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Coexpression of y and B Globin mRNA in Cells Containinga Single Human
Globin Locus: ResultsFrom Studies Using Single-cell Reverse Transcription
Polymerase Chain Reaction
By T. Furukawa, G. Zitnik, K. Leppig, Th. Papayannopoulou, and G. Stamatoyannopoulos
We developed a method detecting globin gene expression in y and j3 mRNA contents. The method was subsequently
in single cells using reverse transcription polymerase chain
used to test whether onlyoneor more than one globin
j3
reaction. C and y globin cDNAs are coamplified by an cy
genes are expressed in cellsthat contain a single human
primer setwhereas y and j3 globin cDNAs are coamplified
globin locus.W e found that about 50%of single cells from
MEL X fetal erythroid cell hybrids containing a single huby a yj3 primer set and the individual globincDNAs are disy and j3 globin mRNA. This
tinguishedby restrictionenzymedigestion.Analysisof
man j3 globin locus coexpressed
finding is best explained by assuming that both y and j3
RNApreparationsfromhuman
fetal liver, neonatal red
blood cells (RBCs), or adult RBCs showed the expected
genes are simultaneously transcribed from
the same j3 glomRNA speciesforeachstageofhumandevelopment.
bin locus implying
that the LCR can simultaneouslyinteract
Analysis of single cells from a human erythroleukemia linewith more than one globin gene promoter.
coexpressing y and j3 globin chains showed heterogeneity 0 1994 by The American Society of Hematology.
H
EMOGLOBIN PRODUCTION in humans is characterized by two major switches, from embryonic to fetal, during the transition from yolk sac to the liver stage of
hematopoiesis and from fetal to adult hemoglobin (Hb)
around theperinatal period.’ Insights on thecontrol of Hb
switching have been obtained with several approaches, especially with studies in transgenic mice. In the mouse, embryonic globin genes are expressed in yolk sac cells whereas
the adult globin genes are expressed only in the cells of definitive erythropoiesis in the fetal liver and the bone
marrow
(BM). Transgenic mice produced using either y or p globin
gene constructs lacking locus control region (LCR) sequences express the humany gene only in embryoniccells
and the B globin gene only indefinitive cells, indicating that
the genes contain those sequences which are responsible for
correct developmental regulation.*” When y or p globin
genes linked to LCR sequences are used for production of
transgenic mice, developmental regulation is lost and these
genes are inappropriately expressed in both the embryonic
and adult stages of
However, correct developmental regulation is restored when a pLCR Ay$@@
cosmid construct9 or other constructs containing LCR sequences linked to both y and p globin genes were used for
production of transgenic
The restoration of developmental regulation of y and ,B
globin genes in pLCR Ay$@/3 transgenic mice has been interpreted by proposing that differential interactions between
globin genes and the LCR are responsible for globin gene
switching during d e ~ e l o p m e n t . ~In” ~theembryonic stage of
From the Divisions ofMedica1 Genetics and Hemutology, the Department ofMedicine, University of Washington, Seattle.
Submitted July 9, 1993; accepted October23, 1993.
Supported by National Institutes ofHealth Grants No. DK30852
and HL20899.
Address reprint requeststo George Stamatoyannopoulos, MD, Dr
Sci, Medical Genetics, RG-25, University of Washington, Seattle,
WA 98195.
The publication COSIS of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with18 U.S.C.section I734 sole1.v to
indicate thisfact.
01994 by TheAmerican Society of Hematology.
0006-4971/94/8305-0013$3.00/0
1412
development, the environment favors the interaction between the LCR and c globin genes and y and p globin genes
are being turned off. In the fetal stage of development, an
interaction between the LCR and y globin genes is favored,
resulting in predominant y gene transcription. In the adult
stage of development, theLCR interacts preferentially with
the p gene and the y globin genes are turned off competitively. One of the questions raised by this model is whether
the LCR interacts with only one globin gene promoter at
any given developmental time or whether it can simultaneously interact with more than one promoter. This question canbe answered by analyzing globin gene transcription
in cells that contain a single p locus.
To address this question we developed a method for single-cell mRNA analysis using reverse transcription polymerase chain reaction (RT-PCR). This method was used to
detect the globin mRNA species present in heterospecific
hybrids produced by fusing MEL cells with human fetal erythroid cells. These hybrids initially express human y globin
but subsequently switch to exclusive p globin formation.
Hybrid cells from the mid y to p switch period were used to
test whether cells containing a single human p globin locus
express only the y or only the p or both the y and the p
globin genes.
MATERIALSAND METHODS
Manipulation of Single Cells
The methods offusion,hybrid maintenance usinga chromosome
l l-specific monoclonal antibody (MoAb),and globin phenotyping
have been described before.” Hybrids were cultured with Iscove’s
modified Dulbecco’s medium (IMDM) supplemented with 20% fetal calf serum (FCS), sodium pyruvate, and antibiotics in a 5% CO*,
highly humidified incubator. Hybrids were induced by dimethyl
sulfoxide (DMSO) at the concentrationof2% vol/vol for 2 to 3 days.
Cells were washed twice in phosphate-buffered saline (PBS) and
after addition of RNAse A (100 ng/mL) were incubated at 37°C for
30 minutes. Drops of the solution containing thecells were placed
on glass slides and single cells were aspirated by gentle suction into
borosilicate glass IM-CS capillaries ( I .O mm OD, Narishige USA,
Greenvale, NY) attached to an Axiovert 35 micromanipulator
(Zeiss, Germany). For prevention of RNA contamination, each cell
was aspirated using a separate capillary. Aspirated cells were transferred individually to 2 pL of lysis buffer (0.8%NP40, 5 mmol/L
dithiothreitol [DTT], 150 U RNase inhibitor (Promega, Madison.
Blood, Vo183, No5(March l), 1994:pp 1412-1419
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1413
SINGLE-CELLGLOBIN rnRNA PCR
WI) in a 0.5-mL tube. The lysate-containingbuffer was immediately centrifuged, frozenby placing into a mixture of methanol and
dry ice,and stored at -70°C until use. A cell-free PBSsolution from
the same drop was usedas a blank.
Table l.Amplified ProductsFrom Human Globin cDNAs
Primer Set
Size
After
Restriction
Products
of Uncut
Product (bp)
Endonucleases
Globin cDNAs
Digestion
(bp)
248,26
191,57,26
156,92,26
263
222,41
263
153,110
263
154,109
RT
Lysates were initially
denatured at 80°C for 5 to IO minutes, then
reverse transcribed by Moloney murine leukemia virus (M-MLV)
reverse transcriptase (GIBCO BRL, Grand Island, NY) using random hexamers (GIBCO BRL)
at a concentration of 2 mmol/L. The
reaction was allowed to proceed for 60 minutes at 42°C. The transcription reaction was terminated by heating at 90°C for IO minutes
and subsequently chillingon ice.
Single-cell PCR
Weused the so-called "hot-start'' PCR method (Perkin-Elmer
Cetus, Norwalk,CT) and high annealing temperatures to avoid the
problem ofprimer dimerization because that most likely wouldoccur in the presence of rare or few target sequencesin a single cell.
PCR was performed in I X PCR buffer(50 mmol/L KCI, IO mmol/
L, Tris HCIpH 8.3, 1.5 mmnl/L MgCl2, 0.001% wt/vol gelatin)
containing 0.02 mmol/L of each dNTP, 100 pmol of each primer,
and 2.5 U of Taq polymerase (Perkin-Elmer Cetus) in a final volume of 100 pL. Two mixtures, separated by a wax barrier, were
placed in eachtube. A mixture of primers, MgCI2,and dNTPs was
on the bottom and a mixture of cDNA,Taq polymerase, and FCR
buffer wason the top. The PCR reaction wasstarted by melting the
wax barrier and blending the two mixtures during an initial denaturation for 3.5 minutes at 94'C. Forty-five cycles ofamplification
were performed the amplification profile of each cycle consistedof
denaturation at 94°C for I minute, primer annealing at a temperature specified for each primer set (see text below)
for l minute, and
elongation at 72°C forl minute.
The primer sets and annealing temperaturesare as follows. t and
y globin:S-GCAAGATGAATGTGGAAGA,(proximal)S-CCCAGGAGCTTGAAGTTC (distal),WC; y and globin: S-TGGACCCAGGTTCTTTGA
(proximal),
S-GCGGTGAA'ITCTTTGCC (distal),63°C.
PCR products were ethanol precipitated, divided into two portions, and each portion was digested with a different enzyme. The
digested products were electrophoresed in 4%NusieveSeakem
GTG (3: 1)agarose gel(FMC Bioproducts, Rockland, ME), stained
with ethidium bromide, and visualized under UV light.
RT-PCR ofRNAPrepared From Erythroid Tissues
Total RNA was isolated from blood, fetal liver,or cultured cells
by the acid guanidium-thiocyanate-phenol-chloroform
extraction
method." Five micrograms oftotal RNA was initially denatured at
80°C for 5 minutes, then reverse transcribed by M-MLV reverse
transcriptase using random hexamers at a concentration of 5
mmol/L for 60 minutes at 42°C in final volume of 20 pL. After
termination of the reaction, 10-pL aliquots were used as templates
for 35-cycle amplification by PCR. PCR was performed
as described in the single-cell RT-PCR method, except that a wax layer
was not used.
Fluorescence InSitu Hybridization (FISH)
Chromosomeswere prepared fromhuman fetal erythroid ( H E )
MEL hybrids using standard techniques. The cells were treated
with colcemid (0.01pg/mL) for 30 minutes, then with hypotonic
solution (0.075 mol/L KCI), the preparations were fixed in methanokacetic acid (3:1 vol/vol) and spread on glass slides. Slides were
baked for I hour at 100°C. FISH was performed as previously deX
scribed." The cosmid cCH 1, containing 39 kb of contiguous DNA
sequences from the Cy gene through the 0 globin gene:'
was biotinylated (Bionick Labeling system; GIBCO BRL)
and used as the
probe. The probe was denatured at 75°C for 5 minutes and allowed
to preanneal for 6 to 8 hours at 37°C. Posthybridization washes were
performed for I5 minutes at 46" to 48°C. Detection of the hybridization signals was performed using fluorescence-conjugated avidin
(Vector Laboratories, Burlingame, CA) that bound to the biotinlabeled nucleic acid probe.
RESULTS
Specificity of Primers
The ty primer set. This primer set is completely homologous to t cDNA but has a base-pair mismatch (in the proximal primer) with y cDNA. Therefore, y cDNA amplified
less efficientlythan t cDNA. There are four mismatches between the ty primer set and ,6 globin cDNA (three in the
proximal, one in the distal primer) resulting in poor p cDNA
amplification. Amplification of t, y, or B cDNA providesa
274-bp product but the three cDNAs speciescan be distinguished from each other by the sizes offragmentsgenerated
after digestion with DpnII (Table l , Fig 1).
The yfl primer set. The yP primer set is completely homologous to y and b globin cDNAs. The primer has four
base-pair mismatches with t cDNA (two in the proximal
primer and two in the distal primer), resulting in poor amplification oft cDNA (Fig 2). Amplification ofglobin cDNA
using this primer set gives riseto a 263-bp product. Distinction between the y and globincDNAs is accomplished
following restriction enzyme digestionusing PvuII and
DruIII (Fig 2, Table I).
Distinction from 6 globin cDNA. The ty primer set has
six base-pair mismatches with the 6 globin cDNA (four in
the proximal primer, two in the distal primer); therefore, it
is unlikely that it can amplify 6 cDNA. The yP primer set
has only a base-pair mismatch with the 6 globin cDNA (in
the distal primer) and should amplify 6 cDNA, though less
efficientlythan p or y. Distinction between 6 and p cDNA
can be achieved by digestionwith DraIII, which cuts fl
cDNA, but not 6 cDNA (Table I). Distinction between y
and 6 cDNA can be achieved by digestionwith BstEII,
which cuts y globin cDNA and BfaI ( M a d ) ,which cuts 6
globin cDNA (Table 1).
Distinction from human genomic globin DNA products.
The y@primer set can amplify y and globin genomic se-
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1414
FURUKAWA ET AL
M 1 2 3 4
274-U
J248-E
7
"191-y
156-p
'92-B
-57"
-26
coexpressesy and p mRNA. All lanes contain the 153-bp and
1 IO-bp diagnostic fragments expected for h w l I digestion of y
cDNA. Digestion with Drulll of the amplified products of the
same cells produces the 154-bp and 109-bpfragmentsexpected for (3 cDNA. Therefore, all cells contain both y and p
mRNAs. The differences in the intensity of p and y cDNA
fragmentssuggests that there is heterogeneity iny and globin
gene expressionamong the cells of this line.
Anai!si.s c!flluman GlobinInRNA in Single Cells From
MEL X Fetal Et:l-tlmid Cell Hybrids
X
Distinctionbetween l w n a n andmurinecDNAs. MEL
fetal erythroid hybrids contain murine as well as human
M 1 2 3 4
Fig 1. Analysis of globin expression byRT-PCR using the ty
primer set. Theamplified cDNAs were digested with Dpnll.The uncut (markedas U) product is 274-bp
long (lane 1). Both C and y globin
mRNA are present in human erythroleukemia cell lines RMlO and
K562 (lanes 2 and 3). y and 6 mRNAs are present in human adult
blood (lane 4);the 274-bpband in lane 4 indicates partial digestion
of the PCR amplification product. 0 Globin is amplified with low
efficiency (lane 4). LaneM is$X174/Haelll size marker.
quences, but cDNA sequences can be distinguished from
genomic DNA sequences by the size of fragments produced
after digestion with RstEII. Plwll. or Dralll (Fig 3, Table 2).
The cy primer set can potentially amplify c and y genomic
DNA sequences. The distal primer spansthe exon ,/exon 3
junction in the cDNAs, IO bp of which can anneal to exon
2 ofthe c and y genes. When large amountsofgenomic template are present. amplification of the c and y genomic DNA
will occur even though there are 7 bp that donot anneal in
Sportion ofthedistal primer. However, because its product
contains the sequenceof intron I , the product is larger than
the product of cDNA amplification (396 bp 274 bp, respectively).
11
-263-U
-1 53>r
-1 10
-263-U
-1 54>p
-109
AnaI!:sis ofRNA Preparations
RNA prepared from human fetal liver, cord blood, and
adult blood was used for RT-PCR analysis. cy Amplification followed by DpnII digestion (Fig4,upper
panel)
showed that both c and y mRNAs are present in fetal liver
RNA samples. BstEll and DraIII digestion of the yp amplification products (Fig 4, middle panels) showed the predominance of y mRNA in liver and cord blood RNA samples and the presence of predominantly p mRNA and of
small amounts of y mRNA in adult blood RNA samples
(Fig 4, lower panel).
The RT-PCR method was subsequently used for analysis
of single cells collected by micromanipulation of drops of
cell suspensions as described in Materials and Methods. Figure 5 (lanes 1 through 6) shows RT-PCR analysis of single
cells of the M902 line. a human erythroleukemia line that
Fig 2. Analysis of globin expression by RT-PCR using the yB
primer set afterdigestion with Pvull (upper panel) and Drelll (lower
panel). Only y globin mRNA is detected in K562 cells (lane 1)
whereasb and y globin mRNAs aredetected in adult blood (lane 2).
Only y globin mRNA can be detected in RMlO cells (lane 3) a human
erythroleukemia cell line that synthesizes considerableamounts of
t and y globin, illustrating the low
efficiency of c cDNA amplification
by the y o primer set. A 263-bp product is amplified from adult
mouse cDNA but it remains uncut with both fvull and Dralll (lane
4). M and U as in Fig 1.
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1415
SINGLE-CELL
likely. There is essentially no homology between the cy
primer set and the other murine
genes. The @hI , pminor,
and
pmajor
cDNAs have two mismatches (out of 20 bp) with the
proximal y@primer and two mismatches (out of I7 bp) with
the distal yP primer. Some amplification of these cDNAs
M 1 2 3
1118,1113
‘l008
Genomic
DNA
M 1 2 3 4 5 6 7 8 9
cDNA
-1
10
Lggk-1118>
Genomic
DNA
-263i
-1 5 4 3 cDNA
-109
M 1 2 3 4 5 6 7 0 9
Fig 3. Differentiation between cDNA and genomic DNA amplification products. yS amplification product from totalRNA (lane 1)
and human genomic DNA
(lane2) and mixture of both (lane3) were
digested with h u l l (upper panel)and Dralll (lower panel) and were
electrophoresed. Note that the110-bpfragment in the h u l l digest
and the 109-bp fragment in the Dralll digest are specific for products of cDNA amplification.
mRNAs. There is a 4-bp difference between the murine ry
mRNA and the 19 bp of proximal ry primer, and 7 bp
difference (out of I8 bp) between the cy mRNA and thedistal ry primer: amplification of ry cDNA is, therefore, un-
Table 2. Amplification Products Obtained Using the
Primer Set
Globin
Size of
Uncut
Product (bp)
BsrEll
Pvull
Ddll
cDNA
Genomic DNA
y cDNA
GyGenomic DNA
Ay Genomic DNA
d cDNA
d Genomic DNA
0 cDNA
0 Genomic DNA
263
1,118
263
1,149
1,129
263
1,161
263
1.1 13
263
1,118
222.41
1.108.41
1.088.41
263
1,161
263
1.1 13
263
1.1 18
153,110
507,489,153
489,487,153
153,110
1,008, 153
263
1,113
263
1,118
263
1,149
1,129
263
1,161
154.109
959,154
t
t
Products After Digestion (bp)
M 1 2 3 4 5 6 7 0 9
1
I
-154>p
-1 09
Fig 4. Globin mRNA species in human fetal liver (lanes 1 to 3).
cord blood (lanes 4 to 6 ) and adult blood (lanes 7 to 9). ty Amplification products were digested by Dpnll (upper panel). y@ Amplification product were digested by BstEll (middle panel) or by Dralll
(lower panel).
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FURUKAWA ET AL
1416
M 1 2 3 4 5 6
-2634
-153>r
-1 10
ing 83 metaphase, 80 had only one hybridization signal (Fig
8), whereas three had two signals. No metaphase had three
signals. These results indicate that 96% of thechromosomes-l l-containing cells had retained only one human
chromosome I 1 whereas 4% of the cells contained two c o p
ies of chromosome 1 I . Therefore, the majority of the cells
that coexpressed y and /3 mRNA contained a single human
p globin locus.
DISCUSSION
We have developed an RT-PCR method allowing the unambiguous detection of human c, y, and 0 globin mRNAs
F-
M 1 2 3 4 5 6 7 8 91011121314
-263-U
C
Fig 5. Analysis of globin mRNA in single humanM 6 0 2 erythroleukemia cells (lanes1 through 6). -yB Amplification productswere
digested with fvull (upper panel) and
Dralll (lower panel).
may take place but the murinecDNAs canbe distinguished
from human y and @ globin cDNAs after digestion with
Pvrdl and Dralll (Fig 2).
Human globin mRNA expression in single HFE X MEL
hyhridcells. To test whether y and @ genes are coexpressed
in a single cell, we analyzed cells of a hybrid undergoing
switching from y to @ gene expression.
Findings inuninduced cells are shown in Fig 6.Several cells
(as those in lanes 2, 4, and 10 of panel A) contain only y
mRNA. Other cells (asthose in lanes 3,5,7,8, and 9 of panel
A) contain only @ mRNA. Cells in lanes 6,11. and 12 ofpanel
A contain both y and @ mRNA. Of 25uninduced cells, 6 contained only y mRNA, 7 only 0 mRNA, whereas 9 cells contained both y and @ RNA (Fig 6 and datanot shown).
We analyzed 24 cells after 2-day induction of the hybrids
with DMSO (Fig 7). In 4 we detected only @ mRNA, in 4
only y mRNA, whereas in I3 cells we detected both y and
@ mRNA. Therefore, over 50% of the cells (uninduced or
induced) of this hybrid coexpressed y and @ human globin
mRNA.
Identification of chromosome l1 bv FISH. To determine the numberof copies of human chromosome1 1 in the
hybrid cells, FISH was performed using a GyAyll,@6@
human
cosmid probe. Of the 1 I I metaphase cells analyzed, 20
lacked hybridization signals, most likely because the cells
have lost human chromosome 1 1 during culture. In eight
metaphases, we could not determine the numberof signals
because ofexcessive fluorescent background. Of the remain-
R
Y
M 1 2 3 4 5 6 7 8 9 1 0 1 1
Fig 6. Globin mRNA expression in uninduced single HFE
X MEL
hybrid cells. y@ Amplification products were digested with h u l l
(upper panel) and with Dralll (lower panel). Lanes 1 through 1 2 in
(A) and lanes1 through 11 in (B) were from single cells. Lane13 is
B positive andlane 1 4 a negative control for
y and B mRNA. Notice
6.1 1,and 1 2 in
the coexpression ofy and 0 mRNA in cells of lanes
(A) and 2 , 4 , 6 , and 7 in (B).
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SINGLE-CELL
mRNA PCR
1417
genes compete with each other for interaction with the LCR
and thatexpression of a globin gene occurs when its interaction with the LCR is favored by the transacting environment.9.10.15.16 one of the questions generated by this model
is whether the LCR interacts with only one globin gene at
any given time. If that is the case and if the LCR/globin
promoter complexes are stable, only one globin gene of the
p locus should be transcribed at any given time.
Testing of this prediction of the competition model can
Fig 7. Globin mRNA expressionin DMSO-inducedsingle HFE X
MEL hybrid cells. yj3 Amplification products were digested with
Pvull (A) and Dralll (B). Lanes 1 through 13 in (A) and lanes 1
through 1 1in (B) were from single cells. Notice the coexpression of
y and B mRNA in lanes 2,3,4,6,9, and 10 of (A) and lanes 1,2,3,
7,8,9, and lOof (B).
and their distinction from murine globin mRNAs and human genomic DNA. We have shown that this method can
be used for the analysis of globin mRNA of single cells and
for asking questions related to the developmental control of
globin genes.
Studies in transgenic mice have shown an autonomous
and acompetitive mechanism ofglobin gene switching. The
autonomous mechanism is exemplified by the turningoff of
the t globin gene.2"24whereas the competitive mechanism
is exemplified by the silencing ofthe p and y globin genes.','0
The competition model assumes that the y and p globin
Fig 8 . Detection of human chromosome 11 in HFE X MEL hybrid cell by FISH. (A) The arrow points to a fluorescent in situ hybridization signal from a '-yA-y+P6j3 globin cosmid (cosmid cCH1).
The chromosomes are
stained with propidium iodine. (B) The metaphase spread is stained with Hoechst/actinomycin D (Sigma, St
Louis, MO) to confirm the presence of human chromosome 11,
which isindicated by the arrow.
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FURUKAWA ET AL
1418
be done only by using cells containing a single ,B globin locus. Such cells are represented by the HFE X MEL hybrids
produced by fusing MEL cells with human fetal erythroid
( H E ) cells. Previous studies of HFE X MEL hybrids using
double immunofluorescent labeling with anti-y and anti-6
globin antibodies conjugated to different fluorochromes
have shown that globin gene switching is accomplished by a
decrease in the frequency of y-positive hybrid cells and an
increase in frequency of &positive hybrid ~ e 1 l s .Using
l ~ our
RT-PCR assay, we detected cells containing only y mRNA,
cells containingonly
mRNA, and other cells that contained both y and p mRNAs. Because the cells we analyzed
were derived from a hybrid undergoing the y to p switch,
the cells containing y mRNA but lacking p mRNA should
be considered as preswitch cells; the cells containing p
mRNA but lacking y mRNA should be considered as cells
in which the y to switch has been completed; the cells in
which the y as well as the p mRNAs are coexpressed should
be considered as cells undergoing the y to 8 switching. The
coexistence of y and p mRNAs in these hybrid cells can be
explained by the following hypotheses.
( 1) The presence of both y and p mRNA in cells containing a single locus may indicate concurrent transcription of
the y and globin genes. Simultaneous transcription of y
and 6 genes would suggest that theLCR can interact simultaneously with the y and the6 globin gene promoters. This
assumption is not unlikely considering the size of the LCR
and the fact that three of the four DNAse I hypersensitive
sites act asenhancers. For example, HS2 may interact with
the y gene promoter whereas HS3 may interact with the P
gene promoter (or vice versa), resulting in simultaneous y
and p gene transcription.
( 2 ) Presence of both y and P mRNA is caused by the prolonged globin mRNA lifespan. Only one gene can be transcribed per locus per cell but a cell can containboth y and p
rnRNAs because it carries globin mRNA species synthesized in the cell’s progenitor. We may, for example, speculate that in the y-positive p-negative cells the LCR interacts
only with the y promoter and only they gene is transcribed.
When the process of y to p switching starts, an adult stagespecifictransacting element is produced resulting in interaction of the LCR with the p globin promoter and only P gene
transcription. However, y mRNA will continue tobe present in such switched cells because the cells inherit mRNA
molecules that were synthesized in the cells’ progenitors before switching. This interpretation is not unreasonable if
one considers that globin mRNA is relatively stable (the
half-life of globin mRNA is at least 17 hours in induced
MEL cellsz’~26and 50 hours in uninduced MEL cells).26
However, this interpretation also implies that the hybrid
cells which coexpress y and p mRNA are those which have
switched recently. Because the half-life of y globin mRNA
is in the range of 17 to 50 hours, and since the generation
time of the hybrids is about 24 hours, cells that contain both
y and mRNA shouldhave switched in the last two to three
cell generations. If 50%of the cells of the hybrids we examined had switched recently, our hybrid should have been
switching at a very fastrate and should complete the switch
within the next few divisions. However, this expectation is
not borne out of our observations. The MEL X fetal erythroid cell hybrids switch with a rather slow rate and take
several weeks to reach a stage in which all the cells express
only human globin.” Therefore, it is unlikely that the hybrid cells which coexpress y and p mRNA are those that
have recently switched and happen to contain the y mRNA
which wassynthesized in an earlier cell.
(3) Both y and p genes are transcribed in a cell that contains a single 6 globin locus, but notsimultaneously. During
switching both fetal-specific and adult-specific transacting
factors may be present in a cell and the LCR may flip-flop
between they or the gene promoter, resulting in transcription of both y and p genes and accumulation of both y and
p mRNA duringa single cell cycle. However, this possibility
is less likely in view of other evidence that enhancer/promoter interactions result in production of stable complexes
producing distinct cellular phenotype^.^'
ACKNOWLEDGMENT
We thank Denise Farrer for technical assistance and ShemBrenner and Bonnie Lenk for preparation of the manuscript. We thank
Ken Peterson for his advice and Doris Morgan for providing the
MB02 cells.
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Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders,
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4. Magram J, Chada K, Costantini F: Developmental regulation
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1994 83: 1412-1419
Coexpression of gamma and beta globin mRNA in cells containing a
single human beta globin locus: results from studies using
single-cell reverse transcription polymerase chain reaction
[published erratum appears in Blood 1994 Aug 15;84(4):1357]
T Furukawa, G Zitnik, K Leppig, T Papayannopoulou and G Stamatoyannopoulos
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