*Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91001-0269; tGendtique.
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4397-4401, May 1994 Immunology
Two Mhc class I and two Mhc class II genes map to the chicken Rfp-Y system outside the B complex MARCIA M. MILLER*t, RONALD GoTo*, ALAIN BERNOTt, RIMA ZOOROB*, CHARLES NAT BUMSTEAD§, AND W. ELWOOD BRILES¶
AUFFRAYt,
*Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91001-0269; tGendtique Mol6culaire et Biologie Ddveloppement, Centre National de la Recherche Scientifique, UPR 420, 94801 Villejuif, France; Institute for Animal Health, Compton Near Newbury, Berks, RG16 ONN, United Kingdom; and IDepartment of Biological Sciences, Northern Illinois University, DeKalb, IL 60115
Communicated by Susumu Ohno, January 7, 1994
ABSTRACT
The Rfp-Y system was encountered coincidentally in a study of B system haplotypes segregating within fully pedigreed families of chickens (ref. 1 and R.G., W.E.B., and M.M.M., unpublished data). The initial observation, similar to that seen earlier by Chaussd et al. (8), was that more than one pattern of restriction fragments could be found when DNA samples from birds of identical serologically defined B genotypes were probed for Mhc class I and Mhc class II gene sequences. Because inheritance of the restriction fragment patterns could be followed within full-sib families, the second genetic system became apparent. The Rfp-Y system was not detected earlier perhaps because Rfp-Y antigens are absent from the surfaces of erythrocytes, the cell type upon which chicken Mhc typing is routinely based. The independent assortment of two sets of chicken Mhc genes was not apparent in earlier DNA studies confined mostly to inbred and Mhc congenic lines since the nature of these lines makes it difficult to recognize that restriction fragments might be originating from different chromosomal regions. In inbred lines selected for B system homozygosity, alleles at other loci would likely become fixed. Even if alleles at a second locus were still segregating, their genetic relationship with the B system would be impossible to determine because of the presence of a single B haplotype already fixed in the line. In lines congenic for the B system, such as the CB and CC lines (9, 10), most alleles at loci distant from the B system are likely to be shared in common and, therefore, would not exhibit restriction fragment polymorphisms as separate from the B system. With this perspective, the lack of polymorphism noted in genomic DNA fragments associated with cosmid cluster II/IV of the chicken Mhc molecular map when tested on the CC and CB congenic lines (4), including the recently described 17.5 lectin gene locus (ref. 11; see also Fig. 1), suggested that this segment of genomic DNA, in which two Mhc class I and two Mhc class II genes have been mapped, might encompass the Rfp- Y system. This hypothesis was further supported by the recent demonstration that the Rff- Y class II gene sequences most closely resemble the B-LBIII gene family members, two of which reside in cosmid cluster II/IV (12, 13, 20). To further test the hypothesis, a 1.6-kb fragment of genomic DNA, called 18.1, adjacent to the polymorphic 17.5 gene in cosmid cluster TT/IV was used in Southern blot hybridizations to examine the pedigreed families in which the Rfp- Y haplotype segregation had been determined previously. Additional tests were made in an attempt to assign Rfp- Yto a linkage group in the current genetic linkage map of the chicken (14).
Gene sequences highly similar to major his-
tocompatibility complex (Mhc) class I and class H genes were recently recognized as mapping to a site in the genome of the chicken separate from the Mhc class I, class II, and B-G genes of the major histocompatibility (B) complex. The present study was undertaken to see whether this complex of Mhc-like genes designated as restriction fragment pattern Y (Rfp-Y) mgt reside in one of three dusters of cosmid clones contained within the molecular map of chicken Mhc genes, since only two of the three clusters can be asigned to the B system. To determine whether the third cluster (cluster il/IV) might contain Rfp-Y, a subclone (18.1) from within duster lI/IV near a polymorphic lectin gene was used to analyze the DNA of families in which Rfp-Y haplotypes are known to be segregating. The restriction fmrgment polymorphisms revealed by the 18.1 probe were found to segregate in parallel with the restriction fragment polymorphisms defining the Rfp-Y haplotypes, thus establishing the location of Rfp-Y within cosmid cluster lI/IV. Two of six Mhc class I genes and two of five Mhc class II genes map to cosmid cluster lI/IV, so a substantial fraction of chicken Mhc genes, including at least one that may be expressed, are located in a chromosomal region separate from theB system. In further linkage analyses, Rfp-Y was found to assort independently from more than 400 markers in the present linkage map of the chicken genome. The recent finding of Briles et al. (1) that certain polymorphic gene sequences detected by cDNA probes for Mhc class I and class II genes assort independently from the major histocompatibility (B) complex located on chicken microchromosome 17 (2, 3) introduces an arrangement of Mhc class I and class II genetic elements hitherto unseen in the study of major histocompatibility genes. Determining whether these gene sequences, designated the Rfp-Y (restriction fragment pattern Y) system, represent functioning Mhc genes becomes particularly important since the presence of a cluster of polymorphic Mhc genes at a second chromosomal site presents a more complex genetic basis for the organization of the Mhc, particularly in terms of the selection of Mhc gene haplotypes within a population and in terms of the coordinate functioning of antigen-presenting genes within the individual. This finding suggests that the evolution of histocompatibility gene complexes in birds may be different from that in mammals. Striking differences have been already noted in the arrangement of Mhc genes in chickens, where a number of genes with no apparent direct function in antigen processing and presentation have been found among the Mhc genes within the B complex (4) and where a large polymorphic family of B-G genes are closely linked to relatively few Mhc class I and class II genes (5-7).
MATERIALS AND METHODS Animals. The animals used in testing for linkage of the Rfp- Y system with cosmid cluster TT/IV are members of fully
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tTo whom reprint requests should be addressed. 4397
4398
Immunology: Miller et al.
Proc. Natl. Acad. Sci. USA 91 (1994)
pedigreed families (C183, D183, A186, and B186) in which three Rfp-Y haplotypes and two B haplotypes have been shown to segregate (1). Large backcross families being used to produce a linkage map for the chicken genome (14) were also tested for the segregation of Rfp-Y haplotypes. The first of these families is the product of a backcross between an F1 (line N and 15I hybrid) hen and a single line 15I cockerel and was comprised of 98 progeny. A further mating between the same cockerel and a second F1 hen (sister to the first) resulted in an additional 15 progeny that were included in this study. Southern Blot Analysis. Genomic DNA (10 pg) from members of the C183, D183, A186, and B186 families was digested in overnight incubations with Bgl I, Pst I, and Pvu II, as was DNA from the parents and the first 55 members of the linkage mapping families. In a separate experiment, the DNA from an additional 43 progeny in the first linkage mapping family and 15 progeny from the second was digested with Bgl I. Electrophoresis and transfer to GeneScreen were as described (1). Hybridizations in 5x SSPE (lx SSPE = 0.18 M NaCl/10 mM sodium phosphate, pH 7.4/1 mM EDTA)/5x Denhardt's solution/1% SDS/salmon sperm DNA (100 ,ug/ml) were carried out at 65°C overnight in a rotating hybridization tube. The filters were washed at 65°C in 0.5x SSC (SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0)/1% SDS for 1 hr. After production of a first set of autoradiograms, the filters were further washed at 650C in 0.1 x SSC/1% SDS and a second set of autoradiograms was produced. Probes. The 18.1 probe is 1.6-kb DNA fragment (BamHIStu I) of genomic DNA subcloned from the 17.5 BamHI fragment of C,17 (4) into pUC18 (Fig. 1). The Mhc gene probes B-F1O (4), B-LBII (15), and bgll (6) were labeled with [a-32P]CTP by random priming. Linkage Mapping. Initial linkage tests made by two-point linkage estimates (16) with data from the Compton, U.K. reference population (14) were followed by similar tests with the East Lansing, U.S.A. reference population (21).
RESULTS Segregation of Retriction Fragment Patterns in Pedgreed Families. DNA samples from members offour fully pedigreed families (A186, B186, C183, and D183) of known B haplotypes and in which Rfp-Y haplotypes 1, 2, and 3 are segregating were analyzed in Southern blot hybridizations with the 18.1 probe. Illustrated in Fig. 2 are the restriction fragment patterns found in DNA of members of the A186 family and a portion of the D183 family prepared with the same three restriction enzymes (Pst I, Bgl I, and Pvu II) used in the earlier analysis of the Rfp-Y alleles (1). The patterns of restriction fragments observed are consistent with the segregation of three alleles at a single locus and are summarized in Table l in the context of the haplotypes at the B and Rfp-Y systems previously assigned to members of these families. In the family A186, three patterns of restriction fragments are found. Two of these, assigned the identifiers A and B, are clearly independent of each other, and the third appears to be the sum the patterns of A and B. Since both dam and sire share the same pattern, the sum of A and B, it appears that Origin of 18.1 probe
B-FV 17.5 17.8 B-FVI B-LBIII -) !-
B-LBV
-
.7
m7
m7
m7 1
r:~:
c1317 clone FIG. 1. Portion of the genes located within the cosmid cluster II/IV indicating the position in the cB17 clone from which the 18.1 probe originates.
D 183 Family
A 86 Fami K . KtD
-. r ;
-
Z,
-
4
:*
9 ,t 6 .f. -9
4
*e:7 q 9.
.4 13r
I I II *tttt11wtIII
,
9 .
: 3
0
Oa a a0
~
~
-*~ 00 0
a
'a
:00 :
-*--
A
0
_
bo
.
3.3 A 2 41 B3 L&
. . E *4 A . e * . ..*i
_9., r Z A-
A
*0** e
~*
-
FIG. 2. Southern blot hybridizations between the DNA of members of the A186 and D183 families digested with Pst I, Bgl I, and Pvu II and the 18.1 probe identifying a polymorphic region within the cosmid cluster II/IV. The size of polymorphic restriction fragments is indicated (in kb) with the 18.1 alleles (A-C) to which they can be assigned.
each is heterozygous for the 18.1 sequence and transmitted to their progeny alleles resulting in a ratio of 3:9:4 for AA:AB.BB genotypes, a ratio consistent with Mendelian inheritance of two alleles at a single locus. In the D183 family, there appears a third 18.1 allele, assigned identifier C, present in both parents along with allele A in the sire and B in the dam. While the C allele shares two restriction fragments (2.7-kb Pst I, and 2.4-kb Bgl I fragments) with the B allele of 18.1, the C allele can be identified as separate from B by the presence of an intense band at 0.68 kb in Pvu II digests and two additional bands at 1.7 and 1.3 kb in Bgl I digests. When the 18.1 allele assignments are compared to the Rfp- Yhaplotypes, complete accordance is found between 18.1 and the Rfp- Y haplotype,
Proc. Natl. Acad. Sci. USA 91 (1994)
Immunology: Miller et al.
4399
Table 1. Segregation of 18.1 restriction fragments presented in the context of B and Rfp-Y haplotypes of members of the A186 and D183 families 18.1 restriction fiagments Rfp- Y alleles
B
alleles Dam 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Sire Dam 1 2 3 4 5 Sire
R9/11 R9/11 R9/11 R9/11 R9/11 R9/R9 11/11 R9/R9 R9/11 11/11 R9/11 R9/11
1/2
1/1 1/2 1/1 1/2 1/2
1/2 1/1 1/2 2/2 2/2 1/2
11/11 11/11 R9/11
1/2 2/2 2/2 1/2 1/2 1/2
R9/11 R9/11 R9/11
Bgl I
8.1
Pst I 3.3 3.2
2.7
2.8
2.5
+ + +
+ + +
-
+ +
+ + +
-
+
+
-
-
+
-
-
-
-
+
+
-
+ + + + +
+ + + + +
+ + + + + -
+ + + + +
-
-
+ +
+ + + -
+ + + + + -
-
-
+ + + + +
-
-
-
+
-
-
-
-
+ +
+ + +
-
-
-
+
-
-
+ +
-
-
-
+ + +
+ + + +
-
+ + +
+ + + + + + +
+
-
-
-
+ +
2.4 1.7 Family A186 + + -
+ + + Family D183 + + + + + +
-
0.65
+ +
-
+ +
-
-
-
-
+ +
-
-
-
+ + + + +
-
-
-
+ +
-
-
+ + +
-
-
6.5
0.92
0.87
-
+ + +
+ + +
-
-
-
Pvu I 0.75 0.70
0.68
1.3
-
+ + +
-
+
-
+
-
-
+ + + + + + +
-
+ + + + + + +
18.1 alleles
A/B A/A A/B A/A A/B A/B A/B A/A A/B B/B B/B A/B A/B B/B B/B
A/B A/B A/B
11/11
2/3
-
-
-
+
-
-
+
-
-
-
-
+
+
+
B/C
R9/11 R9/11
3/3
-
-
-
+
-
-
+
+
+
-
-
-
-
-
+
-
C/C
2/3 2/3 2/3 1/2 1/3
+ +
+ +
-
+ + + + +
+ +
-
+ + + + +
+ + + +
+ + + +
+ +
+ +
-
-
+ + + + -
+ + +
-
+ + + +
+
-
B/C B/C B/C A/B A/C
R9/11 R9/R9
11/11 R9/11
-
Restriction fragments are shown in kb.
in common with B haplotypes previously determined by serology (Table 3) and an additional restriction fragment within the B-L hybridizations assorting independently from the B system haplotypes and, hence, potentially originating from the Rfp-Y system. The results of the analysis of dam, sire, and 55 progeny are summarized in Table 3. Assignments of restriction fragments to B and RJf- Y systems are made on the basis of size and pattern of segregation. The 2.3-kb B-F band, the 4.6-kb B-L band, and the 5.4-, 3.7-, and 2.7-kb B-G bands all segregated with the B21 allele as identified by serology and, hence, are assignable to B21. Since all animals within this backcross family carry B15 haplotype, it is not possible to assign with certainty bands to the B'5; however, it is likely that the 4.6-kb band, a band that is frequently seen in B-L hybridizations and is assignable to the B system alleles, originates from the B'5 haplotype since it is present in all samples. The bands considered to be originating from the Rfp-Y system were similarly assigned. Here again because the family analyzed is a backcross originating from a sire with a pattern of bands indicating that he is likely homozygous for the Rfp- Y3 haplotype (the bands of 4.0 kb in the B-F hybridization and of 9.5 and 5.5 kb in the B-L hybridizations), all
an occurrence highly unlikely on the assumption of independent inheritance of alleles at the two loci. Hence strong evidence has been found for the presence of the Rfp-Y system in close proximity to the 18.1 locus within the DNA cloned within the cosmid cluster II/IV. The patterns of restriction fragments associated with 18.1 alleles A, B, and C are summarized in Table 2 with that of a fourth allele described below. The recent demonstration of the genetic independence of the Rfp-Y system from the B system leads to the conclusion that the genome segment cloned within cluster II/IV must now be removed from the B system molecular map described by Guillemot et al. (4). A revised map is presented in Fig. 3. Linkage Mapping. To assign Rfp-Y to a group within the linkage map of the chicken genome, Southern blot hybridizations were carried out with DNA samples from the families being utilized to establish the map (14). The DNA samples were examined first to determine the pattern of segregation of restriction fragments identified by the B-F, B-L, and B-G probes and the restriction enzyme digestions that allowed the Rfp-Y alleles to be identified in earlier studies, i.e., Pst I, Bgl I, and Pvu II, respectively, for the three probes. In the patterns, it was possible to identify fragments that segregate
Table 2. Restriction fragment patterns identifying the four 18.1 alleles found in this study Pst I Bgl I 6.5 2.4 1.7 1.3 0.92 2.7 2.5 3.2 2.8 18.1 type 8.1 3.3 A B C
+ -
+ -
-
-
+
-
-
-
-
+ +
-
-
-
+ +
+
-
-
+
+
0.87 _
-
-
-
-
Pvu II 0.75 _
0.70 _
0.68 _
0.65 _
-
+
-
+
+ _ _ _ _ _ + + NK + + NK NK NK NK D NK, not known. The D allele has been observed only in heterozygotes in the presence of the C allele. Restriction fragment lengths are presented in kb. -
-
-
-
+
-
/r
Immunology: Miller et al.
4400
>lI
Proc. Natl. Acad. Sci. USA 91 (1994)
by the B-L probe (i.e., the 5.3-kb Bgi I fragment) and jrestriction fragments revealed by the 18.1 probe (3.2-kb Pst I, 2.5-kb Bgl I, 0.87 and 0.75 kb Pvu II fragments) are different ! 'I.-.'/ -'1 1 '/ 1 ! from the bands identified earlier, a fourth allele has been
