATCC
T
4327
Fig 1.
(Cont’d).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
HAN ET AL
848
+
2285
Ref.13
2366
2460
2367
2676
T
0
0
C
A
I
I
I
I
I
Ll1N\
_____
5#{149}
A
(:-
A
I
Eco R II
Hind
i ,550
bp
Ill
Eco R I
Ref. 13
+2730
2746
278830843312
3412
3463
3724
3860
4086
41204125
4127
41414251
4309
4321
Fig 2.
Summary
of sequential
differences
within
the 2.360-bp
EcoRI fragments
isolated
from the DNA samples
of the four subjects
under study as compared
to the published
data.’3
The 5’ segment
of 750 bp is considered
to be the enhancer
element.2
Numbering
is
relative
to the Cap site of the
globin gene. The haplotypes
of the four chromosomes
are: $A or [+
+ + +1; i No 19 or
F
+
+ +];No31
or[+
+ + - + + + + -];Chinese$-HPFH0r[+ + + + + + - +1. Restriction
sites: HincIl 5’
to ; XmnI 5’ to Gy; HindlIl in 07 and A*y; HinclI in ‘fi and 3’ to it; AvaIl in ; HpaI and BamHl 3’ to .
from
his normal
while
chromosome.
The analyses
were
the possibility
that mutations
considering
segment
of
segment
of
DNA,
-750
were
element,2
production
in
Chinese
undertaken
within
this
and particularly
within
the smaller
5’
bp that is considered
to be the enhancer
related
to an increase
in #{176}y
and
A.y chain
the
Saudi
Arabian
55 patient
and in the
subject,
who
is heterozygous
for
the
nd-0”y-”y-
HPFH.
Chinese
and
and
study
differences
additional
2 summarizes
compares
the
these
data
with
for the
four
published
samples
data.
under
All
21
were the same for the four samples,
while one
mutation
(at + 3412, G -‘ T) was present
in the
Five
Ay
chain
production
were
substitutions
simple
in the Arabian
heterozygote.
55
observed
enhancer
the
identical
five
mutations
in
element
isolated
from a chromosome
type
Gelinas
of nd-HPFH
but
normal
chromosome
ing the data obtained
of the same patient.
for our four patients,
that
observed
the
causative
mutations
of the elevations
in
patient
and in the
et al’4 recently
HPFH
the Seattle
observed
These
data make
it unlikely
are related
to an increased
Gy
Chinese
with
Figure
sample.
the 750-bp
enhancer
segment.
that the observed
substitutions
in the
in
and
the
-750-bp
of a patient
not in that
However,
it seems
enhancer
A.y
chain
from
the
consider-
less likely
element
are
production.
REFERENCES
1. Stoming TA, Stoming
GS, Lanclos
KD, Fei YJ, Altay C,
Kutlar
F, Huisman
THJ: An Ay type of nondeletional
hereditary
persistence
of fetal hemoglobin
with a T -‘ C mutation
at position
175 to the Cap site of the A.y globin gene. Blood (in press)
2. Bodine DM, Ley JT: An enhancer
element lies 3’ to the human
A,,., globin
gene. EMBO J 6:2997, 1987
3. Zeng Y-T, Huang
S-Z, Chen B, Liang Y-C, Chan Z-M,
Harano T, Huisman
THJ: Hereditary
persistence
of fetal hemoglobin or (6)#{176}-thalassemia: Three types observed
in South-Chinese
families. Blood 66: 1430, 1985
4. Kutlar A, Hattori Y, Bakioglu I, Kutlar F, Kamel K, Huisman
THJ: Hematological
observations
on Arabian
55 patients
with a
homozygosity
or heterozygosity
for the
chromosome
with haplotype #31. Hemoglobin
9:545, 1985
5. Hattori
Y, Kutlar
F, Kutlar
A, McKie VC, Huisman
THJ:
Haplotypes
of fl’ chromosomes
among
patients
with sickle cell
anemia from Georgia. Hemoglobin
10:623, 1986
6. Liu JZ, Cao Q, Gilman
JG, Bakioglu
I, Huisman
THJ: Four
categories
of low
of ‘y-globin
gene triplications:
DNA
sequence
comparison
and high
triplications.
Blood 72:480, 1988
7. Kaiser
K, Murray NE: The use of phage Lamda replacement
vectors in the construction
of representative
genomic DNA libraries,
in Glover DM (ed), DNA
Cloning,
Vol I. Oxford,
Washington,
DC,
IRL Press, 1985, p 1
O,.,
8. Liu JZ,
containing
three
Huisman
different
THJ:
human
Construction
globin
of two plasmids,
gene
fragments.
each
Hemoglo-
bin 11:13, 1987
9. Efremov GD, Filipce
V, Gjorgovski
I, Juricic
D, Stojanovski
N, Harano
T, Nakatsuji
T, Kutlar A, Kutlar F, Bakioglu I, Huisman
THJ: GyAy(o9)oThalassaemia
and a new form of y globin
gene
triplication
in the Yugoslavian
population.
Br J Haematol
63:17,
1986
10. Maniatis
T, Fritsch EF, Sambrook
J: Molecular
Cloning:
A
Laboratory
Manual.
Cold Spring
Harbor,
Cold Spring
Harbor
Laboratory,
1982
11. Beris Ph, Miescher PA, Diaz-Chico
JC, Han I-S. Kutlar A,
Hu H, Wilson JB, Huisman
THJ: Inclusion
body fi-thalassemia
trait
in a Swiss family is caused
by an abnormal
hemoglobin
(Geneva)
with an altered
and extended
fi chain carboxy-terminus
due to a
modificationin codon fll14. Blood 72:801, 1988
12. Sanger
F, Nicklen
5, Coulson AR: A rapid
method
for
determining
sequences
in DNA by primed
synthesis
with
DNA
polymerase.
Proc Natl Aced Sci USA 74:5463,
1977
13. Shen S-H, Slightom
JL, Smithies
0: A history of the human
fetal globin gene duplication.
Cell 26:191, 1981
14. Gelinas RE, Rixon M, Magis W, Stamatoyannopoulos
G: ‘y
Gene promoter
and enhancer
structure
in Seattle variant of hereditary persistence of fetal hemoglobin.
Blood 7 1: I 108, 1988
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1989 73: 845-848
Identical nucleotide sequences of the 3'A gamma globin gene enhancer
elements from four different chromosomes
IS Han, HJ Huang, YT Zeng, KD Lanclos and TH Huisman
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