GENOMICS

6, 204-211

(1990)

Identification of Two Human Ferritin H Genes on the Short Arm of Chromosome 6 I. J. DUGAST,*

P. PAPADOPOULOS,* E. ZAPPONE,* C. JoNEs,t K. THERIAULT,* G. J. HANDELMAN,* R. BENAROUS,$ AND J. W. DRYSDALE*~’

*Department

of Biochemistry, Tufts University, 136 Harrison Avenue, Boston, Massachusetts 02111; tfleanor Roosevelt Institute for Cancer Research, Department of Biochemistry, Biophysics, and Genetics, School of Medicine, University of Colorado, Denver, Colorado 80220; and Unstitut de Pathologie Mokulaire, CHU de Cochin, Paris, France Received

April 28, 1989;

revised

21, 1989

chromosome 11 (Worwood et al., 1985; Boyd et c-d.,1985; Costanzo et al., 1986; Hentze et al., 1986). Most of the other members appear to be processed pseudogenes (Costanzo et al., 1986; Drysdale, 1988). Some recent results have prompted renewed interest in the possible involvement of ferritin in IH. Ferritin H sequences have been mapped close to the suspected locus of the diseasenear the HLA complex on the short arm of chromosome 6 (McGill et al., 1987; Simon et al., 1987). In addition, two tissue-specific H geneshave been found in other species(Dickey et al., 1987; ColIawn et al., 1987), suggesting that there may also be a second functional human H gene. Therefore, since the ferritin sequenceson chromosome 6 have not been characterized, we thought they might represent a second functional human H gene which affects iron uptake in the intestine. To explore this possibility we have obtained sequencesof ferritin H mRNA from human intestinal mucosa and have cloned a ferritin H gene from chromosome 6.

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.

August

OlSBOAcademicPrer,Inc.

INTRODUCTION

For many years it has been suspected that a defect in expression of ferritin in intestinal mucosa or in reticuloendothelial cells could be responsible for the inherited iron storage disease idiopathic hemochromatosis (IH) (Crosby, 1963; Bothwell et al., 1983). Changes in isoferritin patterns in several tissues and anomalously low levels of ferritin in intestinal mucosa have been found in IH (Powell et al., 1974; Halliday et al., 1978; Whittaker et al., 1989; Fracanzani et al., 1989) but it is not known whether such changes are the cause or consequence of the disease. Ferritin is a complex protein consisting of different proportions of two subunit types, H and L, which generate isoferritin populations and these vary with tissues and with physiological state (Arosio et al., 1978; Drysdale, 1988). The H subunit appears to be important for iron uptake while the L subunit seemsbetter suited to long-term iron storage (Boyd et al., 1985). Both subunits are derived from multiple gene families scattered on many chromosomes (Jain et al., 1985; McGill et al., 1987). So far only one functional gene has been identified for each: an L gene on chromosome 19 (Caskey et al., 1983; Santoro et al., 1986) and an H gene on

MATERIALS

AND

METHODS

Intestinal cDNA Library Human duodenal tissue was obtained surgically from a comatose, normal 20-year-old male at the time of removal of a kidney for transplant. The intestinal tissue was immediately frozen in liquid nitrogen, pulverized, and then lyophilized. Total RNA was isolated by the method of Chirgwin et al. (1979) and a cDNA library was synthesized by the method of Okayama and Berg (1982) using RNase H. The cDNAs were methylated to protect EcoRI sites and then introduced into the EcoRI site of X gtll after EcoRI linkers were added to the cDNA. Genomk Libraries

’ To whom correspondence should bs addressed. Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under Accession No. 504755. 033s7543po

$3.00

Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

A human genomic library constructed in Charon 4A from a partial HaeIII digestion was a gift from Dr. Tom

the

204

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H GENES

Maniatis (Harvard University). A second human genomic library constructed in the cosmid c2XB (Bates and Swift, 1983) was a gift from Dr. Greg Landers of Integrated Genetics (Framingham, MA). This library was prepared from lymphocyte DNA from a normal male, digested partially with MbOII, and cloned as 35to 40-kb fragments into the BumHI site of the cosmid.

Cell Lines Human-hamster hybrid cell lines 836-3A, 640-34A5, and R21-1B and the parent hamster line CHO-Kl (Kao et al., 1976) were used. Line 836-3A contains human chromosome 6q and line 640-34A5 contains human chromosomes Y, 6,10, and 21 (Rashidbaigi et al., 1986). Line R21-1B is a subclone of 640-34A5 with human chromosome Y and the fragment 6pter-6q21 from chromosome 6. These cell lines were grown in Ham 12 medium containing 5% fetal calf serum in an atmosphere containing 5% COz.

Probes Probes for human ferritin H sequences were derived from the essentially full-length liver H cDNA, pHF16 (Boyd et al., 1985). The SacI-KpnI fragment used for screening libraries and for Southern analyses contains most of the coding region and all of the 3’ noncoding region. Other probes were the 5’ and 3’ PstI fragments containing 305 and 495 hp, respectively (Fig. 1). A specific probe, K7, for the cloned gene from chromosome 6 is described in the text. A probe, pSP65, derived from pHLA-A2 (Sood et al., 1981), for class I HLA genes was a gift from Drs. A. K. Sood and S. M. Weissman (Yale University). DNA fragments were isolated by preparative gel electrophoresis in low-melting-point agarose (Bethesda Research Laboratories, MD) and labeled in melted gel slices with 32P by random priming with the multiprime labeling kit provided by Amersham Corp. (Arlington Heights, IL).

Library Screening The cDNA library (3X 10’ PFU/pg) and the genomic libraries were screened by the method of Grunstein 0

100 I

I PstI

200 I SaCI

s'noncoding region

FIG. pHF16

1. (6).

300 I

400 I

600 I

700 I

PstI

800 Base , Pairs

KpnI

coding

Restriction

500 I

map

region

of human

PstI

3'noncoding region

liver

ferritin

H cDNA,

ON

CHROMOSOME

6p

205

and Hogness (1975), as adapted by Benton and Davis (1977) with the 3’ PstI fragment of pHF16. This fragment contains most of the coding region of the human liver ferritin H cDNA (Boyd et al., 1985).

Southern Blots Genomic DNA was prepared and Southern analyses were performed as previously described (Boyd et al., 1985; David et al., 1986).

DNA Sequencing DNA sequencing was performed by the method of Sanger et al. (1977) from cloned m13mp18 using 35S-labeled nucleotides and quenase kit from United States Biochemical

dideoxy DNA in the Se(Ohio).

RESULTS In order to explore whether a second H mRNA was expressed in duodenum, we constructed a cDNA library from human duodenal mucosa and screened it with the liver H cDNA. Due to practical problems with the tissue sample, we obtained only three partial ferritin H cDNA clones with inserts of 200, 250, and 350 bp. All three inserts had sequences identical to the 3’ end of the liver H cDNA, pHF16 (not shown). These results indicate that if there were any differences in intestinal ferritin H mRNA compared with that from other tissues, they were likely to be upstream or that a second H mRNA was not highly represented in this library. In a more direct approach, we searched for the ferritin H gene on chromosome 6. Since the only H gene so far mapped to chromosome 6 lies on an 11-kb EcoRI fragment (Cragg et al., 1985; McGill et al., 1987), we initially looked for such a fragment in a collection of 15 ferritin H genomic clones obtained by screening a human genomic library in Charon 4A with the liver H cDNA. We identified three different clones with ferritin sequences on EcoRI fragments of approximately 11 kb. However, analyses of hybrid cell line 640-34A5, which contains chromosome 6, with subprobes from these three clones indicated that none came from this chromosome. This experience led us to obtain a more definitive characterization of the ferritin H gene on chromosome 6. For these experiments we used a derivative of 64034A5 called R21-lB, which contains chromosome Y and 6pter-6q21 and expresses HLA class I genes (C. Jones, unpublished observations). This line allowed a direct analysis of ferritin H genes in this region since there are no ferritin H sequences on chromosome Y (Cragg et al., 1985; McGill et al., 1987). Initially we examined patterns of H genes from EcoRI digestions

DUGAST

206 EcoRl Kb

Bgl I[

Hind

II

Hind

MspI

ET

AL.

5’

Ill

Kb

23.1

I.&.

3’

Vector

~ii-ii?i-iii?

“u 23.0

9.4 6.6 9.4

EBHMT

-

4.4 $3

=

0.5 -

FIG. 3. Restriction analyses of clone H35. E, EcoRI; B, BgAI; H, HindIII; M, MspI, T, TaoI. Duplicate blots were probed with the 5’ and 3’ PstI fragments of pHF16. The 3’ blot was stripped then reprobed with the cosmid vector.

FIG. 2. Southern analyses of ferritin H genes in genomic DNA from human (Hu), hamster cell line CHO-Kl (Ha), and a humanhamster hybrid R21-1B containing human chromosomes 6pter-6q21 and Y (Ha 6~). The blot was probed with the So&KpnI fragment of human ferritin H cDNA, pHF16, at 42°C in 50% formamide for 18 h and washed twice for 30 min in 2X SSC at 68V (13). An electrophoretic artifact in the Hind111 digests retarded the migration in lane Ha 6p. A better analysis is given in the right-hand panel. Fragments carrying human ferritin sequences from chromosome 6 are marked according to their relative hybridization. The significance of the two Hind111 fragments marked with asterisks is indicated in Fig. 4.

for comparison with previous analyses. We confirmed the assignment to chromosome 6 of an EcoRI fragment of about 11 kb, estimated here at 10 kb, but, to our surprise, found another band of about 9.6 kb also with ferritin H sequences (Fig. 2). These bands are marked in the pattern from the hybrid cell to distinguish them from cross-hybridizing hamster bands in this and the hamster parent line and for tentative identification of these sequencesin other digests. We attribute the new finding of a second hybridizing fragment on chromosome 6 to the superior electrophoretic resolution in the present analysis. Both bands differed in relative intensity of hybridization and had identifiable counterparts in human genomic DNA. Neither was present in cell line 836-3A, whose only human chromosome is 6q (not shown), indicating that both genes lie on 6p, in agreement with the more exact assignment from in situ hybridization (McGill et al., 1987).

An EcoRI band of 5 kb in the hybrid line may also be derived from chromosome 6. There is a faint band at this position both in the human DNA and in the parent hamster line, but its intensity in the hybrid line seems greater than that expected from the parent hamster line alone. The second gene on chromosome 6 may therefore contain an internal EcoRI site. To determine whether the hybridizing fragments on chromosome 6 represented two genes or one gene cut by EcoRI, we examined patterns given by digestions

EcoRl

Bgl

II

HiiIU

MspI Ib 23.1

2.3 2.0

FIG. 4. K7 adjacent

Southern analyses of the blot in Fig. 2 using a probe to the 3’ end of the ferritin sequence in clone H35.

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HUMAN

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FIG. 5. Restriction map and strategy for sequencing the 2-kb BgIII fragment containing the ferritin gene in clone H35. The ferritin gene is in the hatched area. MRR, metal regulatory element in 5’ “noncoding” region. The oligonucleotide 5’ dTGCT”TCAACAGTGCT 3’ and ita reverse complement were taken from the conserved MRE sequence in the liver H mRNA and used to prime sequence reactions in clone H35. Although there proved to be one difference in this region in clone H35, both orientations of the above pentadecamer successfully primed sequence reactions in clone H35. The KpnIBgLII fragment region was used to make the subprobe K7 used in Fig. 4.

with other restriction enzymes. From BgZII patterns we also identified two fragments, approximately 2 and 8 kb in size, containing ferritin H sequences from chromosome 6. The 2-kb fragment hybridized more strongly than the 8-kb fragment and had a clear counterpart in human genomic DNA (Fig. 2). However, the 8-kb band had no obvious counterpart in human genomic DNA since this region contains several H genes on fragments of similar size. A similar analysis with Hind111 digests initially identified only one fragment of about 12 kb that could be assigned to chromosome 6 in the hybrid line (Fig. 2, third panel). However, band alignment was difficult because of an electrophoretic artifact. Another analysis with better resolution confirmed the assignment of the 12-kb fragment and further indicated that a band of about 5.3 kb also contained ferritin sequences from chromosome 6 (Fig. 2, separate panel on right). This band corresponded to a minor band in human genomic DNA migrating just below a cluster of other H genes. The relative intensity of hybridization in this fragment was much lessthan that in the minor hybridizing EcoRI and BgZII fragments mapped to chromosome 6p, suggesting that the 5.3-kb Hind111 fragment represents only a portion of a gene. Finally, analyses of MspI digestions indicated ferritin H sequences from chromosome 6p on two fragments of about 6.6 and 5.7 kb. This agrees with the analyses of Gatti et al. (1987), who mapped two similar-sized MspI fragments to chromosome 6. The difference in intensity of hybridization of the two MspI fragments is similar to that of the EcoRI or BgZII fragments assigned to chromosome 6.

ON

CHROMOSOME

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207

Since all known functional ferritin genes are less than 4 kb in length and we obtained two hybridizing fragments with each of four different restriction enzymes, we concluded provisionally that there are two ferritin H genes on chromosome 6 that can be distinguished by their relative hybridization to the liver H cDNA: one lying on fragments of 10 kb EcoRI, 12 kb HindIII, 2 kb BgZII, and 6.6 kb MspI, marked with asterisks in Figs. 2 and 4, the other on fragments of 9.6 kb (and perhaps 5 kb) EcoRI, 5.3 kb HindIII, 8 kb BgZII, and 5.7 kb MspI (arrowheads, Fig. 2). With this more complete characterization, we searched for ferritin H clones containing these fragments in another human genomic library since no such clones were in the original 15 H clones. We screened a total of about 300,000 cosmid clones with the coding region of the liver H cDNA, pHF16, and obtained 27 different positive clones. Only one, H35, came close to fitting the predicted criteria. This clone contained ferritin sequences on an EcoRI fragment of about 10 kb, a BgZII fragment of 2 kb, a Hind111 fragment of 9 kb, a MspI fragment of about 7.0 kb, and a TuqI fragment of about 13 kb (Fig. 3). All of these fragments seemed to contain most of the ferritin sequences since they were recognized by probes from the 5’ and 3’ ends of the H cDNA. As indicated in the right-hand panel, none of these fragments contained cosmid vector sequences, indicating that they had been derived intact from genomic DNA. The 5’ probe also detected minor bands in some digests, probably because of contaminating plasmid sequences in the probe. We have no explanation for the intensity of hybridization given by the 5’probe with a Hind111 band of 5.7 kb which appears to have vector sequences.From the above, we concluded that clone H35 contains an intact ferritin H gene sequence and that there are therefore two distinct ferritin H genes on chromosome 6p. Efforts to find the second gene with the characteristics described above have so far been unsuccessful. In order to identify the specific genomic fragments containing the gene represented in clone H35, we obtained a subprobe (K7, see Fig. 5) of about 340 bp from the region adjacent to the 3’ end of the ferritin sequences. This KpnI-BgZII fragment was cloned into the KpnI-BamHI site of m13mp18 and excised as a 752-bp AuaII-BgZI fragment. This probe hybridized to unique bands in EcoRI, BgZII, and MspI digests of human genomic DNA (Fig. 4). The hybridizing bands corresponded in size to the major hybridizing bands containing human ferritin H sequences in the hybrid cell line. This result indicates that clone H35 is indeed derived from chromosome 6. (Weak hybridizations of probe K7 with bands in the hybrid and the parent hamster cell lines, but not in human genomic DNA, probably represent sequencesin hamster DNA similar to that in K7.)

208

DUGAST 1

81 161 241

CATCAAAAGA

481

TAACTACATC

AGTAAATATA

TTGCTTTTTT

AAATGCTTTA

CTTCTTATTT

AAAGAGAATT

GATTGTTTAA

ATAAAAATAT

CAATGTAGGC

TACATGCAGT

GGCTCACACC

TATAATCCCA

GCACTTTGGG

GGGTGGATCA

CTTGAGGTCA

GAATGTTCAA

GACCAGCCTG

GCCAACATGG

TGATCTTTGT

GTCTATTAAA

AAATACAAAA

ATAAGCCAGG

CGTGGTGGCG

GGCACCTGTA

ATCCAGCTAC

TCTAAGGCTG

AGGCAAGAGA

TCACTTGAAT

CCAGGAGGCA

GAGGCTGCAG

TCAGCCAAAA

TTACTCCACT

GCCCTCCAGC

CTAGGCAACA

GAGTGAGAAT

CTGTCTCAAA

AATAAAAATA

AAAATAAATC

AATGTAGTAT

CAGACTTATA

TGTAGAAATT

AAATATTTGA

CAAAATAACA

TGAAATTATA GTCTGCAATA

AGGAGAGAAA

TTAAAATTTA

TTATTTTAAG

GTTCTTAATA

TACAAACTAG

TATATTATCA

GTTGAAGATA

ATTTAAAGCT

ATATACTATA

AACACTAAAG

CAACCATTAA

TATAGCAAAA

TTGAAAATAT

GAAGTTAATA

GGGCAACAAA

GGAGATAAAA

TGTAATTTTA

AGAAATATAC

AAATAATTAC

AAAAAGACAG

CATAATAGTA

AAAGAGAATG

AACAGATAAA

ACAATTATAA

AACAAACACC

AAGCTGAGAG

ACTTAAACCT

AATCACACGA

TAATCACACT

AAATGTAAAT

GGTCCAAATA

CTTTCAAATA

GATAATGAAA

ATTCAAGACC

TAACCACAAA

AAAAACACAC

TTAGAATATA

GAGACATTCT C . . . . G....

TCACCAAGAG ..G..G....

TCCTCGGGGT . . . . . ..e..

TTCCTGCTTC . ..... ....

cIiGCCAGCl'GC . . ..G..C..

cccTcaTcA CGTclTcAcT CCATAGCCAG . . . . . . . . . . . . . . . . . . . . .c.......c

561 641 721 801 881

TAAGAAAAGG

AGGCTGAGGT

321 401

ATGAAGAACA

ET AL.

CCCCCATCAA

AAAGGAGAGA

AAGTCACAGA

TAGGGTAAAA

GTGG921*

g61 1041

1118

1185

1248

1311

1374

1435

1498

1561

1622 1689

-CC .A........

-CTTGT . . . . . . . . . . . . ..G..C..

&CCCAT........C

GCACCCTCAG . . . . . . . . G.

ACtACCccAA ..TG......

GGCCCCCACC . . . . . ..G..

GCCGC!CocAG CGccAcGcAG CCACCGC~ CGCAG...CC . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . ..C.CCG..

u ATG AC; . . . .C. ..C

GCA TCC AAC ..G . . . .C.

CATTGCC .GCC... AAC ...

CGC ...

CAG ATC . .. ...

TTT . ..

GAC .. .

CG; . .C

GAT GAT GCG GCT TTG AAG AAC TTT GCC AAA TAC TTT CTT CAC CAA TCT CAT GAG .. . ... ... .. . ... ... ... ... ... ... . .. . .. .. . ... ... . . . . . . .T.

GAG . ..

AGG .. .

GAG* .* A

CAT GCT GAG AAA CTG ATG AAA CTG CAG AAC CAA CAA GGT GGC u . . . . . . ..* *.. .*. . . . .* G . . . ..* *.. . . . .G. . . . . . . C..

CAG ...

GAT ...

ATC ...

AAG AAA CCA GAC T.. . . . . . . . . . . . . .GT

TTA ...

CAT ,..

TTG ...

GAA AAA AAT GTG AAT CAG TCA CTA CTG GAA CTG CAC AAA CTG GCC ACT . . . . . . . . . . . . . . . . . . . . . . . . . . . -.. ... ... . .. ... .. . ...

AAT .. .

GAC .. .

CCC . ..

CAT TTG TGT GAC TTC ATT GAG ACA CAT TAC CTG AAT GAG CAG GTG AAA GCC ATC . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AAA .. .

GAA .. .

TTG . ..

GGT GAA aa C GTG ACC AAC TTG C& . . . ..C CA. . . . . . . . . . . . . .G.

GAA ;. .

TAT .. .

CTC . ..

TTT GAC AAG CAC ACC CTG GGA GAA AGT GAT AAT GAA AGC TAA GCCTCA . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . yL . . . ..G

CCCATAGCTG TACCAAAACA .. .... ....

1849 1919 1989 2059

TCG CAG GTG CGC CAG AAC TAC CAC CAG GAC TCA GAG GCC GCC . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . ..

ATC ...

. . . . . . ..C.

1769

TCTCCTTAGT ,.........

TGGGGTGACT

AAC CTG GAG CTC TAC GCC TCC TAC TTT AAC CTC TCC ATG TCT . . . . . . . . . . . . . . . . . . . . . . . . G . . T....G...............

ATT ..C

GAT GAC TGG GAT AGTt GGG CTG AAT G@!ATA . . . . . . . . . ..G ..C . . . . . . . . . .CA ..G

AAG ATG GGA Gd . . . . . . . . . ..G

TTC CTT .. . ...

GAG TGT GCA ... ... ... GAC

..*

AAA ..,

CCC GAA TCT GGC TTG Gd . . . . . . 1.. . . . . . . ..G

TCCCTGGTCA

CCAAGGCAGT

GCATGCATGT

TGGGGTTTCC

TTTACCTTTT

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

TAC TAC

TCCACTTAAG TTCTTTTGAT TTGTACCATT CCTTCAAATA AAGAAATCT . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . ...*.

GGCTAATTTC .... .... .. CTATAAGTTG . . . . . . . . . .

GGTACCCCCC CCCAAAW ..... ..... ...

=TAGCACAT

GTGAATACAA

ATAAAAAGTA

AGCTACTGTG

GCTACATTCA

TGTCAGACAA

AGTAAATTTC

AGAGTAGAGA

ATATTACCAG

TAATAAATGA

TTATTTCATA

GTGATAAAGG

GGTCAATTCA

TGAAAAAGAC

ATAATCCTAA

ACCTAATACA

ACAGTTCAAA

ATAAATGCAG

CAAAAACTGG

AAGGAGAAAG

AAACAGATCC

TCTAGAGTCG

ACCTGC

TWO

HUMAN

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H GENES

The only discrepancy in the sizes of hybridizing fragments was that the ferritin sequences lay on a Hind111 fragment of about 9 kb in clone H35 but 12 kb in the hybrid cell line. Since the only Hind111 site in the vector is more than 2 kb from the cloning site and the Hind111 fragment with ferritin sequences does not contain vector sequences (Fig. 3), it is unlikely to represent a truncated 12-kb genomic Hi&II fragment. Moreover, the K7 probe hybridized with two fragments of about 9 and 12 kb in human genomic DNA (Fig. 4). This indicates either that these are unrelated fragments with K7-like sequences or that they represent different alleles. Although our analyses here do not show that the 9-kb fragment comes from chromosome 6p, other analyses show that it is indeed a polymorphic fragment of the 12-kb fragment (E. Zappone, I. Dugast, P. Papadopoulos, K. Theriault, J-Y. Legall, L. Powell, and J. Drysdale, submitted for publication). Since most of the ferritin sequences in H35 lay on the 2-kb BglII fragment, we sequenced this fragment using the strategy outlined in Fig. 4. The results (Fig. 6) show that it contains only one ferritin gene sequence and that this sequence has all of the characteristics of a nonfunctional processed pseudogene (Vanin, 1984). It is colinear with the ferritin H cDNA and contains no introns. Immediately preceding the sequence corresponding to the 5’ end of the H cDNA is a sequence of 15 nucleotides which is repeated immediately after a short poly(A) tract at the 3’ flank. Compared to the liver H cDNA, there are 22 substitutions and one deletion of 3 nucleotides in the 203 nucleotides corresponding to the 5’ noncoding region. By contrast, there are only 3 substitutions in the 165 nucleotides corresponding to the 3’ noncoding region. In the sequence corresponding to the coding region of 546 nucleotides there are 24 substitutions and 4 deletions. One substitution 236 nucleotides from the putative initiating ATG results in a translation terminator. None of the cloned DNA in H35 hybridized with a probe, pSP65, derived from pHLA2 (Paul et aZ., 1987) for class 1 HLA genes. A computer search showed that much of the sequence upstream from the ferritin pseudogene represents Ah repeat sequences, particularly in the region between 60 and 700. DISCUSSION These studies were designed to test the hypothesis that an abnormality in ferritin H expression in intes-

ON

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6p

209

tine from the gene on chromosome 6 could be the underlying defect in IH. Three different partial H cDNA clones obtained by screening an intestinal mucosa cDNA library with the liver H cDNA gave sequences identical to portions of the 3’ end of the liver H cDNA, suggesting that the ferritin H mRNA in intestine is the same as that in other tissues. However, this analysis was inconclusive since the largest insert corresponded to only about 35% of the length of the liver H cDNA and would not include likely differences in a message from a second gene (see below). In addition, our cDNA library was derived from all cell types in the duodenum. A library from the absorptive epithelial cells might be more informative since a lesion in ferritin synthesis has recently been found in these cells but not in other duodenal cell types (Fracanzani et al., 1989). In another approach, we characterized the H sequences mapped to the short arm of chromosome 6. To our surprise, we found evidence for at least two ferritin H genes near the HLA region on chromosome 6p, one of which we isolated. We are confident that the lesser hybridizing bands represent a second gene rather than partial digestion products since their sizes in EcoRI, Hi&II, and A4spI digests are all smaller than those of the corresponding genomic bands shown to be represented in clone H35. The relative positions of the two genes are not known but they may be quite far apart in view of the rather broad localization of ferritin sequences to 6~12-21.3 given by in situ hybridization (McGill et aZ., 1987). The two genes differ substantially in their hybridization with the liver H cDNA when washed at high stringency. Perhaps not all fragments of the uncharacterized gene have been identified in the hybrid cells. Perhaps this gene is less homologous to the liver H cDNA, as has been found recently for an apparent second human H mRNA (Moroz et al., 1989). Although the cloned gene in H35 is probably a nonfunctional processed pseudogene, comparison of its sequence with those of the liver H cDNA, pHF16, (Boyd et al., 1985) and of two other H pseudogenes, clones 123 and 133 of Costanzo et al. (1986), raises some interesting issues. The most striking is the similarity in substitutions in all three pseudogenes. For instance, the pseudogene from chromosome 6 has nine substitutions in the putative coding region at positions 1133, 1256, 1319, 1410, 1421, 1422, 1590, 1603, and 1621 which are identical with substitutions in one or both of the other pseudogenes. (Note that the ferritin sequences lie between positions 921 and 1841 in Fig. 6.)

FIG. 6. Sequence of the 2-kb BglII fragment from clone H35. The ferritin sequence lies between bases 921 and 1941 and is compared to that of the expressed liver H cDNA, pHF16, in the lower line. The sequences have been arranged for maximum homology and the numbering is according to the sequence in clone H35. Direct flanking repeats, the metal regulatory element, and potential start and stop codons are underlined. Identical substitutions in more than one human ferritin pseudogene are marked with asterisks.

DUGAST

210

All but one of these mutations are C-T or G-A. Significantly perhaps, the substitution at 1621 in all three human pseudogenesis identical to that in two different mouse H mRNAs described by Torti et al. (1988) and Beaumont et al. (1989) as is that at position 1319 in clones H35 and 133. Also notable is that none of the common substitutions in the human pseudogenes would be likely to disrupt a ferritin structure. Those at 1133,1256,1319,1410,1603, and 1621 would all be silent mutations. Those at 1421/1422 would give a probably neutral substitution of Ala-Val. This residue is not thought to be important for shell assembly or function. Further, the substitution at 1590 which would give Arg-His is also one of the differences in the expressed human H and L chains (Boyd et al., 1985). In addition, there are three identical substitutions at positions 921, 991, and 1053 in the 5’ noncoding region of all three pseudogenes. The finding in three pseudogenes of common substitutions that would not affect the protein structure is intriguing. It seems unlikely that they are coincidences of point mutations on different pseudogeneson different chromosomes. They might have arisen separately from the same error in reverse transcription at an early stage in embryogenesis, from duplication of a single retrotranscript before scattered integration into the genome, or from gene conversion. However, we know of no precedent for these possibilities in other pseudogenefamilies. An alternative explanation is that they all arose from a transcript in germ line cells from a gene other than that described on chromosome 11. If so, the characterization of the second H gene on the short arm of chromosome 6 could still be interesting in terms of IH. ACKNOWLEDGMENTS We thank Dr. Campion, CHU de Rennes, France for providing human duodenal tissue, Dr. Frank Baas, Dana Farber Institute, Boston, for allowing us to screen filters from the cosmid library; Dr. B. David Stollar for the database search; and Ms. Kay Lucey for secretarial assistance. This work was supported by Grant AM 1777514 from the NIH. REFERENCES

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Identification of two human ferritin H genes on the short arm of chromosome 6.

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemo...
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