Cell, Vol. 39, 123-128, November 1984, Copyright 0 1964 by MIT 0092-8674/84/l 10123-06 $02.00/O

The Silent Carrier Allele: ,8 Thalassemia without a Mutation in the @-Globin Gene or Its Immediate Flanking Regions

Gregg L. Semenza, Kathleen Delgrosso, Mortimer Poncz, Padmini Malladi, Elias Schwartz, and Saul Surrey Division of Hematology The Children’s Hospital of Philadelphia Department of Pediatrics and Department of Human Genetics University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104

Summary

A molecular genetic analysis has been performed using as subjects an Albanian family in which the father is a silent carrier, the mother has high Hb AZ- (3 thalassemia trait, and both children have 13 thal- assemia. Nucleotide sequence analysis of the daughter’s paternal @-globin gene and its flanking regions failed to reveal any base changes of known functional significance. When introduced into HeLa cells the gene was expressed at normal levels with proper processing of RNA. Haplotype analysis re- vealed that the affected son and daughter inherited different &3-globin gene clusters from the father. The silent carrier allele is not due to a mutation within the @-globin structural gene or its flanking regions and as such represents a novel form of 8’ thalassemia.

Introduction

Beta thalassemia is a heterogeneous hereditary disease characterized by deficient production of normal ,L%globin chains of hemoglobin (Hb) A. In persons homozygous for p thalassemia, there is an imbalance of globin-chain syn- thesis with precipitation of excess a-globin chains, causing ineffective erythropoiesis and anemia of varying severity. In studies at the molecular level, $ thalassemia alleles have been shown to represent P-globin genes with partial deletions, nonsense mutations, frame-shift mutations, and point mutations at intervening sequence splice junctions. Mutations that cause 0’ thalassemia identified thus far involve the promoter region 5’ to the mRNA cap site, intervening sequences, coding sequences adjacent to splice junctions, and the polyadenylation signal (reviewed by Nienhuis et al., 1984; Collins and Weissman, 1984).

Functional studies, in which cloned genes are introduced into cultured mammalian cells and the transcribed RNAs are analyzed, have confirmed the thalassemic defects identified by nucleotide sequence analysis in a number of @-globin genes. To some extent, it has been possible to correlate the site of mutation with the severity of disease: splice junction mutations abolish normal RNA processing and cause p” thalassemia, while intervening sequence mutations variably decrease the efficiency of processing and are of p’ phenotype (Treisman et al., 1983). All @- thalassemia alleles examined by these structural and func-

tional analyses have been shown to represent mutant p- globin genes.

In addition to efforts to analyze p thalassemia at the molecular level by demonstration of the disease-causing mutations, complementary studies have attempted to es- tablish prenatal diagnosis of this condition by utilizing the presence of linked polymorphisms within the 60 kb of DNA spanning the fl-globin gene cluster (reviewed by Orkin et al., 1983; Boehm et al., 1983). The results of this analysis of haplotypes (patterns of restriction site polymorphism) suggest the origin of different mutations on different chro- mosomal backgrounds (Orkin et al., 1982).

An interesting variant form of /I thalassemia found in the Mediterranean population is the silent carrier allele. In certain families, individuals inherit p thalassemia from par- ents of whom only one is a clinically recognizable carrier. The other parent, the silent carrier of p thalassemia, can only be identified as such genetically by affected offspring and biochemically by detection of a decreased P/a globin synthesis ratio in peripheral erythroid cells. The microcytic, hypochromic erythrocytes containing elevated levels of Hb An, Hb F, or both, characteristically seen in persons het- erozygous for p thalassemia, are not present (Schwartz, 1969). In this report we describe our studies of an Albanian family in which a silent carrier allele of paternal origin and a typical high Hb A2-/3 thalassemia allele of maternal origin have each been inherited by two offspring who are thus compound heterozygotes and clinically are milder than the usual P-thalassemia homozygotes.

Results

Screening for Structural Variants at the DNA and Protein Levels The organization of the p-like globin gene complex in genomic DNA from this family was analyzed by gene- blotting studies following digestion with a variety of restric- tion enzymes. Normal fragment sizes (Flavell et al., 1978; Poncz et al., 1983) were observed with Barn HI, Eco RI, and Pst I, therefore eliminating the possibility of gross rearrangement or deletion resulting in the silent carrier phenotype. Furthermore, digestion with Barn HI revealed no evidence for triplicated a-globin genes (Goossens et al., 1980) therefore minimizing the possibility that imbal- anced p/a globin synthesis ratios in these patients were due to a-globin overproduction caused by the presence of an additional a-globin gene. Results from isoelectric focusing and electrophoresis of hemolysates on triton-acid urea polyacrylamide gels (Rovera et al., 1978) also failed to reveal any abnormal hemoglobins or P-chain structural variants.

Identification of Cloned ,9-Globin Genes of Paternal and Maternal Origins A genomic DNA library was prepared from the daughter’s DNA and P-globin gene clones were isolated, character- ized, and identified as paternal and maternal p genes using

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informative linked polymorphic restriction enzyme sites. The closest such sites to the /3 gene were a pair of polymorphic Hint II sites within and 3’ to the #@globin locus (see Figure 5, and Antonarakis et al., 1982). Both daughter and father were (--/++) while the mother was (--/-+), indicating that the daughter’s paternal chromo- some was (++). Although these sites are too far from the p gene to be included in a single lambda clone, a poly- morphic Taq I site 5’ to the 6 gene, which in Caucasians is in linkage disequilibrium with the Hint II site 3’ to +/3 (Maeda et al., 1983) is sufficiently close to the p gene, and was informative in this family. Clones with the p gene that also encompassed this region were isolated from the daughter, and paternal and maternal clones were identified as being Taq I (+) and (-), respectively, by blotting and directed sequence analysis of this region (data not shown). Confirmation of these assignments was also made using a recently described Rsa I polymorphism (Semenza et al., 1984).

Nucleotide Sequence Analysis of the Cloned Paternal @-Globin Gene A 4.4 kb Pst I fragment from clone KB90, which contained the 1.6 kb paternal @globin gene, as well as 2.2 kb of 5’- and 0.55 kb of 3’-flanking sequence, was subcloned into M13mp9 in both insert orientations. Single-stranded DNA was isolated and annealed to single-stranded DNA from an Ml3 phage containing a reference 4.4 kb Pst I /3-globin gene. Sl nuclease analysis of heteroduplexes demon- strated, in addition to the protected 4.4 kb fragment, two additional pairs of fragments, each of which (a + d, b + c in Figure 1) added up to 4.4 kb, suggesting that two areas of nonhomology identified by Sl nuclease existed between KB90 and the normal /?-globin gene. To rule out cloning artifacts as a cause of nonhomology, the 4.4 kb fragment from KB5, a second paternal clone, was also inserted into Ml 3mp9. KB5/KB90 heteroduplexes digested with Sl nu- clease yielded only the expected 4.4 kb fragment, while heteroduplexes between KB5 and the reference p gene resulted in the same fragments seen using KB90.

The entire nucleotide sequence was determined from 135 bp downstream of the 5’ Pst I site to 130 bp upstream of the 3’ Pst I site, with 60% of the entire region and 75% of the 1.6 kb sequence encompassing the P-globin gene determined on both DNA strands. In addition, sequence information determined from only one DNA strand was confirmed by analysis of insert subclones containing over- lapping regions generated by either random or nonrandom DNA sequencing strategies in Ml3 (see Experimental Procedures), so that all the nucleotides in the sequence were analyzed in at least two overlapping clones,

Within the 1.6 kb from cap site to poly(A) addition site, no nucleotide substitutions other than known polymor- phisms were found. Within the 3’-flanking sequence, a previously unreported A-T transversion was found at 340 bp 3’ to the polyadenylation site (see Figure 2). Within the 5’-flanking region, two sequence changes were found at positions consistent with the fragment sizes observed in

the Sl nuclease heteroduplex assays shown in Figure 1. A series of six ATTTT repeats was found at approximately 1400 bp 5’ to the cap site where the reference @globin gene contained five such repeats. (ATTTT), at this location is a known length polymorphism with n varying between four and six (Spritz, 1981). A second change at -530 bp consisted of a complex rearrangement resulting in the insertion of ATA and deletion of a T residue (Figure 2). Just 5’ to this site an additional T-C transition was found at -554 bp; this creates a new Rsa I site, which we have shown is polymorphic in many populations (Semenza et al., 1984). T to C transitions were also found at -706 bp and -1873 bp.

Functional Analysis of Cloned Paternal and Maternal B-Globin Genes Analysis of reticulocyte RNA shows globin gene-specific, Sl nuclease-resistant fragments corresponding in size to exons 1 and 2 of p and exon 1 of (Y (Figure 3, lanes I-5). Identically sized fragments are also observed when refer- ence p and a globin genes are transiently expressed in HeLa cells (Figure 3, lanes IO, 11, and 12).

Expression of the maternal @-globin gene in the HeLa cell system shows complete absence of protected frag- ments corresponding in size to exon 1, a small amount of label in lower molecular weight bands, and the presence of high molecular weight Sl -resistant fragments at the top of the gel (Figure 3, lanes 8 and 9). This pattern is characteristic of the IVS-1 position 1 splice-junction muta- tion that abolishes normal RNA processing (Treisman et al., 1983). The daughter’s maternal haplotype is the same as that associated with this splice mutation in Mediterra- neans (Orkin et al., 1982a; see below), indicating that the

Figure 1. Heteroduplex Analysis of Cloned p-Globir Genes Usrng Si Nuclease

The 4.4 kb Pst I fragment containing the o-globin gene was subcloned in both orientations in M13mp9. Srngle-stranded DNA of opposite orientation was annealed, digested with Sl nuclease. and fractionated by 0.8% agarose gel electrophoresis. Lanes: (1) KB90 x KB5; (2) KB5 x KBW; (3) HfllS x KB90: (4) HfllS x KB5; (5) KB5 x H@lS. The frrst member of each pair IS in the (+) orientation (coding strand) and the second member is in the (-) orientation. KB5 and KB90 are independently isolated clones of the daughter’s paternal @-globin gene. HfllS is a control fl-globin gene. Lane M IS a marker lane containing a Hind III digest of bacteriophage lambda DNA.

Went Camer Allele of fl Thalassemra 125

maternal P-globin gene has been inactivated by a previ- ously described $ thalassemia mutation.

Expression of the paternal gene (Figure 3, lanes 6 and 7) shows neither qualitative nor quantitative differences

-I 873 -706 -554 -530 +340

Figure 2. Nucleotrde Sequence Analysts of the fl-Globrn Gene and Its Flanking Regrons in Clone KB93

Solid block indrcates posrtion of the fi-globrn gene, from mRNA cap to polyadenylation sites, wrthin the 4.4 kb Pst I fragment analyzed by the drdeoxy chain-termination method. Location of specific nucleotides In the 5’.flankrng region is rdentihed with respect to the cap sate and rn the 3’. flanking regron, wrth respect to the polyadenylation site. Prevrously unde- scribed nucleotrde sequence vanatrons are shown directly above the Irne; + srgnrfies Insertron and -, deletion, of the indrcated nucleotide(s).

e.1 ( . , .> I ,

Frgure 3. Sl Nuclease Mapping of @-Globin RNA: Exons 1 and 2

HeLa cells were cotransfected wrth a recombrnant plasmid containrng a normal mutant, or no P-globrn gene and a plasmid contarnrng a normal a- globin gene. Total RNA was Isolated after 48 hr and hybrrdized to a Bal I fragment spanning the @-globin gene wrth YP-labeling of exons 1 and 2. RNA in lanes 1-5, 7, 9. 11, 12 was also hybndrzed to a Hrnf I fragment contarnrng the al-globin gene labeled rn exon 1. Hybrids were digested wrth Sl nuclease and the reaction products were sized on 8% urea- polyacrylamide sequencrng gels. Lanes: (m) ?labeled size markers (Hae III digest of @Xl 74 DNA precedes lane 1; Hpa II digest of pBR322 DNA follows lane 12); (l-5) 50, 10, 5, 1, 0 ng of reticulocyte RNA: (6, 7) 30, 15 pg of SVpBR KB58 RNA (paternal); (8, 9) 15, 30 pg of SVpBR KBlll@ RNA (maternal), (10, 11) 15, 30 ag of SVpBR HB (reference) RNA; (12) 15 fig of SVpBR328 RNA

when compared to the reference p gene (lanes 10 and 11). Sl nuclease mapping of the 3’ end of fl-globin RNA showed protection of an identically sized fragment (260 nucleotides), corresponding to exon 3 in RNA transcribed from the reference, maternal, and paternal /3-globin genes (Figure 4) thus indicating normal splicing at the 3’ junction of IVS-2 and normal cleavage at the polyadenylation site in RNA. There is no evidence from these assays to indicate that the paternal P-globin gene contains a mutation affect- ing RNA transcription or processing.

Haplotype Analysis of the c&3-Globin Gene Cluster Since structural and functional analysis of the daughter’s paternal P-globin gene failed to reveal a thalassemia mu- tation, the inheritance of the silent carrier allele in relation to the inheritance of linked polymorphic restriction sites (haplotypes) in this family was analyzed to determine whether the allele was in fact linked to the P-globin gene cluster. The daughter and the son inherited haplotype C from the mother and either haplotype A or B from the father (Figure 5). Thus the two children, who are both compound heterozygotes for the maternal and paternal thalassemia alleles, inherited different paternal chromo- somal haplotypes despite the fact that the father is heter- ozygous for the silent carrier allele.

Because the father was homozygous at the polymorphic sites downstream from the Taq I site 5’ to the &globin gene, it is possible that a cross-over 3’ to the Taq site could have resulted in the son and daughter receiving trsfi-globin gene regions from the father that differed 5’ to the Taq I site but were identical 3’ to the crossover site. Thus the haplotype data, while consistent with the hypoth-

Frgure 4. St Nuclease Mapping of fl-Globrn RNA. Exon 3

HeLa cell RNA was prepared as described rn the legend to Figure 3 and rn the text under Experimental Procedures, and hybndrzed to a “P-labeled Rsa I-Pst I fragment spannrng exon 3. Lanes: (1,2) 5, IO pg of SVpBR HP RNA; (3, 4) 5, 10 @g of SVpBR KBlllP RNA; (5, 6) 5. IO fig of SVpBR KB5@ RNA.

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‘V 9 *e 5’dF-e A ,,mcll tdll a Hr 111 4, ttdl To+, 1 2 3 156 7

,A D - b

+ + +-+ + +++ - c+ - - --- -

I i-i -

n A+ - - --- - +++ - B- + - +++ + +++ -

0:; + - _ _ ‘_z +

ii+ - i-f - z

@,“= - - -0- - +++ - - - --- - i-i -

Figure 5. Haplotype Analysis of the Albanian Family

The 60 kb of DNA spanning the @-globin gene cluster is shown, with polymorphic sites labeled 1-l 1 for the various restrictlon endonucleases by which they are identified. The presence or absence of a polymorphic site IS Indicated by + or -, respectively. I refers to the parents and II, to their offspring. Cross-hatched half-kgure indicates a high Hb AZ-@ thalas- semla allele and solid half-figure Indicates a silent carrier allele.

esis that the silent carrier is not linked to the P-globin gene cluster, do not constitute conclusive proof. In either case, the nucleotide sequence and functional analyses demon- strate that the silent carrier allele is not due to a mutation within the @globin structural gene or its immediate flanking regions and as such represents a novel form of p’ thal- assemia.

Discussion

In a Greek family, a silent carrier allele was found to cosegregate with a variant p globin, Hb Knossos (Fessas et al., 1982). The variant was subsequently shown to be due to a point mutation in codon 27 of the P-globin gene, which results in the substitution of serine for alanine (Ma- malaki et al., 1983; Rouabhi et al., 1984). In addition, this nucleotide change causes a P-thalassemia phenotype by the generation of an alternative splice site (Orkin et al., 1984). A 6’ thalassemia has been reported to be present in cis position in this family (Mamalaki et al., 1983). Thus the Knossos silent carrier allele is in fact two presumably closely linked mutations, affecting & and b-globin gene expression.

This model cannot represent the molecular basis for all silent carrier alleles. Three Turkish families have been reported in which offspring were apparently homozygous for a normal Hb A& thalassemia allele (Aksoy et al., 1978). Hb AP in the offspring was elevated by about 30% above normal, indicating that 6’ thalassemia was not present in these families (Kattamis et al., 1979).

The most important conclusion obtained from our results is that the Albanian silent carrier is the first /3’ thalassemia allele in which no mutation has been demonstrable within the immediate P-globin gene region by the standard anal- yses of gene structure and function. In every other case of /3’ thalassemia, a mutation has been detected by nucleotide sequence analysis, either within the @-globin gene or within 100 bp of it. There are cases, however, in which @globin gene expression is affected by distant sequences. A Dutch patient with y&3 thalassemia was

shown to have a deletion that removed the entire gene cluster to 2.5 kb 5’ to the P-globin gene (van der Ploeg et al., 1980). The @-globin gene was intact and when cloned and introduced into HeLa cells was expressed normally; yet in erythroid cells the gene was not expressed, was hypermethylated, and was not sensitive to DNAase I diges- tion (Kioussis et al., 1983). In the case of the Albanian silent carrier allele, however, there is no evidence of a major DNA rearrangement or deletion.

We identified a novel oligonucleotide substitution in a region approximately 530 bp 5’ to the P-globin gene. This region consists of 28 rather than 26 alternating purine- pyrimidine dinucleotides followed by 14 rather than 16 pyrimidine residues (Semenza et al., 1984). In the daugh- ter’s paternal @-globin gene the insertion of an ATA trinu- cleotide and deletion of a T residue has occurred (Figure 2). Of considerable interest is the observation that in supercoiled plasmids containing the human @globin gene a discrete Sl nuclease-sensitive site exists within or close to this purine-pyrimidine tract, and that cleavage at this site is prevented by addition of high mobility group chromo- somal proteins HMG 1 and 2 (Cockerill and Goodwin, 1983). The polymorphic Rsa I site that was identified at -554 bp within the purine-pyrimidine tract is the result of a T-C transition, which preserves the alternating purine- pyrimidine nature of this region, adding support to the hypothesis that maintaining this structure is of functional significance (Semenza et al., 1984). The additional purine- pyrimidine dinucleotides could possibly have a functional effect by facilitating the transition between E?- and Z-DNA (Nordheim et al., 1982). Thus it is necessary to consider the possibility that chromatin changes due to mutations that do not involve gross rearrangement or deletion of DNA in the P-globin gene cluster may represent an entirely new category of /3-thalassemia lesions.

The analysis of DNA polymorphisms in the @-globin gene cluster suggests the possibility that the silent carrier allele may not be linked to the @-globin gene cluster. Precedent exists for transacting factors that affect the expression of genes in the @-globin gene cluster. In some cases of hereditary persistence of fetal hemoglobin (HPFH), haplo- type analysis has demonstrated that the HPFH determinant maps outside the region of the P-globin gene cluster (Old et al., 1982; Gianni et al., 1983). Recently, evidence for a Vans-acting factor affecting /3-globin gene expression at the level of transcription has been presented (Emerson and Felsenfeld, 1984). Presumably, genetic mechanisms exist that serve to maintain balanced levels of LY- and p- like globins in normal individuals. Coordinate regulation could be achieved at any level of gene expression by affecting transcription, RNA processing or stability, and translation or proteolysis. Any mutation that altered the balance to favor a-globin accumulation could cause a P- thalassemia-like syndrome.

In summary, there are multiple potential sites for muta- tions that could affect the ratio of ,& to cu-globin in erythroid cells and result in /3’ thalassemia beyond the mutations within the @-globin gene and its immediate flanking regions

Srlent Carrier Allele of r3 Thalassemra 127

that have been described thus far. A number of experi- mental approaches are presently being pursued in an attempt to elucidate the nature of the defect underlying the silent carrier phenotype at the molecular level. It is likely that this information will significantly add to our understand- ing of normal globin gene expression as well.

Experimental Procedures

Subjects Members of the famrly described rn the original case report of the silent carrier of fl thalassemra (Schwartz, 1969) were studred.

Materials Recombinant plasmrds and phages were generously provided by B. Forget (JWlOi, JW102, JW151) and T. Maniatis (pP3.9, H@lS, HPGl). Clonrng and expressron vectors were the kind gifts of G. Grosveld (SVpBR328) T. Maniatis and R. Treisman (&VHPplac, ?rSVHPol2). and H. Lehrach (EMBL3A). M13mp8 and M13mp9 were obtained from Bethesda Research Laboratones. The pentadecamenc DNA sequencrng primer was from New England Biolabs. E. coli strain 71-18 was a gift from J. Messing. E. coli strarns NM538 and NM539 were the grft of H. Lehrach.

Restriction Analysis of Genomic DNA Hrgh molecular werght DNA was prepared from specimens of perrpheral blood by the method of Poncz et al. (1982b). Restrictron endonuclease drgestron of genomic DNA and blot hybridization were performed as described (Semenza et al., 1984). For analysrs of the 6p- and a-globin gene IOCI, the probes used were a 10.8 kb Xba I genomrc fragment and a 1.2 kb Mbo II a-globrn cDNA fragment isolated from JWlOi, respectively Analysrs of DNA restnction site polymorphisms within the fl-globin gene cluster was performed as described (Antonarakrs et al., 1982; Maeda et al., 1983; Orkrn et al., 1982, 1983; Semenza et al., 1984) using the followrng probes. a 1.3 kb Eco RI-Barn HI genomic fragment containing the c-globrn gene; a 1.6 kb Cfo I y-globrn cDNA fragment Isolated from JWI 51; a 1.7 kb genomic Bgl II-Xba I fragment containrng $0 isolated from pP3.9; a 1.6 kb Bgl II-Eco RI genomic fragment from 5’ to 6; 1.9 kb Barn HI and 550 bp Rsa I genomic fragments from 5’ to 8; a fl-globin cDNA fragment Isolated by Cfo I drgestron of JW102.

Cloning of Maternal and Paternal @Globin Genes High molecular weight DNA from the daughter was prepared, partially drgested wrth Sau 3A, and size-fractionated by sucrose gradrent sedimen- tatron (Manratis et al , 1978). DNA fragments 15-20 kb in size were inserted Into the Barn HI sates of the lambda replacement vector EMBL 3A (Frischauf et al., 1983). Phages were packaged in vrtro (Scalenghe et al., 1981) and 1 X lo6 independent recombinants were screened by filter hybndrzation (Benton and Davrs, 1977) to a p-globrn cDNA probe isolated from JW102. DNA extracts from eight different positrve clones were Isolated after three rounds of plaque purification. Genomic blottrng on DNA from this famrly and directed sequence analysrs of /3-gene-containrng clones showed that KB90 and KB5 were of paternal origrn. whrle KBI 11 was of maternal orrgrn.

Nucleotide Sequence Analysis The 4.4 kb Pst I fragment containing the ,%globin gene was isolated by electrophoresrs on low meltrng point agarose, ligated into Pst l-digested M13mp9 (Messing and Vierra, 1982) and transfected into E. coli 71-18. Bacteria were plated in the presence of rsopropylthiogalactoside and 5. bromo-4-chloro-indolyl-0 D-galactosrde (Messrng et al.. 1977). Srngle- and double-stranded phage DNA was Isolated as described by Poncz et al. (1982a). Subclones for sequence analysis were generated usrng a random sequencing strategy (Sanger et al., 1980) by digestion of the isolated 4.4 kb Pst I o-globin gene fragment with a series of blunt-cutters (Rsa I, Hae III, Hpa I, Alu I) and lrgatron Into the Sma I site of M13mp9. Additional subclones were generated by Barn HI and Eco RI digestron and Insertion Into M13mp9. A thrrd set of clones was generated using a nonrandom sequencing strategy by nuclease Bal 31 digestron (Poncr et al., 1982a). In a modificatron of the establrshed technique, the M13mp9 vector fragment was inactrvated by digestion wrth Hind III, thus eliminatrng the need for purifying the famrly of

Bal 31 -generated varrable-length insert fragments by gel electrophoresrs, allowrng direct ligation of the insert fragments into Pst I-Sma I-digested MlSmp9 at a hrgher efficiency. DNA sequencing was performed by the dideoxy chain-termrnation technique (Sanger et al., 1980). The reaction products were analyzed by electrophoresrs in 6% and 8% urea-polyacryl- amrde gels and sequence readings were independently confirmed by at least two individuals.

Construction of fl-Globin-SV40 Recombinants and Transformation of HeLa Cells The 5.0 kb Bgl II fragment containing the fi-globin gene was Inserted into the Barn HI site of SVpBR328 (Busslinger et al., 1981) wrth the Inserted gene rn the same orientation as the SV40 early transcnption unit. The p- globin gene In thus construction IS expressed from Its own promoter wrth efficrent transcnptron dependent upon the presence of the 72 bp repeat enhancer element adjacent to the SV40 orrgin of replicatron (Grosveld et al 1982; Banerlr et al.. 1981). The paternal @-globin gene was isolated by Bgl II digestron of KB5. The maternal gene was Isolated from KBI 11. Thus latter clone was shown to be Rsa I (-) and therefore of maternal origin (see Frgure 5). The normal P-globrn gene was isolated from the recombrnant phage HflGl (Lawn et al., 1978). DNA was Introduced Into HeLa cells using standard procedures (Wigler et al.. 1977) with the following modifications. Each T75 flask of subconfluent culture was exposed to a calcium phosphate precipitate of 30 pg of pSV@ and 10 pg of aSVHPa2 (Trersman et al., 1983) supercooled DNA for 16 hr, followed by replacement with fresh medium and Incubation for an additronal 48 hr. The cells were then washed twce with Ca+2/Mg’2-free Hanks buffer, trypsinized, centrifuged at 1500 Xg, and the cell pellet was washed once again with Hanks buffer. Total cellular RNA was Isolated usrng hot phenol extractron (Scherrer. 1969).

Sl Nuclease Analysis of Globin RNA Globin RNA was analyzed by Sl nuclease mapping using published procedures (Busslinger et al., 1981; Favalaro et al.. 1980). DNA probes were labeled using T4 DNA polymerase and a-=P-dCTP (O’Farrell, 1981; K. Delgrosso and S. Surrey, unpublished data). The probe for a-globrn RNA was a 1.8 kb genomrc Hinf I fragment containing the al-globin gene (from 18 bp 3’ of the cap sate to 1 kb 3’ of the polyadenylation signal) labeled through exon 1 on the noncoding strand. A 2.1 kb genomic Bal I fragment, whrch spans the fl-globin gene (from 80 bp 5’ of the cap sate to 425 bp 3’ of the polyadenylatron signal), labeled through exons 1 and 2 on the noncoding strand, was used to analyze the r3-globin cap site, IVS-I splrce functrons, and 5’ IVS-2 splice Iunction. A 1.2 kb Rsa I-Pst I genomic fragment (which Includes the last 390 bp of IVS-2 and exon 3) was used to probe the 3’ IVS-2 splice junction and the polyadenylation site.

Acknowledgments

The authors are grateful to Eric Rappaport for hrs assistance in the analysis of the a-globrn gene IOCI and to both him and Carol Way for expert assrstance in the preparatron of the manuscript. We would also like to acknowledge the generosity of B. Forget and T. Manratis, who provrded recombinant plasmids and phages; G. Grosveld, H Lehrach, T. Maniatrs, and R. Treisman for therr gifts of clonrng and expression vectors; and J. Messrng and H. Lehrach for providrng bacterial strains. This work was supported rn part by National lnstrtutes of Health Grants AM16691 and HL28157. a grant from UNICO, and NIH Research Servrce Award HL07150. G. L. S was supported by the Medical Scientist Trarning Program at the Unrversity of Pennsylvania through NIH Grant 5-T32-GM07170.

The costs of publicatron of this article were defrayed in part by the payment of page charges. This arkcle must therefore be hereby marked “advertisement” in accordance wrth 18 U.S.C. Sectron 1734 solely to rndrcate this fact.

Recerved June I, 1984; revised August 15, 1984

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