2x selective media (containing adenine, alanosine, and oua- bain) was added (13). Growth of hybrids was evident 3-4 weeks after fusion. Growing hybrids were ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 780-784, February 1985 Cell Biology
Adult and fetal human globin genes are expressed following chromosomal transfer into MEL cells (fusion hybrid/globin expression/HEL cells/globin gene activation)
TH. PAPAYANNOPOULOU*, D. LINDSLEY*, S. KuRACHI*, K. LEWISON*, T. HEMENWAY*, M. MELIS*, N. P. ANAGNOUt, AND V. NAJFELDt *Department of Medicine, University of Washington, Seattle, WA 98195; tClinical Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20205; and fTumor Cytogenetics Laboratory, Mt. Sinai School of Medicine, New York, NY 10029
Communicated by Clement A. Finch, September 24, 1984
Somatic cell hybridization of mouse erythroABSTRACT leukemia (MEL) cells and HEL cells, a human erythroleukemia ling that produces fetal (y) but fails to express adult (j3) globin, was used to test whether the expression of the two human globin genes is regulated cis or trans. An experimental approach using anti-human globin monoclonal antibodies for detection, efficient cloning, and monitoring of hybrids of interest was employed. Further characterization of hybrids used isoelectric focusing for detection of human globins and S1 nuclease mapping. In contrast to the parental HEL line, all chromosome ll-retaining HEL-MEL hybrids expressed human .3globin, suggesting that the HEL 13-globin genes (i) are transcriptionally competent, (it) become activated in response to a positive trans-acting element within the MEL environment, and (ii) fail to express into the HEL environment because of either the absence of a positive trans-acting element or the presence of a trans-acting inhibitor of 13-globin gene expression. In addition to .3-globin, the primary HEL-MEL hybrids co-expressed y-globin; however, -globin expression segregated by subcloning so that secondary and tertikry clones either expressed only .8-globin or co-expressed v- and /3-globin. The results of subcloning can be explained by assuming that -globin gene expression is controlled by a HEL cell-derived transacting element encoded by a gene not syntenic to chromosome 11 or by postulating that the HEL y-globin genes become randomly modified during the continuous proliferation of hybrids.
trol human globin expression, we formed hybrids between HEL cells and MEL cells. With these experiments we wished, to test whether the absence of f3-globin expression in HIEL cells is controlled cis or trans. Since the parental HEL cells express y-globin, the experiments also tested whether inducible y-globin expression takes place in these hybrids and whether such expression is controlled coordinately or independently of 3-globin expression.
METHODS MEL cells, nearly diploid and deficient in adenine phosphoribosyltransferase, were fused with HEL cells (1:10 cell ratio) in the presence of 50% (vol/vol) polyethylene glycol (Baker). After addition of new medium, the cells were distributed in 20-30 flasks and placed in a 37TC incubator with 5% C02/95% air. Forty-eight hours later, an equal volume of 2x selective media (containing adenine, alanosine, and ouabain) was added (13). Growth of hybrids was evident 3-4 weeks after fusion. Growing hybrids were expanded in the presence of nonselective medium and screened and subcloned as described below. Screening of hybrids for human lactate dehydrogenase A (LDHA) was done by isoenzyme electrophoresis in cellulose acetate as described (14) using lysates of uninduced hybrid cells (0.5-1 x 106 cells). For staining of hybrid cell populations with fluorescent antibodies, an aliquot (-0.5-1 x 106 cells) of hybrid cells was induced with 4 mM hexamethylene bisacetamide (HMBA) for 3 days. Cytocentrifuge smears were fixed in absolute methanol, rinsed in phosphate-buffered saline, then in water, and air dried. A 1:1000 to 1:2000 dilution of mouse ascitic fluid containing the anti-/ or the anti-y chain IgG (15) was applied to the preparation. After incubation for 1 hr, the preparations were washed in phosphate-buffered saline, then in water, and dried. A 1:30 dilution of anti-mouse IgG [F(ab)'2] conjugated to fluorescein isothiocyanate was added and after 40-60 min of incubation the preparations were rinsed as before and viewed with a Zeiss fluorescence microscope with epi-illumination and through the appropriate filters. Cell lysates from [3H]leucine-labeled hybrid cells and controls were subjected to isoelectric focusing for separation of various globin chains, as described (16). Before focusing, human globin was immunoprecipitated by using monoclonal or polyclonal anti-human antibodies. To an aliquot of lysate (-1 ,xg of protein), the following were added sequentially: antibody (-10 pg), anti-mouse IgG (20 Ag; Miles), and (gelatin coated) protein A-Sepharose. Antigen-antibody complexes bound to protein A-Sepharose were washed with 0.2 M borate buffer (pH 9.3) and used for isoelectric focusing in Nonidet P-40/8 M urea gels (16). Gels were fixed and proc-
Cell fusion-mediated chromosomal transfer of human globin genes into mouse erythroleukemia cells (MEL) has been used to study regulatory mechanisms of globin gene expression (1, 2). It has been shown previously that transfer of human chromosome 11 (containing the human f3-globin locus) from bone marrow cells, lymphoblasts, or fibroblasts into MEL cells leads to inducible expression of human globin genes in the hybrids (3-5). However, only the adult (/3) and not the fetal (y) human globin expression program was activated (3-5). The possibility of differential trans control of human 3- and y-globin genes in the MEL environment has been raised by these results. In contrast to murine erythroleukemia cells, which express the adult mouse erythroid program, two human erythroleukemia lines, K562 (6) and HEL (7), both of adult origin, express either a fetal (HEL) or a fetal/embryonic (K562) globin program; in both, expression of adult 3-globin is undetectable (7-11). Since, in these lines, the , locus is intact (11, 12), the reasons for the lack of 8-globin expression are unclear. To further our insight on the regulatory elements that conThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: LDHA, lactate dehydrogenase A; HMBA, hexamethylene bisacetamide.
780
Celi Biology:
Papayannopoulou et aL % cells with human chromosome
Proc. NatL. Acad. Sci. USA 82 (1985)
R-Glohin
781
Globin y- lw_
i
I
FIG. 1. Expression of human f,- and y-globin in HEL-MEL hybrids, after induction. (Left) Staining with anti-P-chain monoclonal antibody in a hybrid clone that had undergone 70 doublings (Upper) and 120 doublings (Lower). (Right) Preparations from the same hybrid labeled with anti-y-chain monoclonal antibody. Note that the proportion of y-positive cells is lower than the proportion of f3positive cells. (The preparations are of similar cell densities.)
essed with EN3HANCE for fluorography, and the fluorograms were scanned in an automatic computing densitometer. For Southern blot analysis, established methods were used (17). For S1 nuclease mapping, total cellular RNA was prepared from 0.5-5 x 107 cells as described (18). M13 templates were provided by T. Ley and A. Nienhuis. Uniformly labeled probes were synthesized from single-stranded templates by the method of Ley et al. (19). Hybridization of probes to various concentrations of RNA (1-20 ,ug) was carried out in 80% (vol/vol) formamide at 50'C (for 3 hr). The hybridized complex was transferred to an S1 buffer containing 280 mM NaCl, 30 mM NaOAc, 4.5 mM ZnSO4, calf thymus DNA at 2 tug/ml, and 60 units of S1 nuclease. After a 30min incubation at 370C and ethanol precipitation, the samples were electrophoresed on 8 M urea/8% polyacrylamide gels and autoradiographed. The p-globin probe yielded a single band of 132 nuclebtides, whereas the y-globin probe yielded two bands, one of 144 nucleotides, corresponding to exon 1, and the other of 172 nucleotides, corresponding to exon 2.
brid clones were analyzed by globin chain isoelectric focusing. In clones that failed to show 9-globin-positive cells, only murine globin chains were detected. Nineteen clones showing p-globin-positive cells were analyzed and all of them showed the presence of globin chains co-migrating with authentic human 8-globin chains (Figs. 2 and 3). Cellular RNA or poly(A) mRNA was purified from HEL cells and HELMEL hybrids that were human -globin-positive by immunofluorescence. S1 nuclease analysis disclosed the characteristic 132-base-pair (bp) fragment protected by correctly initiated and processed 3-globin mRNA (Fig. 4) in all eight hybrids evaluated by that method. No steady-state f3-globin mRNA was detected by S1 nuclease mapping of the uninduced hybrids or of the uninduced and induced HEL cell controls (Fig. 4). The intensity of the 132 bp fragment generated by S1 nuclease mapping of known amounts of hybrid cell RNA was compared with that of the 132-bp fragment from known amounts of adult bone marrow RNA. We estimated that 1000-5000 copies of human P-globin mRNA were present per hybrid cell. a p Gy Ay /
RVSULTS Activation of Human p-Globin Expression in HEL-MEL Hybrids. Two of seven primary HEL-MEL hybrids were LDHA-positive, indicating that they had retained human chromosome 11. In addition, the two hybrids expressed human f3-globin chains as determined by staining of fixed preparations with anti-,-chain monoclonal antibodies. These clones were subcloned by limiting dilution or by plating in methylcellulose and secondary, tertiary, and quaternary subclones were raised. Of the 64 subclones analyzed, 53 were LDHA-positive, 11 LDHA-negative. In the LDHAnegative subclones, no f-positive cells were present after staining with the fluorescent anti-p-chain antibody. Uninduced LDHA-positive subclones were virtually negative in P-globin-positive cells; however, after induction with either HMBA or dimethylsulfoxide, all LDHA-positive subclones showed /-positive cells in high frequencies (Fig. 1). These data suggested that an inducible activation of human -globin genes consistently takes place whenever a HEL-MEL hybrid clone retains human chromosome 11. To verify the production of authentic human 8-globin chains, immunoabsorbed lysates from HMBA-induced hy-
MEL v
2-28-7
R
2-18-10
|mmunoprecpota/ifon with onti 1 + y
2-18-1l it
Cord Blood
(CB)
ontir
$. onti / itontil3+ y
FIG. 2. Detection of human - and p-globin chains in HELMEL hybrids. Monoclonal antibodies were used for immunoprecipitation of the globin chains used for isoelectric focusing. Results in three hybrids (2-28-7, 2-18-10, and 2-18-1) that express 13 and Gy chains are shown. The position of human globin chains is shown in the cord blood control, while the position of murine chains is shown in the MEL control.
782
Cell Biology: Papayannopoulou et al. a
c
/3
GyAy ,t
Proc. NatL Acad Sci. USA 82
A
i
K562
r C 0V -°
8-16 p
I1
6-2
r 1.
18-1-16
I ttt
18-1-8
ff((
(1985)
)
..Zc.
nt
Xz
S
CB
FIG. 3. Expression of human /3-, y-, and a-globin chains in HELMEL hybrids. Monoclonal antibodies and a polyclonal anti-Hb antibody were used for immunoprecipitation of globin chains. Migration of human a, E, Gy, and Ay chains is indicated in control samples from K562 cells and cord blood. Note the presence of human a chains in all hybrid cell lysates as well as the presence of only chains in clone 2-8-16, of mainly At chains in clone 6-2, and of mainly Gy chains in clone 18-1-8. (Mouse globin is partially coprecipitated from hybrid lysates with the polyclonal antibody used.)
-172 -144
132-p
/3,
Activation of Human y-GIobin Expression in HEL-MEL Hybrids. The two primary HEL-MEL hybrids and several subclones expressed human t-globin chains. Hybrid cells were fixed, stained with the anti-y-chain monoclonal antibody, and analyzed by the fluorescence assay (Fig. 1). The number and intensity of -globin-expressing cells increased greatly following HMBA or dimethyl sulfoxide induction of hybrids. Lysates from hybrids that were -globin-negative or -positive in the fluorescent antibody assay were then analyzed by globin chain isoelectric focusing; no -globin chains were present in the former but globin chains co-migrating with authentic y chains were present in the latter (Figs. 2 and 3). Characteristically, in -globin-expressing hybrids, synthesis of either GY (Fig. 2) or Ay (Fig. 3) chains was predominant.
S1 nuclease mapping was used to identify steady-state yglobin mRNA in hybrids that were y positive by the fluorescent antibody or isoelectric focusing assays. After S1 digestion of hybrid cell RNA, mapping using the probe shown in Fig. 5 showed that the expected 144- and 172-bp fragments were protected. Segregation of y-Globin Expression. Staining with anti-p6 and anti-y-globin monoclonal antibodies showed that, in the primary clones and several y-expressing subclones, the proportion of -expressing cells exceeded that of y-expressing cells (Fig. 1). This finding raised the possibility that the ability for y expression was lost in a proportion of cells of a hybrid clone during clonal expansion. Analysis of the phenotypes of subclones showed that hybrid clones that co-express y and 83 chains can generate subclones that co-express the two human globin chains, as well as subclones that express ,&globin chains but fail to express y-globin chains. From primary clone 6, 13 secondary clones were produced by limiting dilution; of those subclones, two had lost human chromosome 11 and the remaining 11 were p positive but 9 failed to show y expression. In the 2 ypositive hybrids, y chains were present in a more or less pancellular fashion. This phenotype has remained stable in >100 doublings. Isoelectric focusing analysis showed that the proportion of y chains exceeded that of p chains (Fig. 3); this relationship and p-globin mRNA was also reflected in the analysis of by S1 nuclease mapping (Fig. 5). From comparison of the intensities of the y-globin fragments with those generated after S1 nuclease digestion of fetal liver RNA, we estimated that 2-5 x 103 copies of y-globin mRNA were present per cell in the two HEL-MEL hybrids.
Exon
B
Probe
3
,13Z 208
3-Globin Gene
FIG. 4. Presence of human /3globin mRNA in HEL-MEL hybrids. (A) Total cellular RNA from HEL-MEL hybrids was studied by S1 nuclease analysis. (B) Probe used and size of the fragment protected by correctly initiated and processed human 3-globin mRNA. Bone marrow control poly(A)' RNA was hybridized with the same f3globin probe, whereas fetal liver [poly(A)+ RNA] and HEL cell (total cellular RNA) controls were hybridized to both the /3-globin probe and the y-globin probe illustrated in Fig. 5B. Note the absence and presence of /-globin mRNA in HEL cells and HELMEL hybrids, respectively. nt, Nucleotides.
From primary hybrid 2, 36 secondary clones were produced by subcloning, and 3 (secondary clones 8, 18, and 28) of them were selected for further subcloning. Of the 29 tertiary clones analyzed from the 3 secondary clones, 5 had lost human chromosome 11. Of the 24 chromosome 11-retaining clones, 21 expressed both y and pglobin chains (y+p+ clones) and 3 failed to express y chains (jy-,p clones). In the y'/3+ clones, the proportion of -positive cells was lower than that of the p-positive cells, suggesting instability in the phenotype of y-globin expression and loss of the ability of yglobin expression in a proportion of cells during clonal expansion. One of the y+f3+ tertiary clones (2-18-1) was subcloned; of the 8 human globin-expressing quaternary clones, 6 clones contained p-, as well as y-, positive cells and y, as well as chains by isoelectric focusing. Two clones completely lacked y-positive cells or -globin chains by isoelectric focusing. y-f3+ clones were studied by anti-y-globin fluorescent antibody staining of their cell populations at various intervals of culture. Such monitoring for >100 doublings failed to identify -positive cells after induction, indicating that loss of the ability of y-globin expression in these clones is permap,
nent.
Segregation of G7 and AVGlobin Chain Expression. The
Cell
Proc. Natl. Acad. Sci. USA 82 (1985)
Biology: Papayannopoulou et aL
783
of secondary clones 2-18 and 2-28 and the quaternary clones from tertiary clone 2-18-1 produced predominantly Gy chains. Hence, in contrast to the parental HEL line in which both Gy and Ay chains are synthesized in almost equal amounts (7), in the HEL-MEL hybrids, differences in expression of the two y-globin chains were present; the phenotypes of predominant Gy or Ay expression were inherited in the hybrid's progeny. To test whether the difference in expression of the two yglobin chains reflected structural anomalies of the p-globin
A
nt
genomic region, DNA from Ay or y-expressing hybrids and from hybrids showing the y-ff' phenotypes was purified and used for Southern blotting analysis. Digestion by several restriction endonucleases and probing with the sequences indicated in Fig. 6 generated only the fragments expected for the normal -globin genomic region (20). Other Studies. A polyclonal anti-hemoglobin antibody that immunoprecipitates all human globin chains was used to test for the presence of a- and embryonic (E and ;) human globin chains in the hybrids. The human a-globin chain was present as expected but e- and 4-globin chains could not be identified by isoelectric focusing. In addition, no t chains could be identified, using a polyclonal anti-4-chain antibody (a gift from D. Chui, Hamilton, Ontario) in an immunoprecipitation assay. In one hybrid, aliquots of cells were used for anti-,3-globin labeling and assessment of human chromosome 11-containing metaphases at two intervals of clonal expansion. The proportion of a-positive cells decreased as the frequency of chromosome 11-containing cells decreased. There were 76% chromosome 11-retaining cells (23 out of 30 metaphases) and approximately the same percentage of,-positive cells after 70 doublings, while the frequency of such cells was about 30% after 120 doublings (Fig. 1).
: t .t~~~~~~~a
-172 .. ^
.:..:.s ...
132-
-144
* .~~~'tow
!
w
60.
B
Exon 2 _____
Exon -- 1
5'
K
/
'3'
172
144, 569
r- Probe
DISCUSSION
FIG. 5. Presence of y- and f3globin mRNA in HEL-MEL hybrids. (A) S1 nuclease analysis. (B) Probe used and size of the fragment protected by correctly initiated and processed human y-globin mRNA. (The f3globin probe is shown in Fig. 4B.) + and -, use of RNA from HMBA-induced and uninduced cells. Note the presence of only ,-globin mRNA in hybrid 2-8-16 and the co-expression of Yand /globin mRNA in hybrids 6-2 and 2-18-10. nt, Nucleotides.
Characteristic of the HEL line is the absence of 8-chain or figlobin mRNA production; however, the HEL ,-globin genes possess both the major and the minor DNAse I-hypersensitive sites (21). Here we report that transfer of chromosome 11 of HEL cells into MEL cells consistently results in inducible activation of the f-globin genes. This result indicates that the HEL ,-globin genes are transcriptionally competent and that the absence of expression of ,3globin genes into the HEL line most likely reflects an abnormality of the HEL environment. This environment may lack a positive regulatory element that is normally required for activation of /3globin expression; if that is the case, this regulatory element
globin chain isoelectric focusing studies disclosed that primary hybrid 6 produced mainly Ay chains. The secondary y+,l+ clones produced by subcloning of this primary clone expressed the same Ayrglobin phenotype (Fig. 3). Primary hybrid 2 co-expressed Gy and Ay chains.§ Of the three secondary clones tested, clone 2-8 produced predominantly Ay chains; clone 2-18 and clone 2-28 produced predominantly tertiary clones generated by subcloning Gychains. The
'y+,+
§This original hybrid was lost because of contamination.
10kb I
Hc IdHd
Hd
Hd
Hd
Hd
x
P
P
B
B
Bg
Bg
Bg
Hp
Bg
BB
P
P B
B
Bg9
BgBg
B 2Hpj
1H.
\/Hp
I~~~
I
B
Bg
P
Bg
jII
Hp
_
_
Gy Ay
3E Hp
E
E
B
BJ JB
O
-4
1.4
1.3
JW151
1.7
JW102
RK29
FIG. 6. Restriction endonuclease mapping of the human ,3-globin locus of HEL-MEL hybrids. The locations of the six probes used are indicated in horizontal bars. B, BamHI; Bg, Bgl II; E, EcoRI; Hd, HindIII; Hp, Hpa I; P. Pst I; X, Xba I. DNAs from normal controls, HEL cells y-,8+ hybrids, Gyp+ and A'yf+ hybrids yielded identical sizes of restriction endonuclease fragments. kb, Kilobase(s).
784
Cell Biology: Papayannopoulou et aLPProc. NatL Acad Sci. USA 82 (1985)
should be different from those chromosomal proteins that, through interaction with globin gene sequences, create the DNase I-hypersensitive sites (22). Alternatively, the HEL environment may contain a negative controlling element that specifically inhibits f-globin gene transcription without interfering with expression of the -globin genes. Irrespective of the nature of the abnormality, our results show that the absence of j-globin gene expression in HEL cells is controlled trans. Similar conclusions have been reached with studies of cloned B3-globin genes from K562 cells (23). Previous work has shown that, although human 3-globin genes become activated into MEL cells, the yglobin genes remain inactive (3-5, 24). This preferential expression of adult human genes within the MEL environment has been interpreted by various postulates (4), including (i) the presence, in MEL cells, of positive trans-acting elements specific for adult P3-globin; (it) the absence of a similar factor(s) for -globin; or (iii) the presence of an environment actually inhibiting yglobin expression. In our experiments, we observed expression of -globin genes in HEL-MEL hybrids, detectable at both the protein and the mRNA level. The difference between our results and previous work can be attributed to the use, in our hybridizations, of human cells that express y chains. However, we found that y and /8 expression segregate independently of each other, since y and fexpressing hybrids can generate progeny that does or does not express 'y chains by subcloning. A likely interpretation of our results is that HEL-MEL hybrids express yglobin chains because they inherit, from the HEL line, a trans-acting element that activates the y globin genes. The presence of that element results in the phenotype of y+f3+ hybrids; its absence, in the phenotype of fly- hybrids. Inherent in this interpretation is the assumption that activation of - and f3-globin genes is under separate controls. Since y expression segregates, the hypothetical y trans-acting element cannot be syntenic to chromosome 11; the segregation of y expression can be attributed to segregation of a human chromosome that encodes the y trans-acting element. The predominant expression of Ay or GY chains in these hybrids could be explained under this hypothesis, if separate trans-acting elements activate each of the two y globin genes; alternatively, the HEL line may have sustained mutations similar to those present in the nondeletion Gy or Ay mutants of hereditary persistence of fetal hemoglobin that allow preferential expression of only one of the two globin genes per chromosome. An alternative interpretation is that the subcloning results reflect only cis effects; in that case, both ,3- and -globin genes are activated in response to the trans-acting element present in MEL cells. Random modifications, such as methylation of the globin genes, may occur during proliferative expansion of the y+,B+ hybrids. If both -globin genes are modified, the subclones will appear as fy/3. If Gy genes are modified, the subclones will express the Ay+ phenotype; if Ay genes are modified, the subclones will express the Gyp+ phenotype. Implicit in this hypothesis, however, is the notion that there is preferential modification of the y and not the 8 human genes in HEL-MEL hybrids. The relevance of these observations to the question of the control of normal yto-/3 switch remains to be determined. It is possible that trans-acting elements specific for y or for ,globin genes are present in fetal or adult erythroid cells and activate each gene in a specific ontogenetic stage; if that is the case, hemoglobin switching is primarily controlled trans. Alternatively, chromosome structure may determine wheth-
er and to what degree each gene of the locus will respond to the activating environment of the erythroblast. Although our findings favor the first alternative, studies of hybrids of MEL cells with erythroid cells at various stages of human ontogeny will be required to determine whether indeed the normal yto-,8 switch is controlled cis or trans. Our results show that the experimental-method we have developed allows application of the somatic cell hybridization approach in studies of hemoglobin switching despite the absence of a selective culture system for hybrids retaining human chromosome 11.
We are indebted to Drs. T. Ley and A. Nienhuis for providing the globin probes and assistance in S1 nuclease assays. We thank Dr. A. Deisseroth for providing the adenine phosphoribosyltransferaselacking MEL cells. This work was supported by National Institutes of Health Grant AM30852. 1. Deisseroth, A., Burk, R., Picciano, D., Mina, J., Anderson, W. F. & Nienhuis, A. W. (1975) Proc. Natl. Acad. Sci. USA 72, 1102-1106. 2. Deisseroth, A. B., Barker, J., Anderson, W. F. & Nienhuis, A. W. (1975) Proc. Natl. Acad. Sci. USA 72, 2682-2686. 3. Deisseroth, A., Velez, R. & Nienhuis, A. W. (1976) Science 191, 1262-1263. 4. Willing, M. C., Nienhuis, A. W. & Anderson, W. F. (1979) Nature (London) 277, 534-538. 5. Pyati, J., Kucherlapati, R. S. & Skoultchi, A. I. (1980) Proc. Nati. Acad. Sci. USA 77, 3435-3439. 6. Lozzio, C. B. & Lozzio, B. B. (1975) Blood 45, 321-334. 7. Martin, P. & Papayannopoulou, Th. (1982) Science 216, 12331235. 8. Rutherford, T. R., Clegg, J. B. & Weatherall, D. J. (1979) Nature (London) 280, 164-165. 9. Benz, E. J., Jr., Murnane, M. J., Tonkonow, B. L., Berman, B. W., Mazur, E. M., Cavallesco, C., Jenco, T., Snyder, E. L., Forget, B. G. & Hoffman, R. (1980) Proc. Natl. Acad. Sci. USA 77, 3509-3513. 10. Rutherford, T., Clegg, J. B., Higgs, D. R., Jones, R. W., Thompson, J. & Weatherall, D. J. (1980) Proc. Natl. Acad. Sci. USA 78, 348-352. 11. Dean, A., Ley, T. J., Humphries, R. K., Fordis, M. & Schechter, A. N. (1983) Proc. Natl. Acad. Sci. USA 80, 55155519. 12. Mueller, R. F., Murray, J. C., Gelinas, R., Farquhar, M. & Papayannopoulou, Th. (1983) Hemoglobin 7, 245-256. 13. Deisseroth, A. & Hendrick, D. (1978) Cell 15, 55-63. 14. Khan, M. P. (1971) Arch. Biochem. Biophys. 145, 470-483. 15. Stamatoyannopoulos, G., Farquhar, M., Lindsley, D., Brice, M., Papayannopoulou, Th. & Nute, P. E. (1983) Blood 61, 530-539. 16. Righetti, P. G., Gianazza, E., Gianni, A. M., Comi, P., Giglioni, B., Ottolenghi, S., Secchi, C. & Rossi-Bernardi, L. (1979) J. Biochem. Biophys. Methods 1, 45-47. 17. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 18. Chirgwin, J. M., Przybyla, A. G., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 19. Ley, T. L., Anagnou, N. P., Pepe, G. & Nienhuis, A. W. (1982) Proc. Natl. Acad. Sci. USA 79, 4775-4779. 20. Orkin, S. H., Kazazian, H. H., Jr., Antonarakis, S. E., Goff, S. C., Boehn, C. D., Sexton, J. P., Waber, P. G. & Giardina, P. J. V. (1982) Nature (London) 296, 627-631. 21. Groudine, M., Kohwi-Shigematsu, T., Gelinas, R., Stamatoyannopoulos, G. & Papayannopoulou, Th. (1983) Proc. Natl. Acad. Sci. USA 80, 7551-7555. 22. Emerson, B. M. & Felsenfeld, G. (1984) Proc. Natl. Acad. Sci. USA 81, 95-99. 23. Fordis, M. C., Anagnou, N. P., Dean, A. & Nienhuis, A. W. (1984) Proc. Natl. Acad. Sci USA 81, 4485-4489. 24. Wright, S., de Boer, E., Grosveld, F. G. & Flavell, R. A. (1983) Nature (London) 305, 333-336.
