Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

Mammalian Genome 6, 809-815 (1995).

"Genome �9 Springer-Verlag New York Inc. 1995

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

J. Hu, 1 N. Bumstead, 4 D. Burke, s F. A. Ponce de Le6n, s E. Skamene , 1 P. Gros, z D. Malo 1'3

1Department of Medicine, McGill University, Montreal, Canada 2Department of Biochemistry, McGill University, Montreal, Canada 3Department of Human Genetics, McGill University, Montreal, Canada 4Institute for Animal Health, Compton, England, UK 5Department of Veterinary and Animal Science, University of Massachusetts, Amherst, Massachusetts USA

Received: 16 April 1995 ! Accepted: 27 June 1995

Abstract. The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nrampl gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chro- mosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.

Introduction

The Nrampl gene has been identified by positional cloning as a candidate gene for the host resistance locus Bcg (Vidal et al. 1993). In the mouse, Beg controls innate resistance to infection with taxonomically and antigenically unrelated intracellular pathogens such as several species of Mycobacterium (Gros et al. 1981; Ska- mene et al. 1984; Goto et al. 1989), Salmonella typhimurium (It?l; Plant and Glynn 1976), and Leishmania donovani (Lsh; Bradley, 1977). The major effect of the Bcg gene is the modulation of the growth rate of these pathogens in cells of the reticuloendothelial system of the mouse early during infection.

Evidence for Nrampl being the candidate macrophage resis- tance gene was based on 1) its map location (Malo et al. 1993a, 1993b); 2) its expression primarily in macrophages, the cell line expressing the Bcg phenotype (Vidal et al. 1993); and 3) the pres- ence of a noncouservative glycine to aspartic acid substitution within transmembrane domain 4 of Nramp~ segregating with the resistance and susceptibility phenotype in 27 inbred strains of mice (Malo et al. 1994). Targeted disruption of Nrampl eliminated nat- ural resistance to infection with Salmonella typhimurium, Myco- bacterium bovis, and Leishmania donavani and formally proved that Nrampl is indeed the Bcg gene (Vidal et al. 1995b). Nrampl is part of a small gene family that contains at least two members

Correspondence to: D. Malo at Montreal General Hospital, 1650 Cedar Avenue, Room Ll1-144, Montreal, QC, Canada, H3G 1A4.

in both mouse (Gruenheid et al. 1995) and human (Vidal et al. 1995a) genomes. Nrampl encodes a novel membrane transport protein with similarities to several transporters, including the CrnA nitrite/nitrate transporter of Aspergillus nidulans (Unkles et al. 1991). Nitrite and nitrate are two by-products of nitric oxide, which is produced by macrophages from c-arginine in response to infection or upon exposure to activation signals such as inter- feron- 7 and lipopolysaccharide. Production of nitric oxide is re- sponsible, at least in part, for the antimicrobial capabilities of activated macrophages (Hibbs et al. 1987; Liew et al. 1990).

Nrampl is located on mouse Chr 1 within an evolutionary conserved chromosomal segment corresponding to human Chr 2q (Schurr et al., 1990), bovine Chr 2 (Barendse et al. 1994), and rat Chr 9 (Mock et al. 1989). Sequence comparisons of the mouse and human proteins revealed a very high degree of conservation be- tween the two species (88% identity and 93% similarity; Cellier et al. 1994). The high degree of conservation of Nrampl among species suggests a pivotal role of this gene in host response to infection with intracellular parasites and opened up the possibility of testing the candidacy of Nrampl in the control of resistance and susceptibility of chickens to infection with Salmonella typhimu- rium.

Human infections with Salmonella are major problems in Eu- rope and North America (Hedberg et al. 1993), and, despite inten- sive control programs, poultry products are a major source of Salmonella food poisoning for humans. Early exposure to invasive serotypes such as S. typhimurium is associated with subsequent development of clinical disease in young chickens (Bumstead and Barrow 1988). Many factors, such as the age of the chicken, en- vironmental stress, and others, affect the susceptibility of chickens to Salmonella colonization. The host genetic background also ap- pears to play a crucial role in resistance and susceptibility of chick- ens to infection with Salmonellae (Bumstead and Barrow 1988). Inbred and partially inbred lines of chickens differ markedly in their susceptibility (mortality rate) to infection with S. typhimu- rium and in their ability to control the proliferation of the bacterial load in their liver and spleen (Barrow et al. 1987; Bumstead N., unpublished data). In susceptible chickens, the mortality rate is higher, and lesions in their reticuloendothelial organs are signifi- cantly larger. Linkage analyses in segregating backcross chicken progeny are compatible with the existence of a major dominant autosomal gene, not linked to the major histocompatibility com- plex in controlling innate resistance to infection with S. typhimu- rium (Bumstead and Barrow 1988).

The identification of a chicken NRAMP1 homolog and its chro- mosomal location are the goals of the present study and represent essential prerequisites to test the possible involvement of NRAMPt

810 J. Hu et al.: Mapping of NRAMP1 in chicken

as one o f the genes control l ing differential res is tance and suscep- tibility o f ch ickens to infect ion with Salmonel la .

Materials and methods

Chickens. The parental lines used in these experiments are derived from the White Leghorn lines N and 15I at the Institute for Animal Health, (Compton, England). Coefficients of inbreeding in line 15I and N have been estimated to 0.8 and 0.5 respectively (Bumstead et al. 1987). The breeding and maintenance of (NX15I)F1 x 15I segregating backcross chickens have been described previously (Bumstead and Palyga 1992). All chickens were maintained in specific pathogen-free environment.

Genomic DNA preparation and Southern blot hybridization. High- molecular-weight DNA was prepared from red blood cells as described by Bumstead and associates (1987) and was tested for the presence of restric- tion fragment length polymorphism (RFLP) with a chicken NRAMP1 probe as described previously (Malo et al. 1994).

Hybridization probes. A chicken genomic DNA library (1.4 x 106 in- dependent clones) constructed in the BamHI site of cosmid vector pWE15 (Stratagene) was screened for the presence of homologous chicken NRAMP1 sequences, with a BamHIIBglII 0.9-kb mouse Nrampl partial cDNA as probe. Duplicated filters were hybridized in 6• SSC, 5x Den- hardt's solution, 0.5% SDS at 50~ Two independent overlapping clones were identified and purified. The restriction map was established for BamHI and rare cutting restriction enzymes (SacII, BssHII, MluI, NotI, and NruI). Two single-copy probes were isolated from these cosmid clones: a 5-kb NotI fragment (MCG1) isolated from the vector adjacent DNA of cos8.1 and a 2.7-kb BamHI fragment containing the 5' end of the chicken NRAMP1 gene (MCG2; J. Hu and D. Malt unpublished). Exon probes E84 and E62, corresponding to Candidates #1 and #2 (Cd#1, Cd#2) respec- tively, have been previously described (Vidal et al. 1993). The villin (VIL) probe is a 250-bp DNA fragment obtained by PCR amplification of chicken genomic DNA with primers CHKVIL1 (5' CACCATGGTG- GAGCTCAGCA 3') and CHKVIL2 (5' CGACAGCAGCACGTAGC- AGT 3').

Linkage analyses. Linkage analysis and haplotype analyses were per- formed with the computer program GeneLink (Montagutelli 1990) and Map Manager version 2.6 (Manly 1993). Gene order was determined by minimizing the number of double-crossover events required to explain haplotype distributions. Calculated genetic distances are reported in cen- timorgans (cM) with the estimated deviation.

Fluorescence in situ hybridization. Primary fibrobtast cell cultures of two White Leghorn embryos were prepared according to standard proto- cols (Ponce de Le6n et al. 1992). The NRAMP1 chicken cosmid clone, cos8.1, was used as probe, after labeling with biotin-16-dUTP by nick translation. Hybridization was performed in 50% formamide, 10% dextran sulfate, lx SSC at 37~ overnight. The probe was detected with fluores- cein-isothiocyanate-avidin D (FITC) in 4x SSC, 1% BSA, 0.1% Tween 20 at 37~ for 30 rain. The slides were then stained with propidium iodide (200 ng/ml) and mounted in p-phenylenediamine at pH 11 (PPD-11). Sig- nals were visualized under a microscope (Zeiss, Axioskop) equipped for epifluorescence microscopy. Propidium iodide and FITC signals were si- multaneously detected with the 546/590 and 450/520 excitation/barrier filter combinations, respectively. A SIT video camera was used to capture images processed with the Image 1/AT software. Photomicrographs of dig- itized images were prepared with a color video printer (Sony, model UP5000).

Pulsed-field gel analyses. The methodology for the isolation of high- molecular-weight DNA and conditions for the enzymatic digestion of DNA in agarose blocks were carried out essentially as described by Malt and associates (1993b). DNA in agarose blocks was prepared from an embryo fibroblast cell line (ATCC CRL 1590 SL-29) of White Leghorn chickens. The cells were maintained in Eagle's MEM medium containing 5% calf

serum at 41~ in 5% CO2. DNA was digested, alone or in combination, with restriction enzymes NruI, NotI, MluI, SacII, and BssHII (enzyme-to- DNA ratio was 5U/gg DNA except for SacII, which was 1U/gg DNA). One-third of a block was loaded per lane on gels consisting of 1% agarose in 0.5• TBE (0.05 M Tris, 0.05 M boric acid, 0.10 mM EDTA). Molecular- weight standards were concatemers of bacteriophage lambda and yeast chromosomes from Saccharomyces cerevisiae strain YNN295 (New En- gland Biolabs, Ltd., Mississanga, Canada). Pulsed-field gel electrophoresis (PFGE) was carried out with a BioRad CHEF mapper system, under con- ditions described in the legend of Fig. 5, at 14~ After electrophoresis, gels were stained in 0.5x TBE with 1 gg/ml ethidium bromide for 30 rain and photographed. High-molecular-weight DNA was UV-irradiated for 4 min, denatured, and renatured prior to transfer to nylon membranes (Hy- bond N, Amersham) by capillary blotting with 20x SSC for a period of 48 h. Prehybridization and hybridization were carried out in either 50% form- amide solution for homologous DNA probes or 35% formamide solution for heterologous DNA probes at 42~ as previously described (Malt et al. 1993a). Blots were washed at increasing stringency up to 0.2x SSC, 0.5% SDS at 65~ for 45 min for homologous DNA probes, and 0.2x SSC, 0.1% SDS at 60~ for 20 rain for heterologous DNA probes.

Results

Isolation and characterization of chicken NRAMP1 cosmid clones. Specif ic probes for genet ic and phys ica l m a p p i n g were obta ined

f rom NRAMP1 cosmid c lones isolated f rom a ch icken g en o m ic D N A library us ing a m o u s e Nrampl partial c D N A as hybr id iza t ion probe. The two cosmids were character ized by d iges t ion wi th re- str ict ion e n z y m e s BamHI, HindIII, BssHII, SacII, MluI, NruI, SalI, and NotI and Southern blott ing (Fig. 1). Since N R A M P is a smal l f ami ly o f related genes , we wan ted to ver i fy that the correct ch icken NRAMP1 clones had been identified. For this, we used the m o u s e Nrampl c D N A clone as a probe on Southern blots co m - par ing the hybr id iza t ion pat terns o f BamHI and HindIII f r agment s obta ined f rom the isolated cosmid c lones and f rom ch icken geno- mic D N A (data not shown) . The m o u s e Nrampl probe revealed hybr id iza t ion pat terns that are identical in both cosmid and geno- mic D N A . In addition, nucleot ide s equenc ing o f subc lones f rom the NRAMP1 cosmids conf i rmed that the ch icken h o m o l o g of m o u s e Nrampl had been cloned. The overall h o m o l o g y be tween m o u s e and chicken N R A M P 1 sequences at the amino acid level was 82% (J. Hn and D. Malo, unpubl i shed) .

B Bs Sa Sa

S Sa NMBsS B BB BBsMN B I I II ~1 I I I ~ ' i l l I

MCG2 MCG1

NRAMP1 CD#2 VIL

Cos8.1

Cos8.2

5kb

Fig. 1. Restriction enzyme map of the cloned NRAMP1 genomic region for enzymes BamHI (B), SalI (S), NruI (N), MluI (M), BssHII (Bs), NotI (N), and SacII (Sa). The two cosmid NRAMP1 clones cos8.1 and cos8.2 are positioned below the restriction map. Probes MCG1 and MCG2 derived from cosmid clone cos8.1 are represented by hatched boxes. The chicken NRAMP1 gene, which is contained in four adjacent BamHt fragments, CD#2, and VIL are identifed by a thick line below the restriction map.

J. Hu et al.: Mapping of NRAMP1 in chicken 811

Linkage analysis. High-molecular-weight genomic DNA samples obtained from N and 15I chickens were digested to completion with numerous restriction enzymes, electrophoresed, blotted, and hybridized to the probe MCG2, which consists of a 2.7-kb BamHI fragment containing the 5' coding portion of NRAMP1 (Fig. 1). No RFLP were detected. The same blot was then hybridized to the genomic probe MCG1, which consists of a 5-kb repeat-free NotI fragment located 4 kb from the 3' end of NRAMP1 (Fig. 1). An RFLP between parental lines 15I and N was observed only with the restriction enzyme PstI (data not shown). Probe MCG1 hy- bridized to seven genomic fragments (2.0 kb, 1.2 kb, 1.1 kb, 0.9 kb, 0.8 kb, 0.5 kb, 0.4 kb) in PstI-digested DNA. The 1.2 kb, 0.8 kb, 0.5 kb, and 0.4 kb fragments were common to 15I and N chicken DNAs, while the 2.0 kb was specific to 15I chicken DNA, and the 1.1-kb and 0.9-kb fragments were specific to N chicken DNA. The segregation of the PstI RFLP identified with probe MCGI was then followed in a panel of 56 backcross progeny from (NX15I)F1 x 15I and compared with the segregation of 270 DNA markers previously assigned to this particular backcross panel (Bumstead and Palyga 1992; Bumstead et al. 1995). Genetic link- age of NRAMP1 with other marker loci was evaluated with the Genelink program (Montagutelli 1990). The results suggested that a likely location for NRAMP1 would be within a linkage group that includes a random genomic clone COM76 (Bumstead and Palyga 1992), a random cDNA clone COM123 (Bumstead et al. 1995), the chicken homolog of the human mitochondrial NADH-coenzyme Q reductase gene NDUFS1 (Bumstead et al. 1995), the chicken ho- molog of the human elongation factor 1 EF1B (Bumstead et al. 1995), and the T cell surface antigen CD28 (Bumstead et al., 1993). At each locus, haplotypes from individual backcross chick- ens exhibited either a homozygous 15I pattern or a heterozygous (NX15I)F 1 pattern. The resulting haplotypes obtained from the 56 backcross progeny are shown in Fig. 2. Assignment of recombi- nant and nonrecombinant classes produced 10 different haplo- types. No recombinant events were detected in 36 backcross chicken, whereas 18 backcross chicken progeny showed single crossover, and two chicken progeny showed haplotypes compati-

ble with double crossover events (Fig. 2). The gene order was established by minimizing the number of crossover events between NRAMP1 and the five loci located within the linkage group (LOD /> 3.0). A highly significant lod score of 4.8 in favor of linkage was calculated between NRAMP1 and CD28. The gene order and distance in centimorgans (+SD) are COM76-8.9 + 3.8-COM123- 3.6 + 2.5-NDUSF1/EFIB-7.3 + 3.5-CD28-20.0 +_ 5.4-NRAMP1. The orientation of the linkage group with respect to the centromere is unknown.

Chromosome localization of the chicken NRAMP1 gene by fluo- rescence in situ hybridization. We next used fluorescence in situ hybridization to determine the chromosomal location of NRAMP1 in the chicken genome. The chicken cosmid clone cos8.1, illus- trated in Fig. 1, was used to probe spreads of chicken metaphase chromosomes. The hybridization signal was detected as symmet- rical spots on both chromatids of the two homologs of the respec- tive chromosome pair. No cross-reactivity of the cosmid probe to other chromosomes was detected. Cytogenetic examination of the hybridized chromosome spreads indicated that the NRAMP1 cosmid clone mapped to macrochromosome 7q13 (Fig. 3).

Physical mapping by pulsed-field gel eIectrophoresis of the NRAMPI region in chicken. We generated a physical map in the vicinity of NRAMPt in the chicken genome with PFGE and probes Cd#1, Cd#2, and Vil known to be closely linked to Nrampl in the mouse genome. Probes Cd#1 and Cd#2 were candidate genes iden- tified in our search for the Bcg gene (Vidal et al. 1993) and were ubiquitously expressed in all tissues examined. These two probes show very strong cross-species conservation and hybridized very nicely to chicken genomic DNA. We also used additional probes (MCG1 and MCG2) derived from the cosmid clone cos8.1 (Fig. 1). To prepare the probes, we first identified in the genomic clones recognition sites for restriction enzymes such as NruI, NotI, MluI, BssHII, and SacII (Fig. 1). Single-copy DNA probes (MCG1 and MCG2) flanking both sides of these rare-cutting restriction sites

A

co=78 � 9 1 4 9 1 4 9 1 4 9 1 4 9 � 9

COM123 �9 [ ] [ ] �9 �9 [ ] �9 [ ] �9 [ ] [ ] �9

NDUSFI/EF1B �9 [ ] [ ] �9 [ ] �9 �9 [ ] �9 [ ] �9 [ ]

c.. m[] m[]mnmmrn m[]

..,.., �9 [] [] �9 [] �9 [] �9 [] �9 �9 []

17 19 2 1 0 0 1 3 3 8 1 1

Chicken Human Mouse

B COM76

8,9

COM123 3.6

NDUSFI/EF1B 2q33-q34

7.3 CD28 2q33-34 1 (30,1)

20.0

NRAMP1 2q35 1 (39.2)

Fig. 2. Segregation analyses of NRAMP1 and flanking markers in 56 (15IXN)F 1 x 151 backcross chicken progeny. (A) Each column represents a chromosomal haplotype identified in the backcross progeny. Each locus is listed on the left. 151 alleles (black boxes) and N alleles (open boxes) are shown. The number of backcross chicken inheriting each type of chromosome is listed at the bottom of each column. Two double crossover haplotypes are listed on the right. (B) Partial genetic map of the conserved linkage group carrying NRAMP1 in the chicken. Recombination distances between loci (in centimorgans) are shown to the left of the chromosome. Position of loci in human (cytogenetic banding; Lafage-Pochitaloff et al. 1990; Duncan et al. 1992; White et al. 1994) and mouse (approximate map positions in cM from the centromere) chromosomes (Howard et al. 1991; Vidal et al. 1993) are shown to the right.

812 J. Hu et al.: Mapping of NRAMP1 in chicken

Fig. 3. Fluorescence in situ hybridization mapping of the NRAMP1 gene on chicken metaphase chromosomes. Specific hybridization signal was seen at Chr 7q13.

were prepared from the cloned DNA. These probes were hybrid- ized to Southern blots prepared from chicken genomic DNA di- gested with the same panel of rare-cutting restriction enzymes, alone or in combination, with the aim of identifying common hybridization fragments (Fig. 4). The fragment sizes from PFGE analyses for the five different enzymes are listed in Table 1, and the summary of the physical map of the chicken NRAMP1 region is presented in Fig. 6A.

Physical linkage of CD#1, CD#2, NRAMP1, and VIL was es- tablished by hybridization of the four probes to common NotI fragment of approximately 100 kb in size. In addition, probes CD#1 and NRAMP1 hybridized to the same MluI fragment, and probes CD#2 and VIL to the same MIuI, SaclI, and BssHII frag- ments (Fig. 4, Table 1). To determine the gene order of the four markers, we used two chicken genomic probes, MCG1 and MCG2, located on either side of a cluster of rare-cutting enzymes (BssHII, SacII, MluI, and NotI). MCGI and MCG2 hybridized to the same 100-kb NotI fragment and to different MluI, SacII, and BssHII fragments in chicken genomic DNA (Table 1). The restriction fragments detected by MCG1 and MCG2 are identical to those recognized by probes Cd#2 and Nrampl, respectively (Table 1). These results indicated that the clustered restriction sites BssHII, SacII, and MluI located between probes MCG1 and MCG2 are not methylated in chicken genomic DNA, while the NotI site is meth- ylated. To further delineate the minimal CD#1-NRAMP1 and CD#2-VIL interval, probes Cd#1, Cd#2, and VIL were hybridized to Southern blots containing restricted NRAMP1 cosmid clones (data not shown). Probe CD#2 and VIL recognized the 5-kb NotI fragment corresponding to MCG 1. Additional restriction mapping showed that probes CD#2 and VIL hybridized to two distinct BamHI, NotlJBamHI fragments, respectively (Fig. 1). Probe CD#1 did not hybridize to NRAMP1 cosmid DNA. The maximum dis- tance separating CD#1 and NRAMP1 is 30 kb and corresponds to the MluI fragment recognized by CD#I and NRAMP1 (40 kb) minus the SacII fragment (10 kb) identified by NRAMP1. The distance separating ArRAMP1 from CD#2 is 2 kb and corresponds to the interval separating the 3' end of NRAMP1 and the cluster of MIuI, SaclI, BssHII sites. The maximum distance separating probes CD#2 and V1L is 5 kb and corresponds to the NotI/BarnHI fragment recognized by these two probes.

Comparison of physical maps of the Nrampl region of mouse Chr t and chicken Chr 7. Comparison of long-range physical maps of

Fig, 4. Physical linkage by PFGE analyses of the CD#1 (A), NRAMP1 (B), CD#2 (C) and VIL (D) loci in the chicken. Genomic DNA was digested with restriction enzymes NotI, MluI, SacII, MluIINotI, NotIINruI, and MluI/ NruI, and the fragments were separated by PFGE. PFGE was performed for 24 h at 200 V with switching times of 50 s. The same blot was hybridized with probes CD#1, NRAMP1, CD#2 and VIL. Fragment sizes in kb were deduced by using Saccharomyces cerevisiae chromosomes and lambda concatemers as size markers and are indicated to the left of each autora- diogram.

the chromosomal segment carrying NRAMP1/Nrampl in chicken and mouse genomes revealed a marked conservation of genomic organization (Fig. 5). The order of the four genes mapped by PFGE in both species was the same: CD#1/Cd#1-NRAMP1/ Nrampl-CD#2/Cd#2-VIL/Vil. Lengths of genomic segments sep- arating each of the genes were calculated from physical mapping data for both species. Comparison of intergene distances revealed striking differences in the mouse and chicken. The overall size estimates of the Cd#l-Vil or CD#1-VIL interval obtained by phys- ical mapping in the mouse and chicken genomes are 175 kb and 50 kb respectively. While the maximum distance between Cd#1 and Nrampl is 110 kb in the mouse genome, the same interval is 30 kb in the chicken genome. The segment delineated by Nrampl and Cd#2 is 5 kb in the mouse genome compared with 3 kb in the chicken genome. The Cd#2-VIL interval is 40 kb in the mouse and only 4 kb in the chicken genome. While striking differences in restriction fragment length were observed, the resultant genomic restriction maps were similar (Fig. 5A and 5B). Probes Cd#l and Cd#2 and their chicken homologs were physically linked to NRAMPI and V1L, respectively, on the basis of commori MluI fragments in both species (MaIo et al. 1993b; Fig. 5). In additiou, conservation of the location of unmethylated CpG residues be- tween the mouse and chicken for this chromosomal segment is suggested by marked similarities in the distribution of cleavage sites for the restriction enzymes NotI, MluI, SaclI, and BssHII.

J. Hu et al.: Mapping of NRAMP1 in chicken

Table 1. List of fragment sizes from PFGE analyses.

813

CD #1 NRAMPI MCG2 CD #2 MCG1 VIL

NotI Ilo0 (175)" 100 (175) 100 (175) 100 (175) 100 (175) 100 (175) [

M,.I [4o 4o 40 ] [45 45 45 1

~a. , 23 [10 10 I 1 4o 40 4o I

~s..I1 23 I lo 10 I 1 40 4o 40 I

Nr., ~00 8oo 80o 8oo 8oo 8o~ I

N o t ~ ' a c . 23 I10 10 I

N o ~ " s . . I I 23 I10 10 I

M,.~Bs~.II 23 I10 10 I

M,.~Not~ [40 40 40 1

NotI/NruI I100 (75) 100 (75) 100 (75)

40 40 40 I

140 40 40 I 14o 4o 40 I

45 45 45 [

100 (175) 100 (175) 100 (175) [

MiuI/NotI [ 40 40 40 ] 45 45 45 I Restriction fragment sizes are given in kb. aThe 175-kb fragments detected by all probes represent partial NotI digests.

A 1 2 3 4

,L l , [ , ,l,

l i b 't' '~

oo#1 . . A , p ~ CD#2 VIL

B 1 2 3 4

,L ,L ~ 4,

~ | I

Cd#1 Nrampl Cd#2 Vii

lOkb

Fig. 5. Comparison of long-range physical maps of the chicken NRAMP1 chromosomal region (A) and the corresponding segment in the mouse genome (B, Malo et al. 1993). The restriction sites for the four enzymes are represented by different symbols: NotI (0), MluI (0), SacII (*), and BssHII (V). Positions of the chicken or mouse genes encoding the natural resistance-associated protein 1 (NRAMP1 and Nrampl respectively), the villin (VIL or Vil respec- tively), and two anonymous cDNAs (CD#1 or Cd#1 and CD#2 or Cd#2 respectively), are shown with hatched blocks below the map. Four clusters num- bered 1 to 4 of two of more CpG-containing rare- cutter restriction sites are identified by arrows on the top of the two maps.

Discussion

Linkage and physical maps are well developed in the mouse and human, and linkage maps for other species including agriculturally important ones such as the cow (Barendse et al. 1994), pig (Rohrer et al. 1994), and chicken (Bumstead and Palyga 1992; Levin et al. 1994) are emerging. These maps are developed as a resource for genetic analysis (maintenance and improvement of breeding stocks) and for the study of evolution of genome organization by comparing linkage relationships of homologous genes or gene clusters (O'Brien et al. 1993). Innate susceptibility to many im- portant diseases, including various types of infection, is controlled by genetic factors (reviewed in Malo and Skamene 1994). The identification and characterization of such genes is easiest in mice because well-defined inbred, recombinant, and mutant mouse strains are available for study. Once a candidate disease gene or

disease region is identified in the mouse, the homologous gene or region in other species can be tested for linkage to the correspond- ing genetic disease (Copeland et al. 1993).

Immunity to Salmonella infection has been studied extensively by use of mice as an experimental model. In the mouse, resistance to infection with Salmonella typhimurium is controlled by the ex- pression of several distinct loci operating via different mecha- nisms: Nrampl Beg (Vidal et al. 1993), Lps (O'Brien et al. 1980, 1984), NF-IL6 (Tanaka et al., 1995), BtU ia (Thomas et al. 1993; Rawlings et al., 1993), and other undefined host genes (O'Brien et al. 1984). Three of these mutant loci, Nrampl "r Lps, and NF-IL6, are involved in the early nonimmune phase of the host response, more precisely, in controlling macrophage function, whereas Btld id modulates humoral immune responses. The natural resistance of chickens to infection with Salmonella typhimurium is also under genetic control (Bumstead and Barrow 1988). To test whether NRAMP1 is involved in the differential resistance of chickens to

814 J. Hu et al.: Mapping of NRAMP1 in chicken

infection with Salmonella typhimurium, we have cloned and mapped the chicken NRAMP1 gene.

The current chicken genetic linkage map has been constructed from two different reference mapping populations (Compton, UK and East Lansing, USA) with at least 461 anonymous loci or cloned genes (reviewed in Burt et al. 1995). We have used one of these reference mapping panels (Bumstead and Palyga 1992) and fluorescence in situ hybridization to identify the chromosomal lo- cation of the chicken NRAMPI homolog. The reference population designed for molecular genetic mapping of the chicken genome was produced by hackcrossing two White Leghorn lines (N and 15I). The chicken genomic map created with this panel consists of 270 loci defining 39 autosomal linkage groups (Bumstead and Palyga 1992; N. Bumstead, unpublished data). NRAMP1 is located within a linkage group that includes two anonymous DNA markers (COM76 and COM123) and three known genes: the mitochondrial NADH-coenzyme Q reductase gene, NDUFS1; the T-cell surface antigen, CD28; and the elongation factor 1, EF1B. To assign this linkage group to a specific chromosome, we have used FISH to probe metaphase chromosome spreads, using a chicken cosmid clone containing the entire NRAMP1 gene; an anonymous cDNA (CD#2), and the 5' end of the villin gene. The NRAMP1 cosmid probe shows hybridization to chicken Chr 7 at band q13. No ad- ditional hybridization sites were detected on the chicken chromo- some, demonstrating that the three genes present in the cosmid clone used as a probe are located on contiguous segments of ge- nomic DNA.

The human CD28 and NDUFS 1 genes have been located on Chr 2 at band q33-q34 (Lafage-Pochitaloff et al. 1990; Duncan et al. 1992) in the vicinity of NRAMP1, which is located on band q35 (White et al. 1994). The murine homolog of Cd28 is located on the conserved syntenic region on mouse Chr 1 carrying Nrampl (Howard et al. 1991). A 35-cM chromosomal region surrounding Nrampl/Bcg on mouse Chr 1, including Cd28 and Ndufsl, has been conserved on the telomeric end of human Chr 2q (Schurr et al. 1990). Current mapping data suggest that the order of at least 13 expressed loci has been conserved between humans and mice in that region (Spurr et al. 1993). The results of the analysis reported here show that the segment of conserved homology between prox- imal mouse Chr 1 and the telomeric end of human Chr 2 is con- served on chicken Chr 7q13.

Comparison of long-range physical maps of the mouse (Malo et al. 1993b), human (White et al. 1994; D. Malo and P. Gros, unpublished) and chicken chromosomal segments carrying NRAMP1 revealed that the genomic organization within this link- age group is conserved through evolution. The gene order for four cDNA markers tested including Nrampl and Vil and the location of at least two CpG islands are precisely conserved between the three species. However, intergene distances revealed striking dif- ferences between the chicken and mouse or human, which were generally smaller in the chicken by a factor of about two. Distances separating all gene combinations examined here were smaller in the chicken than in the mouse and human genomes: the CD#I-VIL interval is 175 kh in the mouse, approximately 250 kb in humans, and only 50 kb in the chicken genome. The results are in good agreement with the overall size of the avian haploid genome, which at 1200 Mb (Oloffson and Bernadi 1983) is smaller by a factor of 2.75 than the mammalian haploid genome size of 3300 Mb.

Linkage conservation has been observed for quite extensive parts of mammalian chromosomes (Nadean and Taylor 1984; Copeland et al. 1993). There are at least 101 segments of con- served homology between the human and mouse (Copeland et al. 1993). The reasons that linkage groups are kept intact over mil- lions of years are not well understood. It is possible that regional chromosomal organization is critical for the activity of the genes in the region (chromosome rearrangements often lead to meiotic dis- turbances and are selected against, Lundin 1993) or that time has

not allowed the disruption of a sufficient number of linkages to cause a random pattern. It has been proposed that chromosomal rearrangement events within conserved regions may provide a mechanism for creating species diversity (Lundin 1993). Linkage conservation has also been reported between chicken and mam- malian chromosomes (reviewed in Burt et al. 1995). With the future development of detailed linkage maps of the chicken ge- nome, the extent of chromosome conservation will be better esti- mated. The significance of conservation of genomic organization within the syntenic groups, as described in this paper, has practical consequences for strategies to map host resistance loci in other species.

Acknowledgments. The authors thank Drs. Joseph H. Nadeau and Dianne Arbuckle for critical comments on the article. This study was supported by research grants to D. Malo, and P. Gros from the Canadian Bacterial Diseases Network (Networks of Centres of Excellence) and National In- stitutes of Health, respectively. J. Hu is supported by a McGill major fellowship. D. Malo is supported by a career award from the Fonds de la Recherche en Sant6 du Qutbec. P. Gros is supported by a scientist award from the Medical Research Council of Canada.

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