Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7395-7399, October 1986 Genetics Polymorphisms in the human cluster: with multiple haptoglobin-related (Hpr) (unequal crossing-over/restriction fragment length polymorphisms/orthogonal pulsed field gradient gel electrophoresis/mouse-human cell hybrids/retrovirus-lke element) NOBUYO MAEDAt, SUSAN M. McEvoyt, HERMAN F. HARRISt, TITUS H. J. HUISMANf, AND OLIVER SMITHIESt tLaboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706; and tDepartment of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912 Contributed by Oliver Smithies, June 18, 1986

ABSTRACT We have found polymorphisms for the num- 3' end of isoleucine tRNA. This RTVL-I element may be ber of tandemly arranged haptoglobin-related (Hpr) genes in responsible for the apparent inability of the Hpr gene to be the haptoglobin gene cluster of Blacks. Genomic mapping and transcribed. nucleotide sequence analysis indicate that two copies of the Hpr Recombinational events involving the two genes, Hp and gene first resulted from unequal but homologous crossing-over Hpr, appear to have occurred frequently in recent human in a region 3' to the haptoglobin (Hp) and the haptoglobin- evolution. At least two gene conversion events have been related genes. Subsequent increases in the number of Hpr loci detected by DNA sequence analysis. One is at the 3' end of have occurred in some chromosomes. Among 25 American the two genes, where 620 bp oftheir nucleotide sequences are Blacks studied (15 were unrelated), 2 related individuals have identical, although other regions of the genes differ by an one extra copy of the Hpr gene and 5 unrelated individuals have average of 6.4% (6). The other gene conversion event more than two extra Hpr genes. None of 26 Whites and one produced the HplF allele, one ofthe common alleles ofthe Hp Oriental studied have extra copies. In one of the Blacks, six locus, as a consequence of transferring a segment of DNA tandemly arranged Hpr genes were demonstrated in one sequence from the Hpr gene to the Hp gene (N.M., unpub- by pulsed field gradient electrophoresis. His other lished data). In the present paper, we demonstrate that chromosome had one Hpr gene. The tandem Hpr genes were homologous but unequal crossings-over between the Hp and found in individuals with the haptoglobin genotypes Hp2/Hp2 Hpr genes have also occurred and have resulted in the (3 of3 tested) and Hp9/Hpl (4 of 11 tested), but none were found formation of chromosomes with multiple tandem Hpr genes. in the Hp'/Hp' individuals (11 tested). Fibroblast cell cultures We describe characterization at the DNA level of several from two Hp2/Hp' heterozygotes were fused to mouse cells to different examples of these chromosomes found in Blacks. obtain cell lines retaining a human on which the haptoglobin gene cluster is located. DNA analysis of the MATERIALS AND METHODS hybrid cells showed that in both individuals the tandemly arranged Hpr genes are linked to the Hp2 allele. These results DNA. DNA was isolated from cultured cells and from suggest that the multiple copies are associated with the Hp2 leukocytes (7, 8). Fifteen unrelated Black individuals were gene. studied. They were not chosen because of their haptoglobin genotypes, but eight had some unusual features in their Haptoglobin, the hemoglobin-binding of plasma, P-globin gene cluster that had led to their initial selection. Ten contains two a and two ,B chains that are synthesized relatives of one individual were also studied. Twenty-six colinearly as a single precursor polypeptide and processed Whites and one Oriental were randomly selected. post-translationally (1-3). In humans, there are three com- Genomic Southern Blot Hybridization. Restriction enzyme mon alleles at the locus Hp, which controls synthesis of digests of genomic DNAs (3 ,g) were electrophoresed and haptoglobin. The HplF and HplS alleles code for a-chain transferred to nitrocellulose (9) and hybridized (10) to the polypeptides of equal length that differ in charged amino following probes: the promoter probe, a 620-bp-Sst I/Bgl II acids making them migrate as fast and slow bands during fragment containing the promoter region and first exon of the electrophoresis in acidic starch gels. The Hp2 allele contains Hp gene; the hp2a probe, a 500-bp Pst I/BamHI fragment of an intragenic duplication of 1.7 kilobase pairs (kbp) and codes Hp2 cDNA (4); the hpB probe, a 650-bp BamHI/HindIII for an a chain that is almost twice as long as those coded by fragment of Hp2 cDNA (4). the Hp' alleles (4). Characterization ofthe Crossing-over Point. Genomic DNA DNA studies of the haptoglobin gene have revealed that it from a Black individual (C.G.) was digested with EcoRI, is duplicated in the , with a haptoglobin- ligated to EcoRI-digested DNA from bacteriophage Charon related gene, Hpr, being located 2.2 kbp downstream of the 32, and packaged in vitro (11). The recombinant phages were Hp gene (5, 6). The Hpr gene appears to code for a screened using radiolabeled hp2a probe. The EcoRI fragment structurally functional protein, although no hemoglobin- that contains the Hpr* gene (see Results) was isolated and binding protein with its expected properties has been de- subcloned into p010A ori (12). The nucleotide sequence ofthe scribed in the literature. In addition, no unequivocal tran- region spanning the crossing-over point was determined by scripts corresponding to the Hpr gene have been detected (5). the method of Maxam and Gilbert (13). In the first intron of the Hpr gene there is a retrovirus-like Orthogonal Pulsed Field Gradient Gel Electrophoresis. element (6), named RTVL-I (retrovirus-like isoleucine) be- Fibroblast cells (-2 x 107) were embedded in 0.5% low- cause it has a potential primer binding site homologous to the melting-point agarose (SeaPlaque, FMC, Rockland, ME) beads (2 ml) by the method of Cook (14). The DNA trapped The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: kbp, kilobase pair(s); RFLP, restriction fragment in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. length polymorphism. 7395 Downloaded by guest on September 27, 2021 73967MProc.Genetics: Maeda et al. Natl. Acad. Sci. USA 83 (1986)

in the beads was digested with Kpn I and subjected to individuals 4, 6, 9, 11, and 12 have more than one copy ofthis orthogonal pulsed field gradient gel electrophoresis in 0.8% gene. A rough estimate by densitometry indicated that agarose using the method of Schwartz and Cantor (15). The individual 6 (C.G.) has four or five and individual 9 (H.F.M.) negative electrodes were 22.5-cm-long linear arrays of 19- has about three copies of Hpr*. diode-isolated platinum wires set up as adjacent sides of a Ten other members of the family of H.N.R. were also right triangle [compare Carle and Olson (16)]. The positive studied. Individuals 1-5 (Fig. lb) are some ofthem. A cousin electrodes were platinum wires placed close to the ends ofthe ofH.N.R. has the same number ofHpr* genes as H.N.R., but hypotenuse. Five-second pulses were applied at 450 V for 5 we could not determine whether the chromosomes involved hr. The buffer was 50 mM Tris HCI/50 mM boric acid/i mM were codescendants. A half-sibling of H.N.R. has three or EDTA, pH 8.3, with HCl. more Hpr* genes (no. 4 in Fig. lb). Eight other individuals in Cell Fusion. Human fibroblast cells (from C.G. or H.F.M.) the family have chromosomes with the usual haptoglobin were fused to mouse LS-24b cells (17), which are defective in gene arrangements, either Hp2-Hpr or Hp'-Hpr. We did not adenine phosphoribosyl transferase, by the method of Da- find any examples of the unusual chromosomes among the 26 vidson and Gerald (18). Twenty-four hours after treatment Whites and one Oriental examined. with polyethylene glycol, cells were trypsinized and redis- Up to Five Hpr* Genes on a Single Chromosome. To tributed in medium containing 0.05 mM azaserine/0.1 mM determine the number of Hpr* genes in each of the chromo- adenine and 0.1 juM ouabain. The medium was changed every somes of C.G. and H.F.M., we used Southern blot analysis 3 days for 3 weeks. Surviving colonies were transferred into of large molecular size DNA separated in agarose gel by new dishes and culture was continued in azaserine/adenine- orthogonal pulsed field gradient gel electrophoresis. Among containing medium. the restriction enzymes that digest outside but not within the haptoglobin cluster, Kpn I was found to be most appropriate RESULTS for our purpose. Fig. 2a shows the ethidium bromide-stained gel and the Southern blot of Kpn I digests of DNA from four Genomic Southern Blot Hybridization. Chromosomes with individuals after hybridization to the hp2a probe. DNA from multiple Hpr genes were first detected by the presence of C.G. shows a hybridizing fragment of =130 kbp as well as a bands with unusual darkness in genomic Southern blots. Fig. 47-kbp fragment. The digest of the control DNA (no. 563 in la shows EcoRI, BamHI, and Bgl II digests ofDNA from two Fig. 2a, a White H2-Hpr homozygote) shows only the 47-kbp individuals, M.K. (White) and C.G. (Black), hybridized to band. Each copy of the Hpr* gene makes the haptoglobin the hp2a probe. Both individuals are Hp2/Hpl heterozy- gene cluster longer by -16 kbp. This result therefore indi- gotes, but a new 10.3-kbp EcoRI band (not separable from the cates that the five copies of the Hpr* genes in C.G. are all 10.1-kbp band corresponding to Hp') was seen in C.G. and tandemly arranged on one chromosome, making it Hp- his 2.4-kbp BamHI band (corresponding to Hpr) was much (Hpr*)5-Hpr. His chromosome has the usual Hp-Hpr ar- darker than the same band in M.K. Subsequently another rangement. The digest of DNA from H.F.M. produced a readily recognizajle hybridizing band of 10.4 kbp was seen in 106-kbp band (four copies of Hpr* on one chromosome) and the Bgl II digest of DNA from C.G. Strongly hybridizing a 47-kbp band; that of H.N.R. produced a 64-kbp band (one bands corresponding to the Hpr gene were also seen (data not copy of Hpr*) and a 47-kbp band. shown) in the Southern blots ofDNA from C.G. digested with Tandemly ArrangedHpr* Genes Are Linked to theH2 Allele. HindIII (3.4 kbp) orXba 1(5.8 kbp) after hybridization to the Chromosomes containing Hpr* genes were only found in hp2a probe, and with BamHI (0.8 kbp) orXba I (2.6 kbp) after individuals having the haptoglobin genotypes W/Hp2 (3 of 3 hybridization to the hpB probe. From these results, we tested) and Hp2/Hpl (3 of 11 tested) but not in Hpl/Hpl concluded that the genome of C.G. contains extra copies of individuals (11 tested). This strongly suggests that the the Hpr gene as illustrated in the lower part of Fig. ic. The tandemly arranged Hpr genes are associated with the Hp2 sizes of the EcoRI band (10.3 kbp) and Bgl II band (10.4 kbp) allele. To confirm this observation, cultures of fibroblasts hybridizing to the hp2a probe and those seen after the from the individuals C.G. and H.F.M., both Hp2/Hpl hybridization to the promoter probes (for example the 4.4- heterozygotes, were fused with cultured mouse cells to kbp BamHI band in Fig. lb) are readily explained if the extra separate the relevant chromosomes. The haptoglobin gene copies of Hpr were formed as a result of unequal crossing- cluster in humans is located on chromosome 16, which also over between the Hp and Hpr genes in their 3' regions (see carries the adenine phosphoribosyl transferase gene. We below). In Fig. ic, we compare a chromosome with the usual therefore fused the human cells with a mouse cell line, Hp'-Hpr arrangement and a chromosome with the Hp2-Hpr*- LS-24b, which is defective in this enzyme, and selected those Hpr arrangement (where Hpr* represents the hybrid gene mouse-human fusion cells that retain the human chromo- formed from Hp and Hpr). We also indicate the sources ofthe some 16 by using a medium containing azaserine and adenine. various hybridizing fragments shown in Fig. 1 a and b. The Genomic Southern blot hybridization ofthe BamHI digests of coding region of the Hpr* gene is identical to that of Hpr- DNA from the resulting hybrid cell lines to the promoter hence, our temporary designation of this hybrid gene as probe is shown in Fig. 2. The 4.4-kbp band corresponding to Hpr*. copies ofHpr* was associated in both C.G. and H.F.M. with The unusual chromosomes are not rare. We found 5 not with the individuals having them in a small study of the haptoglobin the 9.2-kbp band of the Hp2 gene but 7.5-kbp gene cluster in 15 unrelated Black individuals. Surprisingly, band of the Hp' gene. the intensity of the hybridizing bands derived from Hpr* was Characterization of the Crossing-over Events. The 10.3-kbp not the same in all individuals. This is easily seen in Fig. lb, EcoRI fragment, which contains an Hpr* gene, was cloned which presents genomic blots of the BamHI digests of DNA from the individual C.G. All five copies of the Hpr* gene in from 12 Black individuals probed with the radiolabeled C.G. appear to be identical, as judged by the failure of promoter probe. The 9.2-, 7.5-, and 5.0-kbp bands, respec- genomic mapping using 6-base recognition enzymes to show tively, correspond to the Hp2 allele, the Hp' allele, and the any differences between them. In addition, eight independent Hpr gene and are present in individuals (such as M.K.; recombinant phages containing the Hpr* gene showed no White) who have no extra copies ofthe Hpr gene. Individuals recognizable differences in their structures after character- with an extra copy (or copies) of the Hpr gene show a new ization by several 4-base recognition restriction enzymes. We band at 4.4 kbp corresponding to the Hpr* gene (see Fig. lc). therefore infer that any clone of Hpr* can be used to Individual 5 (H.N.R.) has one copy of the Hpr* gene, and characterize the nature of the unequal crossing-over event. Downloaded by guest on September 27, 2021 Genetics: Maeda et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7397 a b BamHI gaCC EcoRI BamHI Bgl 11 h 8 1 a be d x; 0 -I BY1 c Y~ Y.: 12 34 5 6 C 7 8 9 10 1112

9 kb kb kb 11.82 kb S -10.4* 1Qt -O.11 to9.0r,2 t 4w - - - -9.2 2 . .*, -9.2 _751 . ,,,. 7.1 *-a. _* 75 - 040 Aft - 0W dB _5,0O'- 44W 0*- 0 -4A*-

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hp2a probe Promoter probe

C Hp' Hpr 1 234 5 1 23 4 5 III M 0 RTVL-IITI

10.1 E E E E 1-24-1 B ~7.5g 7.1gBB B Bg 90 Bg Bg

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Hp2 ( Hpr*)n Hpr 1 234 56 7 1 234 5 1 23 4 5 N ..kI m =-M- - I MN,11 RTVL-I VL-I v *i Ha 11.8 EE ~~~~~~~E E 10.3 E E 8.3 E B B 1B-2.4- B'-2A-' Bg 88 Bg I9.0I 10.4Bg Bg Bg

I------9.2 - - - -- B 67-5-0-1B -- 4-4--1B FIG. 1. (a) Southern blots ofEcoRI, BamHI, and Bgi II digests of genomic DNA from C.G. and M.K. hybridized to the hp2a probe. Bands derived from the Hp2, Hp', and Hpr genes are superscripted 2, 1, and r, respectively. Bands corresponding to the Hpr* gene are marked with an asterisk. (b) Southern blot of BamHI digests of genomic DNA from 12 Black individuals and from a control individual M.K. (also marked C). The blot was hybridized to the promoter probe. Nos. 1-4 are family members of no. 5. Nos. 5-12 are unrelated. (c) Organization of the haptoglobin gene cluster. A chromosome with the usual Hp'-Hpr arrangement (Upper) and a chromosome with Hp2 associated with multiple Hpr genes (Lower) are shown. Exons are numbered. The Hp2 allele has two more exons than the Hp' allele. The a-chain coding region and the 3-chain coding parts in the last exon of all the genes are shown with black bars and slashed bars, respectively. The retrovirus-like element (RTVL-I) in the first intron of the Hpr gene is represented by a dashed line with long terminal repeats at both ends (black arrows). The EcoRI, BamHI, and BgI II fragments, which hybridize to the hp2a probes (a), are shown in c by horizontal bars labeled E, B, and Bg with their sizes shown (kbp). The BamHI fragments, which hybridize to the promoter probe (b), are shown in c by dashed lines with their sizes in kbp. Not all sites are shown. The mapping data (summarized in Fig. 1c) and sequence It was suggested (6) that the 620-bp region of identity is analysis (summarized in Fig. 3) indicate that the crossing- probably the result of a gene conversion event between the over occurred homologously but unequally between mis- Hp and Hpr genes. Interestingly, this region was also aligned Hp and Hpr genes within a region =1 kbp 3' to the apparently involved in the crossing-over event that generated genes. The nucleotide sequences enable the crossing-over the Hpr* gene. The nucleotide sequence of the region point to be defined to within 4 bp, as shown in Fig. 3. The containing the putative crossing-over point (Fig. 3) has no nucleotide sequence of the Hpr* gene is identical except for obvious features that would make the region a recombina- one nucleotide (see below) to that of the Hpr gene in the tional "hot spot." We note, however, that the region imme- region upstream of these 4 bases, while it is identical except diately 5' to the crossing-over point is pyrimidine-rich (22 for one nucleotide (see below) to that of the Hp2 gene in the cytosine and thymine versus 6 adenine and guanine) and that region downstream of these 4 bases. The crossing-over point the region immediately 3' is purine-rich (22 adenine and is very close to the 3' end of the 620-bp region over which the guanine versus 5 cytosine and thymine). Possibly, this local nucleotide sequences of the Hp and Hpr genes are identical change of strand asymmetry could be related to initiation of (6). the crossing-over event or to resolution of a heteroduplex Downloaded by guest on September 27, 2021 7398 Genetics: Maeda et al. Proc. Natl. Acad. Sci. USA 83 (1986) a b

N LUN 2 2 CM (0 LL to Z C) (DO LJ LL iCn u 0 1) 1 10 10 u,:rx I I I I I

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FIG. 2. (a) Orthogonal pulsed field gradient gel electrophoresis of genomic DNA samples digested with Kpn I. After electrophoresis, the DNA was stained with ethidium bromide (Left), transferred to nitrocellulose, and hybridized to the hp2a probe (Middle). Ligated X phage DNA [Charon 3A ALac; (7)] was used as a size marker. (Right) Blot of another gel. The DNA used for this panel had random nicks causing artifactual hybridization to smaller-sized DNA in addition to the full-sized fragments. (b) Separation ofthe two chromosomes 16 from C.G. and from H.F.M. in mouse-human hybrid cells. DNA from the parental human cells (C.G. and H.F.M.), the parental mouse cells (LS-24b), and the hybrid cells (C.G.-2a, C.G.-5a, H.F.M.-12a, and H.F.M.-5a) were digested with BamHl. The gel blot was hybridized to the promoter probe. The filter was washed at astringency sufficient to remove hybridization to mouse DNA. after branch migration. More data on the characterization of position 236 to histidine instead of the aspartic acid found in similar crossing-over events are needed to determine the both Hp and Hpr in previous sequence analyses (5, 6). There significance of the strand asymmetry. is a cytosine at this position in Hpr* that makes the gene Restriction Fragment Length Polymorphisms. The different resistant to digestion with the restriction enzyme BstNI, numbers of Hpr genes in the unusual chromosomes we have whereas the guanine in Hp and Hpr (positions 7246 and 17450 described create several restriction fragment length polymor- in figure 2 ofref. 6) makes both genes susceptible to digestion. phisms (RFLPs) ofthe haptoglobin gene cluster as illustrated Another base difference was found 3' to the Hp, Hpr, and in Fig. 1. In addition, two possible RFLPs resulting from base Hpr* genes, where a nucleotide cytosine in the Hpr* gene is substitutions were found from nucleotide-sequence compar- different from an adenine at the equivalent position ofthe Hp2 isons. gene (position 7894 in figure 2 of ref. 6) but is identical to the The sequenced Hpr* gene of C.G. has a base substitution cytosine found in the Hpr gene sequenced (6). Cytosine was in the P3-coding region that would change the amino acid at also found at the equivalent position in the Hp's gene (N.M., 236 ,t Rsa I (Asp) AATAAA G _ Hp ak"Oh"I__im_I --_A III I I III (His) II I I I lill -IIII III 11 I~~~~~~~~~~Ju Hpr G (Am P) ,- RsaI 100LJbp

+- Hp GGTCTGTTTGATACAAACTCAGAGGTCCrTiCACTGCA'TaCTGTTGCCTC'CTCAAAGTGAATTAGGAGAAAAGGCATGGGCCTGGTTGAGGAAGAGGCTA Hpr* GGTC TGrTTGA TAcAAAC TCAGAGGTCCTTTecacTGCATGCTrGTTGCC TC TTAAAGTGAATTA^GGAGAAAAGGC ATGGGCCTvGGTTGAGGAAGAGGCTAt Hpr GGTC TGTTTGATAC AAAC TCAGAGGTC C TTTCAC TGC ATGCTrGTTGC C TC TTTAAAGC GAA TTAGGAGAAAAEGGCA^TGGGCCTTGGTrGAGGAAGAGGCTA

FIG. 3. Nucleotide sequence of the crossing-over point. About 1.3 kbp of the nucleotide sequence of the Hpr* gene was determined (thick horizontal line) and compared to the equivalent regions of the Hp gene (upper horizontal line; positions 7001-8320 from figure 2 of ref. 6) and the Hpr gene (lower horizontal line; positions 17205-18519 from figure 2 of ref. 6). Nucleotide differences between Hp and Hpr sequences are shown by vertical long bars and length differences by short vertical bars between the two horizontal lines representing the two genes. The -chain coding regions are indicated by slashed bars and the poly(A) addition signal sequence is indicated. The nucleotide sequence of Hpr* is identical (except for one nucleotide) to the sequence of Hpr in the 5' half of the sequenced region; therefore, the thick line representing Hpr* is placed close to the lower thin line. The sequence of Hpr* in its 3' half is identical (except for one nucleotide) to the sequence of Hp, and so the thick line is placed close to the upper thin line in this region. The sequences of the three genes close to the crossing-over point are compared in the lower part of the figure. Within the 100 bp given, the sequences upstream of the 4 bases marked by brackets are identical in Hpr* and Hpr but the sequence of Hp differs in two bases. Hp and Hpr* are identical downstream of the bracket but Hpr differs from the other two genes in 3 bases. These differences are shown by vertical bars. Two nucleotide differences, which appear to be due to polymorphisms (see text), are indicated in the upper part of the figure. Downloaded by guest on September 27, 2021 Genetics: Maeda et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7399

unpublished data). Because cytosine makes the position ofthree individuals, two carrying one extra Hpr gene and one susceptible to digestion with the restriction enzyme Rsa I, we carrying three or more extra copies. The amounts of tested cloned genes from several individuals for the presence haptoglobin in their sera show no significant differences from of this site. It was present in each of two independent clones the controls, as judged by staining hemoglobin-haptoglobin of the Hp's, HplF, and Hpr genes, and in one isolate of the complexes with benzidine after gel electrophoresis. Nor was Hp2 gene, but absent in the Hp2 gene whose sequence was any unbalanced expression ofthe two alleles observed in one determined previously. The Rsa I site is therefore present in individual who is an Hp2/Hpl heterozygote and has one extra some but not all Hp2 genes. Hpr gene. It is therefore unlikely that the extra Hpr genes are Further studies are needed to determine whether these affecting the expression of the upstream Hp locus under BstNI and Rsa I site differences are common RFLPs in normal conditions. human populations and to find the frequencies ofthe RFLPs, The reciprocal product of the unequal crossing-over event so far seen only in Blacks, due to differences in the number characterized in this paper is a chromosome with a single of Hpr genes. haptoglobin-type locus. Because the expression of the Hpr gene is doubtful (5), loss of the Hpr gene would be expected DISCUSSION to be harmless. Yet we have not found such a chromosome in humans. Possibly, chromosomes with a single Hp locus are Our data show the existence of polymorphisms for the detrimental. Possibly, they would be detected by a larger number of haptoglobin-related genes in the human genome. survey. On the other hand, our finding, in a limited survey, We have identified chromosomes with one, four, and five of chromosomes with up to seven loci raises the interesting extra copies of the Hpr gene. These extra copies are found speculation that multiple Hpr genes could be advantageous. linked to the Hp2 allele but none has yet been seen linked to Especially intriguing is the fact that multiplication of the Hpr the Hp' allele. This suggests that the subsequent increases in gene also increases the number of retrovirus-like elements the number ofHpr* genes were caused by unequal crossings- (RTVL-I) in the haptoglobin gene complex. The significance over involving Hp -Hpr*-Hpr chromosomes. To generate of this element is not known, but if it has some positive seven loci, the maximum yet seen, from the normal two loci, function then chromosomes with multiple copies of the Hpr the same chromosome must have been involved in unequal gene may be selectively advantageous. crossover at least three times. Some individuals are naturally more prone to sister chromatid exchanges than are others, as We are greatly indebted to many individuals who gave us blood is the case with individuals with Bloom syndrome. E. Hirsch samples or tissues samples and to many collaborators for supplying (University of Minnesota) kindly tested a fibroblast culture DNA to us. For these samples we thank Drs. B. Forget, G. Dover, S. Orkin, M. Lewis, W. Bias, D. Barnett, E. Schwartz, R. DeMars, from C.G. for the frequency of sister chromatid exchanges. D. Mager, P. Henthorn, P. Powers, K. Lyons, and A. Metzenberg. The culture did not differ from other normal fibroblast We thank Dr. E. Hirsch for cytological tests, Dr. R. Kucherlapati and cultures. Nevertheless, there remains a remote possibility P. Broeker for LS-24b mouse cells and helpful advice on cell fusion that C.G. (or one of his forebears) has some overall predis- experiments, and N. Borenstein for her technical help and great position for events involving chromosomal misalignments. patience. This work was supported by National Institutes of Health This possibility arises because C.G. was originally studied by Grants GM-20069 (O.S.) and HLB-15168 (T.H.J.H.). This is paper us (19) because he is homozygous for chromosomes with a no. 2895 from the Laboratory of Genetics, University of Wisconsin- fetal Gy-Gy.globin gene arrangement instead of the usual Madison. gene arrangement. One other individual we Gy-Ay-globin 1. van der Straten, A., Herzog, A., Jacobs, P., Cabezon, T. & Bollen, A. studied, who has three or four extra copies of the Hpr gene, (1983) EMBO J. 2, 1003-1007. also has an unusual arrangement, Ay_Ay, of fetal globin 2. Yang, F., Brune, J. L., Baldwin, W. E., Barnett, D. R. & Bowman, genes. A pairing of misaligned genes is probably necessary to B. H. (1983) Proc. Nati. Acad. Sci. USA 80, 5875-5879. generate genes and also to generate or 3. Raugei, G., Bensi, G., Colanluoni, V., Romano, V., Santoro, C., Hpr* GYGy Ay_Ay Constanzo, F. & Cortese, R. (1983) Nucleic Acids Res. 11, 5811-5819. chromosomes. 4. Maeda, N., Yang, F., Barnett, D. R., Bowman, B. H. & Smithies, 0. Some of the Black individuals we have studied were (1984) Nature (London) 309, 131-135. originally selected because they had unusual hemoglobin 5. Bensi, G., Raugei, G., Klefenz, H. & Cortese, R. (1985) EMBO J. 4, phenotypes, including four ofthe five unrelated individuals in 119-126. 6. Maeda, N. (1985) J. Biol. Chem. 260, 6698-6709. whom we found extra Hpr genes. Yet the haptoglobin gene 7. Blattner, F. R., Blechl, A. E., Denniston-Thomson, K., Faber, H. E., cluster is localized on chromosome 16, while the f-globin Richards, J. E., Slightom, J. L., Tucker, P. W. & Smithies, 0. (1978) gene cluster is on chromosome 11. Further studies are needed Science 202, 1279-1284. to determine unbiased frequencies of chromosomes with 8. Poncz, M., Solowiejczyk, D., Harppel, B., Mory, Y., Schwartz, E. & Surrey, S. (1982) Hemoglobin 6, 27-36. extra Hpr genes and to see whether they are significantly 9. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. correlated with hemoglobin phenotypes. 10. Jeffreys, A. J. & Flavell, R. A. (1977) Cell 12, 429-439. Changes in the numbers of genes as a consequence of 11. Horn, B. (1979) Methods Enzymol. 68, 299-309. unequal but homologous crossing-over have been reported 12. Maeda, N., Bliska, J. B. & Smithies, 0. (1983) Proc. Natl. Acad. Sci. USA 80, 5012-5016. frequently. In the human globin gene family, for example, 13. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, chromosomes with a single adult a-globin gene, with a single 560-564. fetal y-globin gene, and with a single 8-/3 Lepore fusion gene 14. Cook, P. R. (1984) EMBO J. 3, 1837-1842. have all been found [see Maeda and Smithies (20) for a 15. Schwartz, D. C. & Cantor, C. R. (1984) Cell 37, 67-75. These chromosomes cause 16. Carle, G. F. & Olson, M. V. (1984) Nucleic Acids Res. 12, 5647-5664. review]. deficiency phenotypic 17. Tischfield, J. A., Trill, J. J., Lee, Y. I., Coy, K. & Taylor, M. W. (1982) effects that range from mild to severe in homozygous indi- Mol. Cell. Biol. 2, 250-257. viduals. When the number of genes is increased, the pheno- 18. Davidson, R. L. & Gerald, P. S. (1976) Somatic Cell Genet. 2, 165-176. typic changes usually are milder, and individuals having an 19. Powers, P. A., Altay, C., Huisman, T. H. J. & Smithies, 0. (1984) extra copy or copies of the a- and t-globin genes are Nucleic Acids Res. 12, 7023-7033. essentially asymptomatic (21). Four haploid copies of the 20. Maeda, N. & Smithies, 0. (1986) Annu. Rev. Genet. 20, 81-108. 21. Nakatsuji, T., Carver, J., Wilson, J. B., Lam, H., Reese, A. L., Miwa, fetal y-globin gene have been found (22, 23). S. & Huisman, T. H. J. (1983) Am. J. Hematol. 14, 121-132. Extra copies of the Hpr gene, including its retrovirus-like 22. Hill, A. V. S., Bowden, D. K., Weatherall, D. J. & Clegg, J. B. (1986) element, do not appear to affect the expression of the Blood 67, 1611-1618. upstream Hp gene. We examined the haptoglobin phenotypes 23. Shimasaki, S. & luchi, I. (1986) Blood 67, 784-788. Downloaded by guest on September 27, 2021