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From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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- From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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). From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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). From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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. REFERENCES 1. Stamatoyannopoulos G, Nienhuis AW: Hemoglobin switching, in Stamatoyannopoulos G, Nienhuis AW, Varmus H (eds): Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders, 1993, p 107 2. Chada K, Magram J, Costantini F: An embryonic pattern of expression of a human fetal globin gene in transgenic mice. Nature 319:685, 1986 3. Kollias G, Wrighton N, Hunt J, Grosveld F Regulated ex- pression of human Ay-, P-, and hybrid ?/&globin genes in transgenic mice: Manipulation of the developmental expression patterns. Cell 46:89, 1986 4. Magram J, Chada K, Costantini F: Developmental regulation ofa cloned adult P-globin gene in transgenic mice. Nature 3 15:338, I985 5. Townes TM, Lingrel JB, Chen HY, Brinster RL, Palmiter RD: Erythroid-specific expression of human P-globin gene in transgenic mice. EMBO J 4:1715,1985 6 . KolliasG,Hurst 3 , deBoer E, Grosveld F:Thehuman P-globin gene contains a downstream developmental specific enhancer. Nucleic Acids Res 155739, 1987 7. Behringer RR,Hammer RE, Brinster RL,PalmiterRD. Townes TM: Two 3’ sequences direct adult erythroid-specific expression of human P-globin genes in transgenic mice. Proc Natl Acad Sci USA 84:7056, 1987 8. Enver T, Ebens AJ, Forrester WC, Starnatoyannopoulos G: The humanP-globin locus activation region alters the developmental fate of a human fetal globin gene in transgenic mice. Proc Natl Acad Sci USA86:7033, 1989 9. Enver T, Raich N. Ebens AJ. Papayannopoulou Th, Costantini F, Stamatoyannopoulos G: Developmental regulatlon of human fetal-to-adult globin gene switching in transgenic mice. Nature 344:309, 1990 10. Behringer RR, Ryan TM, Palmiter RD, Brinster RL. Townes TM: Human 7-to &globin gene switching in transgenic mice. Genes Dev 4:380, 1990 1 I. Lloyd JA, Krakowsky JM, Crable SC, Lingrel JB: Human Yto P-globin gene switching using a mini construct in transgenic mice. Mol Cell Biol 12: I 56 I , 1992 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. SINGLE-CELLGLOBIN mRNA PCR 12. StrouboulisJ, Dillon N, GrosveldF Developmental regulation of a complete 70-kb human @-globinlocus in transgenic mice. GenesDev6:1857, 1992 13. Peterson KR, Clegg CH, Huxley C, JosephsonBM, Haugen HS, Furukawa T, Stamatoyannopoulos G. Transgenic mice containing a 248 kb human @ locus yeast artificialchromosome display proper developmental control of human globin genes. Proc Natl Acad Sci USA90:7593,1993 14. 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Aviv H, Voloch Z , Bastos R, Levy S: Biosynthesis and stability of globin mRNA incultured erythroleukemic Friend cells. Cell 8:495, 1976 26. Lowenhaupt K, Lingrel JB: A change in the stability of globin mRNA during the induction of murine erythroleukemiacells. Cell 14:337, 1978 27. Weintraub H: Formation of stable transcription complexes as assayed by analysis of individual templates. Proc NatlAcad Sci USA 853819, 1988 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 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 Updated information and services can be found at: http://www.bloodjournal.org/content/83/5/1412.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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