Cell, Vol. 7, 323-329,

March

1976,

Copyright

% 1976

by MIT

Absence of Messenger RNA and Gene DNA for ,&Globin Chains in Hereditary Persistence of Fetal Hemoglobin B. G. Forget, D. G. Hillman, H. Lazarus, E. F. Barell, and E. J. Benz, Jr. Children’s Hospital Medical Center and Sidney Farber Cancer Center Harvard Medical School Boston, Massachusetts 02115 C. 1. Caskey Baylor College of Medicine Houston, Texas 77025 T. H. J. Huisman Medical College of Georgia Augusta, Georgia 30902 W. A. Schroeder California Institute of Technology Pasadena, California 91125 D. Housman Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary The relative amounts of (Y- and j&globln mRNA and globin gene DNA were measured in reticulocyte RNA and lymphocyte DNA of an individual with homozygous hereditary persistence of fetal hemoglobin whose red blood cells contain 100% fetal hemoglobin (Hb F: (y2y2). Molecular hybridization assays used as probes full-length DNA copies of human CY- and /3-globin messenger RNA. The results of these hybridization assays demonstrated the expected amounts of a-globin mRNA and gene DNA, but absence of ,&globin mRNA and absence of ,&globin gene DNA. In the individual studied, hereditary persistence of fetal hemoglobin is associated with total deletion of the ,&globin structural gene. Introduction On the basis of numerous genetic studies, the presumed number and chromosomal arrangement of the normal human globin genes can be schematically represented as in Figure 1. A number of human hereditary blood disorders are characterized by absent or diminished synthesis of the 01or /3-globin chains of normal adult hemoglobin, Hb A (c&). These disorders can be classified into two general categories which are genetically distinct: the thalassemias and hereditary persistence of fetal hemoglobin (HPFH) (Weatherall and Clegg, 1972). In the thalassemias, there is poor compensation by the red cell for the deficit and imbalance of globin chain synthesis, and there results an anemia characterized by small, poorly hemoglobinized red cells. In HPFH, on the other hand, the deficit of P-chain

synthesis is associated with very high levels of ychain synthesis of normal fetal hemoglobin (Hb F: azy2), so there results little or no imbalance between (Y- and non-a-chain synthesis, and little or no deficit in intracellular hemoglobin content; no anemia results. In the thalassemias, the deficit of LY- or ,&chain synthesis is associated with a deficit of the corresponding chain-specific globin messenger RNA (Housman et al., 1973; Kacian et al., 1973; Forget et al., 1974). In the Oriental type of a-thalassemia which is associated with total absence of a-chain synthesis, a deletion of a-globin genes has been demonstrated by hybridization of total cellular DNA of these individuals with II- and ,&globin DNA copies (cDNAs) synthesized from globin mRNA by viral reverse transcriptase (Taylor et al., 1974; Ottolenghi et al., 1974; Ramirez et al., 1975; Kan et al., 1975a). On the other hand, studies using DNA from individuals with ,8-thalassemia associated with total absence of ,&chain synthesis and ,&chain mRNA have failed to demonstrate a P-globin gene deletion (Ottolenghi et al., 1975; Tolstoshev et al., 1976). Normal amounts of /3-globin gene DNA are present in DNA of patients with /3+-thalassemia, which is associated with some synthesis of P-chains, but in reduced amounts (Ramirez et al., 1975). Recently, Kan et al (1975b) have reported hybridization experiments using DNA from cultured fibroblasts of a Black individual with homozygous HPFH, which demonstrated decreased hybridization of this DNA to /3-globin cDNA. We have studied a second unrelated Black individual who is homozygous for HPFH of the type which is most common. This condition is characterized by total absence of Hb A and absence of the normal minor hemoglobin, Hb AZ ((~~82)~ but is associated with intact synthesis of both normal a H.-M

H..

a

GY AY ,----I..+.--+..-,..

a

fl

F-Y

C

N

Hb A: Figure

6

a2f12

1. Schematic

N

Hb A*: Representation

C

a&i, of Human

Hb F: Globin

a,Y, Genes

The detailed genetic evidence for this model has been summarized by Forget and Kan (1974) and Weatherall and Clegg (1972). The (Y- and non-n-globin genes are situated on separate chromosomes (Deisseroth et al., 1976), and the non-a-globin genes are clustered together on the same chromosome. The intergene distances are represented by dots, and their size relative to the structural genes is unknown. N and C indicate the extremities of the genes coding for the N terminal and C terminal sequences of the globin chains.

Cell 324

types of Hb F (with either glycine (Gy) or alanine (“y> at position 136 of the y chain) (Huisman et al., 1971). Total cellular RNA isolated from peripheral blood cells of this individual had the expected amounts of cY-globin mRNA, but lacked @globin mRNA when analyzed by hybridization of the RNA with (Y- and ,&cDNA. DNA was isolated from cultured lymphocytes of this individual and hybridized with (Y- and ,&cDNA: the DNA hybridized to (Y-cDNA at the expected Cot,,,, but there was no significant hybridization of the DNA to ,&cDNA. On the other hand, DNA from cultured lymphocytes of a normal individual hybridized with both (Y- and ,&cDNAs at roughly the same Cot,. These results indicate absence of /3-globin genes in the DNA of the individual with homozygous HPFH.

Results Genetic and Hematologic

Studies of the Case

The individual with homozygous HPFH is a newly discovered case, not related to the three other Black homozygous individuals cited in the world’s literature (Weatherall and Clegg, 1972). She is a healthy 8 year old American Black female who was discovered as a result of a hemoglobin screening program for sickle cell disease. Her hematologic values are: hemoglobin, 14.8 g/100 ml blood (normal 13.7 & 1); hematocrit or packed red cell volume, 44.3% or 44.3 ml/100 ml blood (normal 40.953); red blood cell count, 6.26 x 106/mm3 blood (normal 4.51 10.36 x 106); MCV (mean corpuscular volume): 72 ~3 per red cell (normal 90.4 * 4.8); MCH (mean corpuscular hemoglobin): 24.7 pg per red cell (normal 30.2 f 1.9); MCHC (mean corpuscular hemoglobin concentration): 33.8 g/100 ml red cells (normal 33.6fl .l). Her hemoglobin consisted of 100% Hb F; Hb A and Hb A2 were absent when the hemoglobin was analyzed by cellulose acetate and starch gel electrophoresis and by DEAE-Sephadex column chromatography. The y-chain of her Hb F was purified, and the y CB-3 peptide isolated and analyzed for its glycine and alanine content; the glytine content was 0.61 and the alanine content 2.41 residues. Family studies demonstrated that the individual’s mother, one of three siblings, two maternal aunts, and one of their three children are heterozygous for HPFH; their hematologic values are normal, but hemolysates of their red cells contain 2030% Hb F with a glycine/alanine content in y CB-3 peptide of 0.46-0.52/2.6-2.49 (the usual gly/ala ratio found in the Hb F of adults). The other two siblings are normal hematologically with normal levels of Hb A2 and Hb F, and no stigmata of heterozygous thalassemia. The father was unavailable for study. In view of the family studies, the only other possible diagnosis for this individual with 100% Hb

F, other than homozygous HPFH, would be the doubly heterozygous state for HPFH and SD-thalassemia, a combination not yet reported in the literature, but which would be expected to be associated with definite signs of thalassemia (enlarged spleen, anemia, and hypochromic microcytic red cells) which are absent in this case. Intact peripheral blood red cells were incubated in the presence of 3H-leucine, and the globin chains of the hemolysate subsequently separated by carboxymethylcellulose column chromatography in the presence of 8 M urea, as shown in Figure 2. The results show that the reticulocytes from the individual with homozygous HPFH synthesize roughly twice as many a-chains as y-chains (y/n synthetic ratio is 0.52). Similar results were described in another case of homozygous HPFH by Charache et al. (1975, Clinical Research 23, 397A). Thus although homozygous HPFH is not associated with anemia and other features of thalassemia, synthetic studies do indicate a certain degree of imbalance between 01- and non-a (y)-chain synthesis in these cases. The degree of imbalance is similar to that seen in heterozygous /3-thalassemia, but is less severe than that seen in homozygous /3-thalassemia. On the other hand, synthetic studies in heterozygous HPFH have shown that a- and non-a (y + p)chain synthesis is balanced in these cases (Natta et al., 1974; Sofroniadou et al., 1975).

Globin Messenger RNA Analysis by RNA-cDNA Hybridization Total cellular RNA isolated from peripheral blood red cells of the individual with homozygous HPFH was hybridized with human (Y- and ,G-globin cDNA (Figure 3) in saturation-hybridization assays using constant amounts of full-length cDNA (Figure 4) and variable amounts of RNA, as shown in Figure 3. The results demonstrate that the RNA achieves a plateau of over 90% hybridization with the ar-cDNA 0.6

r

FRACTION NUMBER

Figure

2. Globin

Chain

Synthesis

in HPFH

Peripheral blood cells from the individual homozygous for HPFH were incubated with V-l-leucine, and 40 mg of globin from the cell hemolysate were fractionated by carboxymethylcellulose column chromatography as described in Experimental Procedures. The column chromatogram is shown: (-0) AzaO; (O-----O) cpm/ml of each fraction.

[+Globin 325

Gene

Deletion

in HPFH

probe at a low RNA input, whereas a plateau of only approximately 10% hybridization is achieved with the P-cDNA. Even at an RNA input 100 times greater than that required to achieve half-saturation of hybridization with the a-cDNA, there is no increase over the baseline of 10% hybridization of the RNA with the ,GcDNA. The /3-cDNA probe is the DNA copy of mRNA from a patient with a-thalassemia (Hb H disease), which contained approximately 85% pmRNA and 15% a-mRNA (Housman et al., 1973). The 10% hybridization plateau of the HPFH RNA with the P-cDNA can therefore be attributed to hybridization of a-mRNA with the small amount of 01cDNA present in the ,&cDNA probe. Normal (nonthalassemic) reticulocyte RNA achieved 80-90% hybridization with both (Y- and P-cDNA probes at approximately equal inputs of RNA (data not shown). The results indicate that /3-mRNA is absent from the reticulocyte RNA of the patient with homozygous HPFH, because there is no significant hybridization of this RNA with the /I-cDNA probe. There may also be absence of the S-chain mRNA of Hb AZ, because S-mRNA would be expected to crosshybridize with &cDNA due to the close homologies of the S- and P-chain amino acid sequences and, presumably, of their mRNAs. In patients homozygous for ,@-thalassemia associated with intact synthesis of Hb AZ but absence of Hb A and P mRNA, there is some significant hybridization of reticulocyte RNA with ,&cDNA at high RNA inputs, and this finding has been attributed to &mRNA-PcDNA cross-hybridization (Forget et al., 1974; Ottolenghi et al., 1975). This low degree of hybridization

with P-cDNA at high RNA inputs is not observed with RNA from patients with homozygous GP-thalassemia (Forget et al., 1974) and HPFH (Figure 3) in which there is no synthesis of Hb AZ. Reticulocyte RNA from the case of homozygous HPFH studied by Kan et al. (1975b) gave a similar hybridization pattern with /3-cDNA, indicating absence of p- (and presumably S-) mRNA in that case also (data not shown).

Figure 3. Hybridization and @DNA

Figure 4. Polyacrylamide in the Presence of 99%

of HPFH

Reticulocyte

RNA with

Human

n-

Saturation hybridization curves were obtained as described in Experimental Procedures. The cDNA input was 540 cpm of u-cDNA and 538 cpm of V-f-/3-cDNA. After digestion with Sl nuclease, the blank (minus RNA) reaction mixtures contained 24 cpm (a-cDNA) and 17 cpm (/3-cDNA) above a background of 20 cpm. (-0) n-cDNA; ( -) /3-cDNA.

Hybridization of Cellular DNA with (Y- and ,B-Globin cDNA Lymphoid cell cultures were established from peripheral blood lymphocytes by transformation with virus from marmoset lymphoblastoid cells and served as a source of total cellular DNA. These cells remain diploid while in continuous culture and contain a normal complement of 48 chromosomes. DNA isolated from these cells was hybridized with CI- and P-cDNA in conditions of DNA excess to quantitate its relative content of (Y- and ,&globin gene DNA. In these studies, as in Figure 3, we used cDNA which was synthesized in the presence of a high concentration (0.2 mM) of radioactive dCTP to obtain full-length cDNA copies of the globin mRNA (Efstratiadis et al., 1975). When globin cDNA is synthesized in the presence of lower concentrations of deoxynucleotide triphosphates, it consists of a mixture of different sized cDNAs, many of which are shorter than the globin mRNA (Efstratiadis et al., 1975). Absence of hybridization to full-length cDNAs would imply absence (deletion) of the entire structural gene rather than deletion of only that portion of the structural gene which is represented in the shorter cDNAs. Figure 4 shows the results of polyacrylamide gel electrophoresis, in the presence of 99% formamide, of the /I-cDNA probe used in

Gel Electrophoresis Formamide

of Human

B-cDNA

Electrophoresis was carried out as described in Experimental Procedures The position of the RNA markers, run on the same gel, are indicated by the brackets, and were visualized directly by staining with methylene blue. The gel was cut into 2 mm thick transverse slices and assayed for radioactivity as described in Experimental Procedures.

Cl?ll 326

the experiments described here. The cDNA migrates as a homogeneous band with a mobility slightly slower than the 10s globin mRNA marker; a DNA fragment of 700 nucleotides in length obtained by endonuclease digestion of SV40 DNA was analyzed on a parallel gel and migrated slightly slower than the cDNA (data not shown). The (YcDNA gave a similar pattern (data not shown). These results indicate that the cDNAs used in these experiments are a representation of virtually the entire length of the respective globin mRNAs and their structural genes. The results of hybridization of normal and HPFH lymphocyte DNA to the (Y- and P-cDNAs are shown in Figure 5. The normal DNA hybridizes equally well with the (Y- and ,&cDNA, and achieved 75-80% hybridization with both probes at a Cot consistent with the hybridization kinetics of single-copy DNA (Figure 5). The HPFH DNA hybridizes with the a-cDNA in the same manner and with the same kinetics as does normal DNA (Figure 5). However, hybridization of the HPFH DNA to the ,8-cDNA gives a plateau of only 30% hybridization (from 10% at 0 time). In the case of homozygous HPFH described by Kan et al. (1975b), fibroblast DNA hybridized with ,i3cDNA to a plateau of 35% hybridization (from 0% at 0 time). Some of this hybridization is no doubt due to hybridization of a-gene DNA with the small amount of a-cDNA present in the ,B-cDNA probe, but there is probably also some additional hybridization, the nature of which is unknown and which is considered to be nonspecific. Similar levels of presumed nonspecific hybridization have been observed when DNA from infants with homozygous a-thalassemia, due to deletion of the a-genes, is hybridized with a-cDNA (Taylor et al., 1974; Ottolenghi et al., 1974; Ramirez et al., 1975; Kan et al., 1975a). We conclude therefore that there is probably no significant hybridization of the HPFH DNA with pcDNA, and that the ,&globin structural gene is deleted in this individual with homozygous HPFH. Discussion In the type of HPFH which is most common in Blacks, there is absence of Hb A and Hb AZ, but both the Gy and Ay forms of Hb F are present in the ratio usually found in adults (Huisman et al., 1971; Weatherall and Clegg, 1975). In the individual with this condition that we studied, reticulocyte RNA totally lacks P- (and probably 6-) globin mRNA, as demonstrated by absence of significant hybridization of this RNA with P-globin cDNA. Cellular DNA from this individual also fails to hybridize significantly with ,&cDNA, indicating deletion of the pglobin genes. The deletion probably involves the entire ,B-structural gene because the /3-cDNA used

20

B

40

!i E E

60

ae 60 I

I

102

103

100

CONC

A

x TIME : Cot

‘\

60

\

t SO

t

100 1, Ibl

B Figure

5. Hybridization

I

I

102

103

a-cDNA ‘.

‘*

--*-VT.

cot of Lymphocyte

lo4

DNA with w and b-cDNA

DNA excess hybridization was carried out as described in Experimental Procedures. (A) normal lymphocyte DNA; (B) HPFH lymphocyte DNA. (o----o) WCDNA; (u) p-cDNA.

as a hybridization probe was a full-length copy of the /3-globin mRNA, and this P-cDNA failed to give any significant hybridization with the HPFH cellular DNA. We are unable to conclude that the d-chain gene is also deleted, because it is not yet possible to obtain pure &cDNA to test for S-gene DNA specifically, and it is not known to what degree /3-cDNA cross-hybridizes with S-gene DNA in the experimental conditions used. It is not clear how the presence of a P- (or ,l3+ S-) globin gene deletion results in a continued high level of activity of the y-globin genes, which are presumably situated to the N terminal coding side of the S- and ,Gglobin genes. Concomitant deletion of adjacent y-chain control genes is one possible explanation, but these would be situated to the C terminal coding site of the y-structural genes. This hypothesis, however, is highly speculative and awaits direct confirmation. Not all forms of HPFH are associated with deletion of the p- (and S-) structural genes. In the other types of HPFH seen in Blacks, the Hb F may be only of the Gy type or predominantly of the Ay type,

[I-Globin 327

Gene

Deletion

in HPFH

and it is generally thought that in these conditions also the 6- and @globin genes are inactive in cis. However, in rare Blacks with various types of HPFH, activity of the P-gene in c/s has been demonstrated (Huisman, Miller, and Schroeder, 1975; Stamatoyannopoulos et al., 1975; Friedman and Schwartz, 1976). In the Greek (Ay type), British, and Swiss types of HPFH, there is also most probably activity of the P-globin gene in cis to the HPFH gene (Weatherall and Clegg, 1975). It is not possible therefore to devise a unified theory of the molecular basis of HPFH based on deletions of the ,8- and Sstructural globin genes alone. Experlmental

Procedures

Measurement of Giobin Synthesis in intact Ceils 1.5 ml of peripheral blood cells were washed 3 times in KrebsRinger’s phosphate (KRP) buffer (pH 7.4), then resuspended in 1 ml of KRP. and 1 ml of AB. Rh positive plasma which had been extensively dialysed against KRP. To the mixture were added a solution of 19 amino acids, minus leucine (final concentration 15 pM), 2 mg/ml of D-glucose, and 100 $i of 3H-leucine (New England Nuclear, 81.3 Ci/mmole). The cells were incubated for 2 hr at 37”C, then washed 4 times with saline, and lysed as described previously (Benz, Swerdlow, and Forget, 1973). Globin was prepared from the membrane-free hemolysate by acid acetone precipitation and fractionated by chromatography on a column of carboxymethylcellulose (CM-52) as previously described (Kan, Schwartz, and Nathan, 1968). 1 ml samples of each fraction were assayed for radioactivity in a liquid scintillation counter after addition of 10 ml Insta-Gel (Packard Instrument Co.). RNA isolation and cDNA Synthesis Reticulocyte RNA was prepared from membrane-free red cell hemolysates by detergent and phenol extraction as previously described (Benz and Forget, 1971; Benz et al., 1973; Benz, Swerdlow, and Forget, 1975). Globin mRNA was isolated from the total reticulocyte RNA by sucrose gradient centrifugation (Benz and Forget, 1971; Benz et al., 1973) and/or oligo(dT)-cellulose column chromatography (Aviv and Leder, 1972). Human ol-globin mRNA was purified from nonthalassemic globin mRNA by polyacrylamide gel electrophoresis in the presence of 99% formamide (Forget et al., 1975), and was shown by rerunning on gels and by RNA-cDNA hybridization assays to be 95% pure. The human P-globin mRNA was the reticulocyte globin mRNA of a patient with a-thalassemia (Hb H disease), and was 85-90% P-mRNA as determined by RNAcDNA hybridization (Housman et al., 1973) and by polyacrylamide gel electrophoresis in the presence of 99% formamide (Forget et al., 1975). Human a- and /I-globin cDNA were synthesized from the a- and fi-mRNAs by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus (AMV) in the presence of high concentrations of dNTPs to obtain full-length cDNAs (Efstratiadis et al., 1975). The reaction mixtures contained in 100 (I: 50 mM Tris-HCI (pH 8.3), 60 mM NaCI; 6 mM magnesium acetate; 10 mM dithiothreitol; 0.6 mM dGTP, dATP, dTTP; 0.2 mM ‘H-dCTP (New England Nuclear, 24.8 Ci/mmole); 100 pg/ml actinomycin D; 20 pg/ml oligo (dT)12--18 (Collaborative Research); 5 pg globin mRNA; and 7 units of AMV DNA polymerase. The AMV DNA polymerase was prepared by Drs. D. Beard. and J. W. Beard by the method of Kacian et al. (1971) and was provided through the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute. After phosphocellulose chromatography, the enzyme was dialysed against 50% glycerol, 0.2 M potassium phosphate (pH 7.2) 2 mM dithiothreitol, and 0.2% Triton X-100. It was stored frozen at -8OOC until used, at a concentration of 1390 units per ml. One unit of

enzyme activity is defined as the amount of enzyme required to incorporate into acid-insoluble material 1 nmole of dTMP at 37°C in 10 min using as substrate poly(rA-dT)ll. After incubation at 37°C for 1 hr, the reaction mixture was hydrolysed for 4 hr at 37°C in the presence of 0.3 M NaOH. After neutralization with HCI, the cDNA was isolated from the mixture by gel filtration through a 1 X 45 cm column of Sephadex G-150 equilibrated with 0.1 M ammonium bicarbonate. To the fractions containing the cDNA were added 100 pg E. coli tRNA, 0.1 vol of 20% sodium acetate (pH 5.4) and 2 vol of 95% ethanol. After remaining overnight at -2O”C, the cDNA was recovered by centrifugation (100,000 x g for 1 hr), dried under a stream of nitrogen, then dissolved in H20 and kept frozen at -20°C until use. The size of the cDNA was determined by polyacrylamide disc gel eiectrophoresis in the presence of 99% formamide (Forget et al., 1975) with nonradioactive RNA markers run on the same gel. After electrophoresis initially for 1 hr at 1 ma per gel, then 2 hr at 2 ma per gel, at room temperature, the gels were stained with methylene blue to identify the RNA markers (Forget et al., 1975) cut into 2 mm slices which were digested overnight with 0.5 ml 30% Hz02 at 6O”C, and counted for radioactivity in a liquid scintillation counter after addition of 10 ml Insta-Gel (Packard Instrument Co.). RNA-cDNA Hybridization Assay Saturation hybridization curves were obtained as previously described (Housman et al., 1973). Each assay contained approximately 500 cpm of cDNA corresponding to approximately 0.07 ng of cDNA, at a counting efficiency of 18% for 3H (on millipore filters in toluene-PPO-POPOP). The fixed amount of cDNA was incubated with variable amounts of RNA, in 0.2 M sodium phosphate (pH 6.8) and 0.5% SDS, for 40 hr at 78°C. in these conditions, limiting amounts of RNA will hybridize to completeness with the cDNA, and there is no cross-hybridization of v-chain mRNA with P-chain cDNA(Housman et al., 1973, 1974; Forget et al.. 1974). The amount of hybridization is determined by measuring the percentage of the cDNA which becomes resistant to digestion by the Sl nuclease of Aspergillus oryzae (Housman et al., 1973). In the case of the HPFH RNA, total unfractionated reticulocyte RNA was hybridized to these cDNAs, rather than sucrose gradient or oligo (dT)cellulose-Durified RNA. Lymphoid Ceil Cultures Peripheral blood was collected in preservative-free heparin (Weddel Pharmaceuticals, Ltd., London), and lymphocytes were separated by Ficoll-Hypaque sedimentation (Boyum, 1968). Primary cultures were seeded at a density of 2 x 106 cells per ml in 10 ml cultures, and were fed twice a week by allowing the cells to sediment at unit gravity for 1 hr, followed by removal of 3 ml of the supernatant and replacement with 3 ml of fresh medium. Primary cultures were initiated and maintained in medium composed of 100 ml RPM1 1629 (Iwakata and Grace, 1964) 20 ml fetal bovine serum, 1 ml horse serum, 1 ml 100 mM sodium pyruvate, 1 ml of 100X Eagle’s “nonessential” amino acids (Eagle, 1959) 1 ml of 1 mM mercaptoethanol [Lazarus, Barell, and Oppenheim, 1974, In Vitro, 9. 370 (Abstract)], and 1 ml PSG: 1 x 104 units per ml sodium penicillin G, 1 x 104 pg per ml streptomycin sulfate, and 0.2 M L-glutamine. Preparations of transforming virus were obtained from the Marmoset lymphoblastoid cell line 895-8 (Miller and Lipman, 1973; Robinson and Miller, 1975). A starter culture was provided by Dr. George Miller, Yale University School of Medicine. 0.1 ml of virus-containing medium was added per ml of culture. After 4-6 weeks, exponential growth occurred, and the cultures were fed by addition of medium: 100 ml S-MEM (Eagle, 1959) 10 ml fetal bovine serum, 1 ml of 100X “nonessential” amino acids (Eagle, 1959), 1 ml of 100 mM sodium pyruvate, and 1 ml PSG. Large cultures were grown in the same medium in Erlenmeyer flasks on a magnetic stirrer. The cell lines described here maintain

Cell 328

a viability, as measured by dye exclusion (Nigrosin), in excess of 90%. They have a population doubling time of 24-48 hr and attain a maximum density of 2-3 x 106 cells per ml. Cultures were examined periodically for mycoplasma and have consistently been found to be negative. DNA lsolatlon from Lymphold Cell Nuclei Lymphoid cells were collected by centrifugation, washed 3 times with saline, then resuspended in 4 vol of a solution of 10 mM TrisHCI (pH 7.6), 40 mM KCI. 5 mM MgC&. (TKM), 1 mM dithiothreitol. and 0.1% Nonidet P-40, and allowed to lyse for 20 min at 4°C. The cells were then homogenized manually with 20 strokes in a Teflon pestle tissue grinder, and the nuclei sedimented by centrifugation at 630 x g for 5 min. Cell lysis was monitored by microscopic examination. The nuclei are then washed 3 times with TKM containing 1 mM dithiothreitol and isolated by centrifugation through a 3 ml cushion of 2 M sucrose containing 0.1 M EDTA, 150 mM NaCI, and 20 mM Tris-HCI (pH 7.6) at 38,000 x g for 45 min at 4°C using a SW-40 rotor. The nuclei are resuspended in 20-25 vol of a solution of 0.1 M EDTA (pH 8.0) and 0.15 M NaCI; 10% sodium dodecyl sulfate is then added to a final concentration of 0.5%, and the suspension is stirred for 10 min at 60°C. Sodium perchlorate is added to a final concentration of 1 M, and the solution is spun at 5000 x g for 10 min at 4°C. The supernatant is then repeatedly extracted with an equal volume of chloroform:isoamyl alcohol (5OO:l) until there is a clear interface. To the aqueous phase is added 5 M NaCI, to a concentration of 0.15 M, and 2 vol of 95% ethanol. The DNA is spooled out of the solution using a glass rod, then dissolved in 0.1 x SSC by stirring overnight at 4°C. The DNA solution is treated with pancreatic RNAase (previously boiled for 5 min at a concentration of 1 mg/ml) at an enzyme concentration of 50 gg/ml at 37°C for 30 min, followed by treatment for 2 hr at 37°C with proteinase K (E-M Laboratories) at a final enzyme concentration of 250 gg/ml in IO mM Tris-HCI (pH 7.6) 0.1 M NaCI, 1 mM EDTA, and 0.5% SDS. The treated DNA solution is then extracted with chloroform-isoamyl alcohol until the interface is clear, and the DNA is alcohol-precipitated, spooled out, and redissolved in 0.1 x SSC as described above. The DNA concentration is adjusted to 1 mg/ml, and the DNA is sheared by passage through a French Pressure Cell at 40,000 psi. The sheared DNA is precipitated by addition of 1 /lO vol sodium acetate (pH 5.4). and 2 vol of 95% ethanol. After standing overnight at -20°C. the DNA is recovered by centrifugation, dissolved in H20, and desalted by passage through Sephadex G-25. The A&AZsO and A2&AZ3,, of the lymphocyte DNA used in the experiments described here were 1.7-l .4 and 2.5-3, respectively. The size of the sheared DNA was similar to that of the cDNA when analyzed by alkaline sucrose gradient centrifugation (5-20% sucrose in 0.3 N NaOH, 0.9 M NaCI, 5 M EDTA, 35,000 rpm for 17 hr at 4°C in a SW-40 rotor). However, when analyzed by polyacrylamide gel electrophoresis in 99% formamide (Forget et al., 1975) the DNA migrated as a broad band between the 7s and 10s RNA markers. 3.75 mg of DNA were lyophilized to dryness in individual tubes for each Cot curve hybridization experiment. DNA-cDNA Hybridization Assays Sheared lymphoid cell DNA was hybridized to globin cDNA in conditions of cellular DNA excess. The hybridization mixture contained, in a volume of 250 ~1: 3.75 mg cellular DNA, 2000 cpm (approximately 0.28 ng) of ‘H-aor /3-globin cDNA, 50% formamide (Eastman X565) and 3 x SSC (0.45 M NaCI, 0.045 M sodium citrate) (Deisseroth, Velez, and Nienhuis, 1976). Replicate samples of 25 pl were sealed in individual capillary tubes, boiled for 10 min, then incubated at 49°C for various lengths of time, from 10 min to 46 hr. After incubation, the contents of the capillary tubes were expelled into 0.5 ml of 0.05 M sodium phosphate buffer (pH 6.8) and frozen at -20°C until assayed. The percentage of hybridization was determined by hydroxylapatite batch elution. Each individual reaction was incubated with 1 g of hydroxylapatite (Bio-Rad, DNA

grade) in 5 ml of 0.05 M sodium phosphate buffer (pH 6.8) at 65°C. The resin was then pelleted by centrifugation and successively washed with 0.16 M and then 0.5 M sodium phosphate. The percentage of hybridized (double-stranded) material is expressed as the TCA-precipitable cpm (X 100) of the cDNA which eluted at 0.5 M sodium phosphate divided by the total TCA-precipitable cpm eluted at both 0.16 M and 0.5 M sodium phosphate. Acknowledgments We thank Drs. D. Nathan, H. Abelson, A. Deisseroth. and L. Schnipper for helpful discussions and technical advice. R. Gorer and D. Paci provided skilled technical assistance. We thank Drs. D. Beard and J. W. Beard for the AMV DNA polymerase; Dr. G. Miller for the starter culture of 895-8 cells; S. Drost for the SV40 DNA marker; and Dr. S. Charache for providing blood from the second case of homozygous HPFH for mRNA analysis. B. G. F. is the recipient of a USPHS Research Career Development Award. This work was supported in part by a contract from the National Cancer Institute and by grants from the NIH. Received

November

25, 1975;

revised

December

30, 1975

References Aviv, H., and Leder. P. (1972). Purification of biologically globin messenger RNA by chromatography on oligothymidylic cellulose. Proc. Nat. Acad. Sci. USA 69, 1408-1412.

active acid-

Benz, E. J., Jr., and Forget, B. G. (1971). Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia. J. Clin. Invest. 50, 2755-2760. Benz, E. J., Jr., Swerdlow, P. S., and Forget, B. G. (1973). Globin messenger RNA in hemoglobin H disease. Blood 42, 825-833. Benz, E. J., Jr., Swerdlow. P. S., and Forget, B. G. (1975). Absence of functional messenger RNA activity for beta globin chain synthesis in ,@-thalassemia. Blood 45, l-10. Boyurn, marrow.

A. (1968). Separation Stand. J. Clin. Lab.

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