J Mol Evol (1997) 44(Suppl 1):S147-S154

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�9 Springer-Verlag New York Inc. 1997

Genomic Characterization of the Region Between HLA-B and TNF: Implications for the Evolution of Multicopy Gene Families

Silvana Gaudieri, 1 Chanvit Leelayuwat, l'z David C. Townend, 1 Jerzy K. Kulski, 1'3 Roger L. Dawkins I

Department of Clinical Immunology, Centre for Molecular Immunology and Instrumentation, Royal Perth Hospital, University of Western Australia, Perth Western, Australia 2 Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand 3 Department of Microbiology, Royal Perth Hospital, University of Western Australia, Perth Western, Australia

Received: 7 August 1996 / Accepted: 2 September 1996

Abstract . The major histocompatibili ty complex (MHC) contains genes which confer susceptibility to nu- merous diseases and must be important in primate evo- lution. In some instances, genes have been mapped to the region between human histocompatibility leukocyte an- tigen (HLA)-B and tumor necrosis factor (TNF) but pre- cise localization has proven difficult especially since this region is subject to insertions, deletions, and duplica- tions.

Utilizing computer similarity searches and coding prediction programs, we have identified several potential coding sequences between HLA-B and TNF. Three of these sequences, PERB 11.2, PERB 15, and PERB 18, are similar to members of multicopy gene families that are located in other regions of the MHC. The identification of numerous fragmented and intact retroelements (L1, Alu, LTR, and THE sequences) flanking the PERB11 and PERB 15 genes suggests that these retroelements are involved in the duplication process. The evaluation of candidate genes for disease susceptibility within the MHC is complicated by their similarity to other members of multicopy gene families. The determination of se- quence differences within and between species provides

Publication number 9631 of the Centre for Molecular Immunology and Instrumentation, University of Western Australia, Department of Clini- cal Immunology, Royal Perth Hospital and Sir Charles Gairdner Hos- pital, Perth Western, Australia Correspondence to: R.L. Dawkins; e-mail: dawkins@erythema.dci. uwa.edu.au

a strategy with which to investigate the candidate genes between HLA-B and TNF.

Key words: HLA-B - - TNF - - Multicopy gene fami- lies

Introduction

Candidate genes for several human diseases have been mapped to the region between human histocompatibility leukocyte antigen (HLA)-B and tumor necrosis factor (TNF) in the major histocompatibility complex (MHC). The relevant diseases associated with this region include insulin-dependent diabetes mellitus (IDDM), myasthenia gravis (MG), Behcet's disease, IgA deficiency, and an- kylosing spondylitis (Degli-Esposti et al. 1992a,b; Mi- zuki et al. 1995; French and Dawkins 1990). For IDDM and MG, recombinant mapping studies have shown that irrespective of any other MHC influences such as those contributed by class II genes, a locus near HLA-B modu- lates autoantibody titers (Degli-Esposti et al. 1992a). A major influence on generalized MG is marked by the allele B8 rather than A1 or DR3 (Degli-Esposti et al. 1992b). Ankylosing spondylitis is associated with B27, and Behcet's disease with B5 and/or a gene in the region between HLA-B and TNF (Mizuki et al. 1995). Rapid progression to AIDS following HIV-1 infection has been mapped to the region between HLA-B and C4 or TNF (Mallal et al. 1990; Cameron et al. 1992).

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H U M A N 5 7 . 1 A H

BATI

LT-[~. /

TNF-oc~ I N~-KI3

I ! I | | |

lambda clone

C H I M P A N Z E E

I n s e r t i o n / d e l e t i o n

57J10-10

PERB 11.2

PERBI3 /p)R PERil5

PERB18 / B19~ J

I I I I I I e l I I I I

/ / I

57B1-18-1 7W4-1

TNF-~ BATI PERB 11.2

I ? 7 17 7 1 7 I I i I I i i I i i i I I I i l J

Fig. 1. Genomic organization of the region between HLA-B and TNF (57.1 AH). There are several characterized genes within the region between HLA-B and TNF, namely TNF-~, LT-[3, NF-K[3, PERB11.1, PERB 11.2, and HLA-B, which are illustrated as black boxes; also, there are several potential coding sequences determined by homology and GRAIL (thin lines). Pulse field gel electrophoresis fragment sizes and YACs have been used to position the PERB sequences and thus the ordering of particular sequences may be reversed in order. The posi-

PERB6 PERB 14 ~ERB 7

/ PERBII'I i N / \ I "

I

I li I

57X-8-4

PERB6

PERB5 PERB 1

I I I I I

57PS5-1

PERB 1

PERB 17 Patr-B

I ? ? / / I I I I I

I I I I f I

tions of the lambda clones are shown as black rectangles. The corre- sponding map of the chimpanzee is shown below; the deleted region is indicated by a forward slash and the region is also flanked by dashed lines. Several probes have not been mapped onto the chimpanzee and therefore have been shown as a question mark. Patr-B in chimpanzee is the homolog of HLA-B in humans. Other chimpanzee haplotypes need to be tested to determine the presence or absence of PERB 11.1.

Several genes within this region have been described p rev ious ly inc luding P E R B l l . 1 , BAT1, TNF, and HLA-B (Marshall et al. 1993; Leelayuwat et al. 1994).

PERB 11.1 is a member of a mult icopy gene family within the MHC and has been proposed as a candidate disease susceptibility gene. The presence of mult icopy gene families complicates the evaluation of candidate disease genes. To address this problem we have investi-

gated nonhuman primates. Humans and chimpanzees share a high level of iden-

tity at the DNA level (Diamond 1988). However, we have previously shown that there is a large ( -50%) length difference of approximately 130 kb in the region between HLA-B and TNF (Leelayuwat et al. 1993). In- terestingly, chimpanzees infected with HIV-1 do not ap- pear to progress to AIDS or develop autoantibodies in the same way as humans (Goudsmit et al. 1988; Watan- abe et al. 1991). In contrast, macaque monkeys progress rapidly to disease following simian (S)IV infection from the African green monkey isolate (Lewis et al. 1992; Simon et al. 1992). To evaluate the candidate suscepti- bil i ty genes between HLA-B and TNF we have com- pared humans and chimpanzees at the sequence level and by pulse field gel electrophoresis (Leelayuwat et al. 1993). The genes which are deleted/transposed in the chimpanzee should be critical in the pathogenesis of the autoimmune and infectious diseases mapped to this re- gion. Therefore, we expect the macaque monkey and human to possess the candidate susceptibi l i ty genes which have been deleted/transposed in the chimpanzee.

In this paper, we describe new potential coding se-

quences in this region, their relationship to multicopy gene families within the MHC, and their possible asso-

ciation with several diseases.

M a t e r i a l s a n d M e t h o d s

Cell Lines. The cell lines used for the construction of lambda genomic DNA libraries have been described previously (Leelayuwat et al. 1992). The libraries were derived from the 57.1 (designated by D6S105[126]; D6S3061241]; D6S464[199]; MOGA[127]; MOGB[122]; MOGC[160]; HLA-A1; HLA-Cw6; HLA-B57; PERB1B; TNFBb5; TNFAa2; TNFNL; C2C; BfS; C4A6; C4B1; HLA- DRB1-0700; HLA-DQA-0201; HLA-DQB-03032; TAP1A; TAP2A; HLA-DPA1-01; HLA-DPB1-0401) and the 7.1 ancestral haplotypes (designated by D6S105[115, 126]; D6S3061239, 246]; D6S4641212, 218]; MOGA[129], MOGB[129]; MOGC[160]; HLA-A3; HLA-Cw7; HLA-B7; PERB1A; TNFBb4; TNFAall; TNFNL; C2C; BfS; C4A3; C4B 1 ; HLA-DRB 1-1501; HLA-DQA-0102; HLA-DQB-0602; TAP1A; TAP2A; HLA-DPA1-01; HLA-DPB1-0401) (Tay et al. 1996, submitted).

Genomic Cloning and Sequencing. The following lambda clones were used in this study, 57BATl-18-1, 7W4-1, 57X-8-4, 57PS5-1, and 57J10-10 (Fig. 1). These clones were isolated using the probes BAT1, WR9A, XR17A, PS-5, and JAB, respectively (Wu et al. 1992; Leel- ayuwat et al. 1994).

The lambda clones 57BATl-18-1, 7W4-1, 57X-8-4, and 57PS5-1 were subcloned into pBCKS+ (Stratagene, La Jolla, CA, USA), soni- cated into random fragments, and then further subcloned into pBCKS+ and sequenced using the ABI Dye primer sequencing chemistry (ABI, Foster City, CA, USA). The resultant chromatograms were edited and aligned using the program SeqEd vl.03 (ABI). The lambda clone 57J10-10 was subcloned into pBK-CMV and digested using PstI (Pro- mega Corporation, Madison, WI, USA). The resultant fragments were subcloned into pBK-CMV and sequenced as above.

Table 1. Description of the putative and known coding sequences obtained within the region between HLA-B and TNF

PER Description/GenBank Gene family Assignment homology in MHC Reference

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PERBt PERB5 PERB6 PERB7

PERB 11 PERB 13 PERB 14

PERB 15 PERB 17 PERB 18 PERB 19

Fibroblast Growth Factor Receptor (FGFR) FGFR3, FGFR5 HLA-17 (exon 6, 7, 8) HLA TRAP, CD40L Fas ligand TNF P5-1 (5' UTR, exon 1, 3' UTR) Found within CL-10-30 kb cemromeric of P5

HLAB Non mammalian class l, IgG FcR Zn alpha-2 glycoprotein PERB 11 Cyclophilin A endogenous retroviral sequence shares protein motif with the pol region of endogenous

Moloney and Foamy retroviruses retrovirus 98% homology to P5-1, 70% with PERB7 Located centromeric of PERB7 P5 dihydrofolate reductase located 10-20 kb centromeric of HLAB ribosomal protein LI0 HLA class I HLA

Leelayuwat et al. 1994 Marshall et al. 1993 Marshall et al. 1993 Marshall et al. 1993

Leelayuwat et al. 1994

Gaudieri et al. 1996

Genomic Analysis. Sequence analysis of the genomic sequence was performed using the neural network programs GRAIL and GE- NEPARSER to identify potential coding sequences (Uberbacher and Mural 1991; Synder and Stormo 1993). The programs BLASTn and BLASTx were used to identify similar sequences with the databases GenBank and SwissProt, respectively. Sequence alignments were pro- duced using ClustalW. The programs FindPatterns and PatternSearch were used from GCG version 8 (Wisconsin Sequence Analysis Pack- age) to search for motif patterns within the GenBank and SwissProt

databases.

Results

Identification of Novel Sequences in the Genomic Region Between HLA-B and TNF

The novel sequences detected by similarity searches and analyses of coding sequences by GRAIL include: PERB 11.2; PERB 13 (a cyclophilin-like sequence, in the region between HLA-B and TNF, has also been de- scribed by Nalabolu et al. 1996), PERB14; PERB15; PERB17; PERB18; and PERB19 (Table 1). Figure 1 shows the relative position of the sequences within the region between HLA-B and TNF in the 57.1 human MHC ancestral haplotype.

Similarity of Sequences to Members of Multicopy Gene Families Within the MHC

Several of the sequences are related to members of mul- ticopy gene families within the MHC (Table 1). PERB 15 is a member of the P5 family (Vernet et al. 1993), PERB 11.2 of the PERB 11 (MIC) family (Leelayuwat et al. 1994; Bahram et al. 1994; Babram and Spies 1996), and PERB18 is a ribosomal protein-like sequence. This ribosomal protein-like sequence may form part of a new multicopy gene family with the localization of two re- cently described ribosomal protein-like sequences near HLA-C (Mizuki et al. 1996, iia preparation) and HLA-A (Totaro et al. 1996).

The P5 Family At least nine sequences related to the P5 family have

been described in the MHC (Marshall et al. 1993; Vemet et al. 1993; Avoustin et al. 1994; Venditti et al. 1994). PERB7 and PERB15 are located 30 kb and 100 kb cen- tromeric of HLA-B, respectively. PERB15 shares ap- proximately 98% identity with P5-1 and 70% with PERB7 and P5-3 (Fig. 2B). PERB15 is very similar to the putative coding sequence of P5-1 except that it con- tains a four-base pair insertion in the open reading frame. This frameshift adds a proline residue and increases the length of the putative product translated. PERB15 is a duplicated copy of the P5-1 sequence which has been mapped to a telomeric position near HLA-E (Vernet et al. 1993) (Fig. 3). P5-3 has been mapped by the same group near HLA-A. The relative locations of the de- scribed P5 genes in the MHC are shown in Fig. 3.

A sequence which shares >95% identity with a cDNA clone termed 3.8-1 by Venditti et al. (1994) lies approxi- mately six kilobases upstream of PERB15. This cDNA sequence is also part of a multicopy gene family within the MHC.

The PERB 11 Family The nucleotide sequence of PERB 11.2 is very similar

to PERB 11.1 in most domains except in the transmem- brane region. Nucleotide differences in the transmem- brahe region result in a number of nonsynonymous sub- stitutions at the amino acid level (Fig. 2A). DNA and amino acid comparison of the PERB 11 family shows that P E R B l l . 2 appears to be in te rmedia te be tween PERB 11.4, which is located near HLA-A, and the more closely located PERBll .1 (Fig. 3).

Ribosomal Protein-Like Sequence PERB18 exists several kilobases downstream of the

BAT1 gene and shares over 90% identity with the human L10 ribosomal protein sequence. At the nucleotide level there are many insertions and deletions, causing frame- shifts which result in premature stop codons.

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Fig. 2. DNA and amino acid polymorphism of the PERB 11 and P5 multicopy gene families in the MHC. A DNA and amino acid com- parison of the PERB11 family. The comparison of three members of the PERB 11 gene family (PERB 11.1, 2, and PERB 11.4) reveals a high level of identity between all of the members. Based on the residues, PERB11.2 is an intermediate between the other two members of this family at both the DNA and amino acid level. PERB 11.4 has an early putative stop codon in the alpha 2 domain, but the DNA sequence following this codon is still very similar to the other two members. B DNA comparison of the members of the P5 family. At the DNA level, P5-1 and PERB 15 are highly similar to each other and both share about 70% identity with the other two members of the P5 family, PERB7 and P5-3. The boxed-in area represents the sequence difference between

the different members of P5, which causes a change in the putative amino acid sequence shown in C. Both members within the beta block share this insertion, although only PERB15 contains the open reading frame corresponding to the amino acid sequence. P5-1 and PERB 15 are obviously most closely related but the other two members, PERB7 and P5-3, are intermediate and share residues with all the different mem- bers. C Amino acid translation of P5-1 and PERBI5. The putative translation of the open reading frame in P5-1 and PERB15 shows the difference in amino acid sequence following the insertions in PERB 15, which is indicated by vertical arrows. Dashes indicate identity, an asterisk in the DNA sequence indicates a deletion and a stop codon in the amino acid sequence, and a hash box indicates a deletion in the amino acid sequence.

S151

Fig. 2. Continued.

S152

Fig. 2. Continued.

Fig. 3. Organization of the PERB 11, P5, and HLA multicopy gene families within the MHC. The locations of multicopy gene family members within the telomeric end of the MHC are illustrated by ver- tical lines. Each member of a family is aligned to the family name,

which is boxed on the left. Several HLA fragments and pseudogenes within the MHC have not been shown for simplicity. The position of the PERB 11 genes have been described by Leelayuwat et al. (1994) and Pichon et al. (1996) and P5 members by Avoustin et al. (1994).

Retroelements Flanking Potential Coding Sequences

Intact and fragmented Alu, L1, LTR, and other inter-

spersed repeat sequences such as MERs have been found

within and surrounding the novel sequences PERB 11.2,

PERB13, PERB14, and PERB15. Also, there is a large

open reading frame corresponding to a LINE near the

retroviral-like sequence PERB14.

Discussion

We have identified new candidate genes for disease sus- ceptibility in the region between HLA-B and TNF. Sev-

eral of these sequences are members of multicopy gene

families located in the MHC (Table 1 and Fig. 3).

In the region of the MHC containing the duplicated

genes C4 and CYP21, there are haplotype and species

differences in assortment and copy number (Zhang et al.

1990; Tokunaga et al. 1991). We postulate that disease

susceptibility and progression are due not only to the

gene itself but also to the copy number. In addition, the

sequence variation between duplicated sequences in the

region between HLA-B and TNF and the telomeric re-

gion of the MHC may be relevant in disease associations

with hemochromatosis (Totaro et al. 1995; Yaouanq et

al. 1994). The candidate disease susceptibility gene PERB 11.1

is particularly attractive as a key molecule in the patho-

genesis of autoimmune and infectious diseases. It ap-

pears to be deleted in the chimpanzee and is part of the

large ancestral family of class I-like molecules which

includes HLA class I, neonatal IgG FcR molecule

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(FcRn), Zn-alpha2-glycoprotein, MR1, and HLA-H (po- tential Hemochromatosis gene) (Leelayuwat et al. 1994; Hashimoto and Kurosawa 1995; Feder et al. 1996) (Fig. 1). This large family is involved in binding and trans- portation of peptides, viral proteins, and possibly heavy- metal-binding receptors. Sequence analysis of the trans- membrane region of PERBll .1 in several different haplotypes reveals an insertion causing a frameshift and an early termination codon resulting in a putative trun- cated product (Leelayuwat et al. 1996). Currently anti- body and disease population studies are underway to investigate the association of the PERB11 gene family with IDDM, psoriasis, Behcet's disease, and IgA defi- ciency.

Sequence analysis of the region between HLA-B and TNF has also revealed numerous fragmented and intact retroelements. Comparative genomic sequence analysis suggests that retroelements have a role in duplication, transposition, recombination, and deletion of gene seg- ments (Erickson et al. 1992). The presence of retroele- ments flanking the members of multicopy gene families is suggestive of a role in the duplication of the genes within the telomeric end of the MHC. The evolution of multicopy gene families within the MHC may be an important mechanism for providing functional diversity of surveillance genes and protection from deleterious ef- fects of mutations in a single-copy gene. In this context, although the role of the P5 family genes is unknown it may have an important influence in the disease process because of its many copies and the variation in copy number between species.

Acknowledgments'. We wish to thank Louise Taylor, Elisa McIntyre, Laila Gizzarelli, Guan Tay, Campbell Witt, and Gerhard Saueracker for their technical assistance and advice. We also wish to thank Michael Chorney from Penn State University, Hershey, USA, and Hidetoshi Inoko from Tokai University, Kanagawa, Japan. This work was sup- ported by the National Health and Medical Research Council and the lmmunogenetics Research Foundation.

References

Avoustin P, Ribouchon MT, Vernet C, N'Guyen B, Crouau-Roy B, Pontarotti P (1994) Non-homologous recombination with the Major Histocompatibility Complex creates a transcribed hybrid sequence. Mamm Genome 5:771-776

B ahram S, Spies T (1996) Nucleotide sequence of a human MHC class I MICB cDNA. Immunogenetics 43:230-233

Bahram S, Bresnaban M, Geraghty DE, Spies T (1994) A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci USA 91:6259-6263

Cameron PU, Mallal SA, French MAH, Dawkins RL (1992) Central MHC genes between HLA-B and complement C4 confer risk for HIV-1 disease progression. In: Tsuji K, Aizawa M, Sasazuki T (eds) HLA 1991, vol 2. Oxford University Press, New York, pp 544-547

Degli-Esposti MA, Abraham L J, McCann V, Spies T, Christiansen FT, Dawkins RL (1992a) Ancestral haplotypes reveal the role of the central MHC in the immunogenetics of IDDM. Immunogenetics 36:345-356

Degli-Esposti MA, Andreas A, Christiansen FT, Schalke B, Albert E,

Dawkins RL (1992b) An approach to the localisafion of the sus- ceptibility genes for generalised myasthenia gravis by mapping recombinant ancestral haplotypes. Immunogenetics 35:355-364

Diamond JM (1988) DNA based phylogenies of the three chimpanzees. Nature 332:6854586

Erickson LM, Kim HS, Maeda N (1992) Junctions between genes in the haptoglobin gene cluster of primates. Genomics 14(4):948-958

Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel GS, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayna DT, Risch NJ, Bacon BR, Wolff RK (1996) A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13:399408

French MAH, Dawkins RL (1990) Central MHC genes, IgA deficiency and autoimmune disease. Immunol Today 11:271-274

Gaudieri S, Kulski JK, Dawkins RL (1996) The central region of the Major Histocompatibility Complex contains a motif of amino acid sequence with homology for the pol region of Moloney retrovirus- es. Immunogenetics 44(2): 157-158

Goudsmit J, Debouck C, Heloen RH, Smit L, Bakker M, Ashers DM, Wolff AV, Gibbs CJ, Gajdusek DC (1988) Human immunodefi- ciency virus type I neutralization epitope with conserved architec- ture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc Natl Acad Sci USA 85:4478-4482

Hashimoto KH, Kurosawa M (1995) A gene outside the human MHC related to classical HLA Class 1 genes. Science 269:693

Leelayuwat C, Abraham LJ, Tabarias H, Christiansen FT, Dawkins RL (1992) Genomic organisation of a polymorphic duplicated region centromeric of HLA-B. Immunogenetics 36:208-212

Leelayuwat C, Zhang W J, Townend DC, Gaudieri S, Dawkins RL (1993) Differences in the central MHC between man and chimpan- zee: implications for autoimmunity and acquired immune defi- ciency development. Hum Immunol 38:30M1

Leelayuwat C, Townend DC, Degli-Esposti MA, Abraham LJ, Dawk- ins RL (1994) A new polymorphic and multicopy MHC gene fam- ily related to non mammalian class I. Immunogenetics 40:339-351

Leelayuwat C, Gaudieri S, Mullberg J, Cosman D, Dawkins R (1996) Proceedings of the 12th IHWE genetic diversity of HLA: functional and medical implications, vol. 2. Conference 1996. EDK

Lewis MG, Zack PM, Elkins WR, Jahrling PB (1992) Infection of rhesus and cynomolgus macaques with a rapidly fatal SIV (Siv smm/phj) isolated from sooty mangabeys. AIDS Res Hum Retro- viruses 8(9):1631-1633

Mallal SA, Cameron PU, French MAH, Dawkins RL (1990) MHC genes and HIV infection. Lancet 335:1591-1592

Marshall B, Leelayuwat C, Degli-Esposti MA, Pinelli M, Abraham LJ, Dawkins RL (1993) New MHC genes. Hum Immunol 38:24-29

Mizuki N, Ohno S, Sato T, Ishihara M, Miyata S, Nakamura S, Naruse T, Mizuki H, Tsuji K, Inoko H (1995) Microsatellite polymorphism between the tumor necrosis factor and the HLA-B genes in Beh- cet's Disease. Hum Immunol 43:129-135

Miznki N, Ando H, Kimura M, Ohno S, Miyata S, Yamazaki M, Tasbiro H, Watanabe K, Ono A, Taguchi S, Sugawara C, Fnkuzumi Y, Okumura K, Goto K, Ishibara M, Kikuti YY, Shina T, Lei C, Ando A, Ikemura T, Inoko H (1996) Nucleotide sequence analysis of the HLA class I region spanning the 237 kb segment around the HLA-B and -C genes. (in preparation)

Nalabolu S, Shukla H, Nallur G, Parimoo S, Weissman S (1996) Genes in a 220-kb region spanning the TNF cluster in human MHC. Genomics 31:215-222

Pichon L, Hampe A, Giffon T, earn G, Legall JY, David V (1996) A new non-HLA multigene family associated with the PERB 11 fam- ily within the MHC class I region. Immunogenetics 44:259-267

Simon MA, Chalifoux LV, Ringler DJ (1992) Pathologic features of SIV-induced disease and the association of macrophage infection with disease evolution. AIDS Res Hum Retroviruses 8(3):327-335

S154

Synder EE, Stormo GD (1993) Identification of coding regions in genomic DNA sequences: an application of dynamic programming and neural networks. Nucleic Acids Res 21:607-613

Tokunaga K, Zhang WJ, Christiansen FT, Dawkins RL (1991) The genomic structure of two ancestral haplotypes carrying C4 dupli- cations. Immunogenetics 34:247-251

Totaro A, Grifa A, Roetto A, Lunardi C, D'Agruma L, Sbiaz L, Zelante L, DeSandre G, Camaschella C, Gasparini P (1995) New polymor- phisms and markers in the HLA class I region: relevance to heredi- tary hemochromatosis (HFE). Hum Genet 95:429-434

Totaro A, Rommens JM, Grifa A, Lunardi C, Carella M, Huizenga JJ, Roetto A, Camaschella C, De Sandre G, Gasparini P (1996) He- reditary hemochromatosis: generation of a transcription map within a refined and extended map of the HLA Class I region. Genomics 31:319-326

Uberbacher EC, Mural RJ (1991) Locating protein-coding regions in human DNA sequences by a multiple sensor-neural network ap- proach. Proc Natl Acad Sci USA 88:11261-11265

Venditti CP, Harris JM, Geraghty DE, Chorney MJ (1994) Mapping and characterization of non-HLA multigene assemblages in the human MHC class I region. Genomics 22:257-266

Vemet C, Ribouchon M, Chimini G, Jouanolle A, Sidibe I, Pontarotti

P (1993) A novel coding sequence belonging to a new multicopy gene family mapping within the human MHC class I region. Im- munogenetics 38:47-53

Watanabe M, Ringler DJ, Fultz PN, MacKey JJ, Boyson JE, Levine CG, Letvin NL (1991) A chimpanzee-passaged human immunode- ficiency virus isolate is cytopathic for chimpanzee cells but does not induce disease. J Virol 65:3344-3348

Wu X, Zhang WJ, Witt CS, Abraham LJ, Christiansen F/', Dawkins RL (1992) Haplospecific polymorphism between HLA-B and tumour necrosis factor. Hum Immunol 33:89-97

Yaouanq J, Perichon M, Chorney M, Pontarotti P, Le Treut A, E1 Kahloun A, Mauvieux V, Blayau M, Jouanolle AM, Chauvel B, Moirand R, Nouel O, Le Gall J, Feingold J, David V (1994) Anony- mous marker loci within 400 kb of HLA-A generate haplotypes in linkage disequilibrium with the hemochromatosis gene (HFE). Hum Genet 54:252-263

Zhang WJ, Degli-Esposti MA, Cobain TJ, Cameron PU, Christiansen FT, Dawkins RL (1990) Differences in gene copy number carried by different MHC ancestral haplotypes: quantitation after physical separation of haplotypes by pulsed field gel electrophoresis. J Exp Med 171:2101-2114