Polymorphisms in the human immunoglobulin x locus
Eur. J. Immunol. 1991. 21: 1829-1835
Walter Pargent, Karlheinz F. Schable and Hans G. Zachau Institut fur Physiologische Chemie der UniversitGt Miinchen, Munchen
Polymorphisms and haplotypes in the human immunoglobulin x locus* By comparing the restriction patterns of the DNA from 23 unrelated individuals 16 polymorphisms were defined which allowed us to differentiate between the duplicated copies Op, Ap, Lp and Od, Ad, Ld of the 1c locus (p for the C, proximal, d for the distal copy). Some of these duplication-differentiating polymorphisms or DDP revealed also allelic differences between individuals; they are therefore restriction fragment length polymorphism (RFLP) markers at the same time. Three RFLP in the single copy B-J,-C, region were included into the study.Three basic haplotypes were derived from the combined genotype data, haplotypes N, G and 11.The latter haplotype in which the whole distal copy of the 3t locus is missing was found three times among the 46 haploid genomes studied. The genotypes of the family members of an individual who is homozygous for haplotype 11are consistent with Mendelian inheritance. Haplotypes N and G are distinguishedfrom each other by eight RFLP markers. Six additional haplotypes, which were found in one or severalindividuals each, can be derived from the basic haplotypes N and G by hypothetical recombination and/or mutation events.
1 Introduction The human Ig 1c locus comprises two very similar but not identical copies: the C, proximal (p) copy which contains, in addition to the C, gene segment and 5 J,, 40V, genes and pseudogenes within a 500 kb contig, and the distal (d) copy that comprises 35 V, genes and pseudogenes within 400 kb (reviews [ l , 21). In elucidating the structure of the locus we were frequently confronted with the task to decide whether a newly isolated cosmid or phage h clone belongs to the p or d copy. If the new clone differed from an existing one at one or a few restriction sites, this could mean that it belonged to the other copy of the locus or the differences could be allelic. A decision was usually reached by studying the regions around the restriction site in question in blot hybridization experiments with the DNA of a number of unrelated individuals. If all or most individuals yielded both types of restriction fragments, the ones related to the new and the old clones,this was considered proof that the clones are derived from homologous positions of the two copies of the locus. This way a number of duplicationdifferentiating polymorphisms (DDP) were established along the locus. DDP probes ideally recognize in DNA digests from unrelated individuals both copies as restriction fragments of different length ( e . g [3, 41). As could be expected the hybridization patterns were complicated by allelic differences: the cosmid and phage h clones covering the 3t locus were derived from the DNA of a number of different
[I 93931
*
1829
This work was supported by Bundesministerium fiir Forschung und Technologie, Center Grant 0316200A, and Fonds der Chemischen Industne .
Correspondence: Hans G. Zachau, Institut fiir Physiologische Chemie, Schillerstr. 44,D-8000 Munchen 2, FRG Abbreviations: 0, A, L, B regions: V, gene-containing contigs of the human x locus DDP Duplication-differentiatingpolymorphism 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
individuals and the DNA of the individuals used for assaying turned out to belong to different haplotypes. In this paper we describe several differences between the duplicated copies and the character and extent of polymorphism of the human x locus.
2 Materials and methods In addition to the materials and methods (Sect. 2) described in the preceding report [5] the following methods were employed. DNA from white blood cells and cell lines were prepared according to [6].The 23 unrelated Caucasian individuals include ind.1-19, placentas AF and N, the prostate carcinoma cell line PC-3, and the lymphoid cell line GM607 [7].The parents and brother of ind.11 are designated 11M, 11V and 11B. All blots were prepared by the alkali method [S]; hybridization experiments were in phosphate buffer; washing was in 40 m~ sodium phosphate [9]. Scanning of autoradiograms was performed with an LKB (Bromma, Sweden) Ultrascan XL using the software package GelScan XL, version 1.20.
3 Results 3.1 The prevalent haplotype N and a haplotype from which half of the x locus is absent In Fig. 1and Table 1the 19 DDP and RFLP studied in the human 1c locus are compiled, and in Fig. 2 a typical example of a blot hybridization is given. About half of the 23 unrelated individuals, placenta DNA and cell lines studied here are homozygous for haplotype N which is defined as yielding with most DDP probes two bands of equal intensity. Already a while ago, a healthy individual (ind.11) was detected who is homozygous for the absence of the d copy of the 3t locus (ind.11; [4]). In DNA digests from this individual only the bands characteristicfor the p copy of the locus were detectable with DDP probes. In pulsed-field gel electrophoresis (PFGE) experiments the bands characteristic for the d copy were absent [lo].
+
0014-2980/91/0808-1829$3.50 .25/0
Ad
Od
Figure 1. Polymorphisms in the human Ig x locus. The boxes designating the V, genes are not to scale. The triangle indicates a region absent from the p copy 141. The distance between 01 and 011 is approximately 800 kb and the two copies of the locus are arranged in opposite 5', 3' polarity [7]. DDP and RFLP probes are indicated with arrows pointing to the respective polymorphic sites. The characteristicsof the probes are described inTable l.TheV, genes have been or will be described: for the 0 regions in the accompanying report [5],for the A regions ([4]and A. Lautner-Rieske et al., in preparation), for the L regions ([3] and C. Huber et al., in preparation, where in addition the designations of the L region genes will be described), and for the B regions [ll]. Many of the pseudogene sequences will be in K. E Schlble et al., in preparation.
The DNA of ind.11, his parents and brother were then studied systematically. It turned out that one parent was homozygous (ind.llM), the other one and the brother heterozygous for the absence of the d copy (Fig. 3). There was also one individual in our group of 23 DNA donors who is unrelated to the ind.11 family and who is heterozygous for haplotypes N and 11 (ind.17, Fig. 2). The DDP blot hybridization patterns of the heterozygous individuals (ind.11 family, ind.17) are characterized by two bands of unequal intensity. Quantitation of the band intensities, e.g. by densitometry, showed the expected ratio of 2 : 1for the bands characteristic for the p vs. the d copy [12].The finding of 3 haploid genomes without d copy among the 46 haploid
INDIVIDUALS
Ekb 1 5.2 4.5
AP Ad
Figure3. Inheritance of haplotype 11. About 10 pg of DNA of ind.11 family members and two placenta DNA were digested with BglII, electrophoresed and hybridized with the A region probe m659-2.
INDIVIDUALS and 12 others
[kbl
(A)
2 .o 1.7
genomes of the 23 individuals (4 of 48 haploid genomes if the non-inherited alleles of the parents of ind.11 are included) indicates that the ind.11 haplotype may not be very rare. It would be interesting to determine its frequency in different populations.
3.2 RFLP within DDP sites
4.0
(B)
2.5
1.7
(C)
1.15
Figure 2. Characterization of polymorphisms in the 0 regions by blot hybridization.The majority of the individuals yielded the same patterns as placenta N with two bands of equal intensity.The other DNA samples yielded only one band or bands of unequal intensity (see text). The following polymorphisms are shown: (A) p151-4, AccIBamHI. BamHI; (B) m143-1, RsaI; (C) m146-5,6,
Individuals with haplotypes N and 11 show in digests of their DNA the same situation at most DDP sites: two bands of equal intensity for N, a single band for the homozygous ind.11, and 2 : 1band intensity ratios for the heterozygotes. There is a number of individuals, however, whose DNA have haplotype N characteristics at some DDP sites but exhibit only one band or a band intensity ratio larger than 2 : 1 at other DDP sites. Such intensity ratios could in principle be due to local amplifications or deletions, to local sequence divergence or to point mutations in the restriction sites that define the DDPThe first two possibilities could be made unlikely in a number of cases by restriction mapping (cf. Table 2), by hybridization experiments at different stringencies, and by the use of homologous probes from both the p and the d copies [lS].We, therefore, explain the situations by the third possibility, i.e. by RFLP superimposed to the DDP sites (see below).
Eur. J. Immunol. 1991. 21: 1829-1835
Polymorphisms in the human immunoglobulin II locus
1831
Table 1. Polymorphisms in the human x locus Probes"'
Type of polyrn.h)
Nuclease(s)c' Nd
~207-6 mlU-2 111144-2 111113-2 ml.13-2 pljl-4 m14.7-1 m 146-5.6 111237-I ~313-1') p3 13-1') m142-2 PS 16-1M' 1n6S9-2~' rn656- 1 mHK134-6i) m l?7-3k) m21-1
mJIL-17 AF2-I0
pc-2
DDP(N) DDP(G) DDP(N) DDP DDP DDP(N) DDP(N) DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP RFLP RFLP FWLP
Hind IIlrSam HI ECORI Bgl I1 KpnIlBgl I1 KpnI/Sac I Bam HI Rsa I Acc UBam HI Bal I1 E& RI Bam HI Sac I/Kpn I Kpn I Bgl I1 Bam HI Bgl I1 Eco RI Bgl I1 Bgl I1 Hind 111 Sac I
1.2 10 7.7 7.2 3.5 1.7 2.5 1.7 5.6 0.8 5.2 3.3 7.6 4.5 4.7 3.5 2.4 4.0 -
Fragment sizesd)(kb) NP 0.8 10 6.9 6.9 9 2.0 4.0 1.15
15 0.5 4.9 6.4 8.3 5.2
-
3.8 3.5 3 .o 2. I 7.6
4.9
Region -
Described in"
0
151 113. 141 [5. 141
0 0 0 0 0 0
0
[ S . 141
(5. 141
l jI IS, 141
A A A A A A A
1151 [I51 I41 I121 1.1. Ihj
L L L B J C
(17. 181 1191 [31 120,211 12 I , 221 123.241
I4
a) The probes are listed in 5 ' , 3' direction (Fig. 1). Cross-hybridizations were observed for p207-6 and the L region probes. p313-1 detects an Alu element insertion in the d copy with several restriction cleavages. p516-16 was tested only with five individuals. b) DDP(N) and DDP(G) refers to duplication-differentiatingpolymorphisms which can be used only for haplotype N or G, respectively (see text). c) In double digests the polymorphic sites are underlined. d) Data on the distal (d) and proximal (p) copies of haplotypes N and G are given. Equation marks (=) indicate that the sizes are identical for Nd and Gd and for Np and Gp, respectively. e) References are to the description of the probes and/or to initial polymorphism studies. f) The clone p313-1 is a 0.5-kb EcoRI fragment cloned in Bluescript SK- [15]. g) The clone 516-16 is a 0.96-kb BamHI/HpaI fragment cloned in pUC19; as probe a 360-bp AccI fragment from this clone was used t121. h) The clone m659-2 is a 1.2-kb Sau3A fragment cloned in M13mplO [16]. i) The clone HK134-6 is a 0.8-kb BamHIlEcoRI fragment cloned in M13mplO [18]. k) The clone m127-3 is a 1.1-kb Sau3A fragment in M13mplO [19].
The haplotype which is maximally different from haplotype N is called haplotype G after the cell line GM607. It is defined by yielding at various DDP sites just one band which corresponds either to the p or the d situation of haplotype N. Accordingly, there are DDP which are valid for both haplotypes studied and DDP which are useful only for haplotypes Nor G [DDP(N)or DDP(G) inTable 11.The latter two DDP types can be regarded also as RFLPThe cell line GM607 is homozygous for the allele G at seven out of eight RFLP sites (see below). The structures of the two cosmid clones cos 607/7 and cos 607/30 which were isolated from a library prepared from GM607 DNA [5] are consistent with this definition of haplotype G. In Fig. 4 blot hybridizations of DNA digests are shown which were prepared from individuals with different combinations of haplotypesN, G and 11in their genomes. Since the densitometry does not always allow an exact determination of the band intensity ratios, additional data on such ratios were provided by dilution experiments of the type shown in Fig. 5. Additional restriction cleavages were also helpful in certain cases: in BglIIKpnI-digested DNA of individual 5 the presence of both haplotypes N and G can be directly visualized by the appearance of three bands with
the DDP probe m144-2 (Fig. 4).The band intensity ratio of 1:2 : 1is as expected from the restriction map of the region (Fig. 2 of [ 5 ] , preceding report) with the intensities of the separated fragments of the proximal copies being half of the one of the two co-migrating distal copy fragments. The figure also illustrates the finding that the father (and also the brother, Table2) of ind.11 carries a rare haplotype which was found for the germ-line allele GM607 (Tables 2 and 4). Ind.17, on the other hand, carries haplotype N in combination with haplotype 11 (Tables 3 and 4). The 14 DDP probes used in the current work are shown in Fig. 1and Table 1; since m144-2 recognizes three differentiating restriction sites, 16 DDP sites are being discussed. Five of them are RFLP sites at the same time belonging t o the DDP(N) or DDP(G) categories. In addition, three RFLP probes from the non-duplicated B-J,-C, part of the x locus were studied which are also shown in Fig. 1 and Table 1.The experimental results obtained at the DDP and RFLP sites are compiled in n b l e 2; for the DDP sites the relative signal intensities of the p or d copy derived fragments are given, and for the three RFLP sites the genotypes are noted.
Eur. J. Immunol. 1991.21: 1829-1835
W.Pargent, K. F. Schtible and H. G. Zachau
1832
Table 2. Results of the blot hybridizations of DNA digestsa)
DNA"
p107-h Hind 111
rnl44-2 Eco RI'I'
in 144-2 d =p d=p d=p
s+0
d=p
d'
AF
d=p d p' d' > p' d' = p' d'
d p'
P P NI) d P
P
All') DDP
Allc1
d=p d=p d=p d=p d=p d
Eur. J. Immunol. 1991. 21: 1829-1835
Walter Pargent, Karlheinz F. Schable and Hans G. Zachau Institut fur Physiologische Chemie der UniversitGt Miinchen, Munchen
Polymorphisms and haplotypes in the human immunoglobulin x locus* By comparing the restriction patterns of the DNA from 23 unrelated individuals 16 polymorphisms were defined which allowed us to differentiate between the duplicated copies Op, Ap, Lp and Od, Ad, Ld of the 1c locus (p for the C, proximal, d for the distal copy). Some of these duplication-differentiating polymorphisms or DDP revealed also allelic differences between individuals; they are therefore restriction fragment length polymorphism (RFLP) markers at the same time. Three RFLP in the single copy B-J,-C, region were included into the study.Three basic haplotypes were derived from the combined genotype data, haplotypes N, G and 11.The latter haplotype in which the whole distal copy of the 3t locus is missing was found three times among the 46 haploid genomes studied. The genotypes of the family members of an individual who is homozygous for haplotype 11are consistent with Mendelian inheritance. Haplotypes N and G are distinguishedfrom each other by eight RFLP markers. Six additional haplotypes, which were found in one or severalindividuals each, can be derived from the basic haplotypes N and G by hypothetical recombination and/or mutation events.
1 Introduction The human Ig 1c locus comprises two very similar but not identical copies: the C, proximal (p) copy which contains, in addition to the C, gene segment and 5 J,, 40V, genes and pseudogenes within a 500 kb contig, and the distal (d) copy that comprises 35 V, genes and pseudogenes within 400 kb (reviews [ l , 21). In elucidating the structure of the locus we were frequently confronted with the task to decide whether a newly isolated cosmid or phage h clone belongs to the p or d copy. If the new clone differed from an existing one at one or a few restriction sites, this could mean that it belonged to the other copy of the locus or the differences could be allelic. A decision was usually reached by studying the regions around the restriction site in question in blot hybridization experiments with the DNA of a number of unrelated individuals. If all or most individuals yielded both types of restriction fragments, the ones related to the new and the old clones,this was considered proof that the clones are derived from homologous positions of the two copies of the locus. This way a number of duplicationdifferentiating polymorphisms (DDP) were established along the locus. DDP probes ideally recognize in DNA digests from unrelated individuals both copies as restriction fragments of different length ( e . g [3, 41). As could be expected the hybridization patterns were complicated by allelic differences: the cosmid and phage h clones covering the 3t locus were derived from the DNA of a number of different
[I 93931
*
1829
This work was supported by Bundesministerium fiir Forschung und Technologie, Center Grant 0316200A, and Fonds der Chemischen Industne .
Correspondence: Hans G. Zachau, Institut fiir Physiologische Chemie, Schillerstr. 44,D-8000 Munchen 2, FRG Abbreviations: 0, A, L, B regions: V, gene-containing contigs of the human x locus DDP Duplication-differentiatingpolymorphism 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
individuals and the DNA of the individuals used for assaying turned out to belong to different haplotypes. In this paper we describe several differences between the duplicated copies and the character and extent of polymorphism of the human x locus.
2 Materials and methods In addition to the materials and methods (Sect. 2) described in the preceding report [5] the following methods were employed. DNA from white blood cells and cell lines were prepared according to [6].The 23 unrelated Caucasian individuals include ind.1-19, placentas AF and N, the prostate carcinoma cell line PC-3, and the lymphoid cell line GM607 [7].The parents and brother of ind.11 are designated 11M, 11V and 11B. All blots were prepared by the alkali method [S]; hybridization experiments were in phosphate buffer; washing was in 40 m~ sodium phosphate [9]. Scanning of autoradiograms was performed with an LKB (Bromma, Sweden) Ultrascan XL using the software package GelScan XL, version 1.20.
3 Results 3.1 The prevalent haplotype N and a haplotype from which half of the x locus is absent In Fig. 1and Table 1the 19 DDP and RFLP studied in the human 1c locus are compiled, and in Fig. 2 a typical example of a blot hybridization is given. About half of the 23 unrelated individuals, placenta DNA and cell lines studied here are homozygous for haplotype N which is defined as yielding with most DDP probes two bands of equal intensity. Already a while ago, a healthy individual (ind.11) was detected who is homozygous for the absence of the d copy of the 3t locus (ind.11; [4]). In DNA digests from this individual only the bands characteristicfor the p copy of the locus were detectable with DDP probes. In pulsed-field gel electrophoresis (PFGE) experiments the bands characteristic for the d copy were absent [lo].
+
0014-2980/91/0808-1829$3.50 .25/0
Ad
Od
Figure 1. Polymorphisms in the human Ig x locus. The boxes designating the V, genes are not to scale. The triangle indicates a region absent from the p copy 141. The distance between 01 and 011 is approximately 800 kb and the two copies of the locus are arranged in opposite 5', 3' polarity [7]. DDP and RFLP probes are indicated with arrows pointing to the respective polymorphic sites. The characteristicsof the probes are described inTable l.TheV, genes have been or will be described: for the 0 regions in the accompanying report [5],for the A regions ([4]and A. Lautner-Rieske et al., in preparation), for the L regions ([3] and C. Huber et al., in preparation, where in addition the designations of the L region genes will be described), and for the B regions [ll]. Many of the pseudogene sequences will be in K. E Schlble et al., in preparation.
The DNA of ind.11, his parents and brother were then studied systematically. It turned out that one parent was homozygous (ind.llM), the other one and the brother heterozygous for the absence of the d copy (Fig. 3). There was also one individual in our group of 23 DNA donors who is unrelated to the ind.11 family and who is heterozygous for haplotypes N and 11 (ind.17, Fig. 2). The DDP blot hybridization patterns of the heterozygous individuals (ind.11 family, ind.17) are characterized by two bands of unequal intensity. Quantitation of the band intensities, e.g. by densitometry, showed the expected ratio of 2 : 1for the bands characteristic for the p vs. the d copy [12].The finding of 3 haploid genomes without d copy among the 46 haploid
INDIVIDUALS
Ekb 1 5.2 4.5
AP Ad
Figure3. Inheritance of haplotype 11. About 10 pg of DNA of ind.11 family members and two placenta DNA were digested with BglII, electrophoresed and hybridized with the A region probe m659-2.
INDIVIDUALS and 12 others
[kbl
(A)
2 .o 1.7
genomes of the 23 individuals (4 of 48 haploid genomes if the non-inherited alleles of the parents of ind.11 are included) indicates that the ind.11 haplotype may not be very rare. It would be interesting to determine its frequency in different populations.
3.2 RFLP within DDP sites
4.0
(B)
2.5
1.7
(C)
1.15
Figure 2. Characterization of polymorphisms in the 0 regions by blot hybridization.The majority of the individuals yielded the same patterns as placenta N with two bands of equal intensity.The other DNA samples yielded only one band or bands of unequal intensity (see text). The following polymorphisms are shown: (A) p151-4, AccIBamHI. BamHI; (B) m143-1, RsaI; (C) m146-5,6,
Individuals with haplotypes N and 11 show in digests of their DNA the same situation at most DDP sites: two bands of equal intensity for N, a single band for the homozygous ind.11, and 2 : 1band intensity ratios for the heterozygotes. There is a number of individuals, however, whose DNA have haplotype N characteristics at some DDP sites but exhibit only one band or a band intensity ratio larger than 2 : 1 at other DDP sites. Such intensity ratios could in principle be due to local amplifications or deletions, to local sequence divergence or to point mutations in the restriction sites that define the DDPThe first two possibilities could be made unlikely in a number of cases by restriction mapping (cf. Table 2), by hybridization experiments at different stringencies, and by the use of homologous probes from both the p and the d copies [lS].We, therefore, explain the situations by the third possibility, i.e. by RFLP superimposed to the DDP sites (see below).
Eur. J. Immunol. 1991. 21: 1829-1835
Polymorphisms in the human immunoglobulin II locus
1831
Table 1. Polymorphisms in the human x locus Probes"'
Type of polyrn.h)
Nuclease(s)c' Nd
~207-6 mlU-2 111144-2 111113-2 ml.13-2 pljl-4 m14.7-1 m 146-5.6 111237-I ~313-1') p3 13-1') m142-2 PS 16-1M' 1n6S9-2~' rn656- 1 mHK134-6i) m l?7-3k) m21-1
mJIL-17 AF2-I0
pc-2
DDP(N) DDP(G) DDP(N) DDP DDP DDP(N) DDP(N) DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP RFLP RFLP FWLP
Hind IIlrSam HI ECORI Bgl I1 KpnIlBgl I1 KpnI/Sac I Bam HI Rsa I Acc UBam HI Bal I1 E& RI Bam HI Sac I/Kpn I Kpn I Bgl I1 Bam HI Bgl I1 Eco RI Bgl I1 Bgl I1 Hind 111 Sac I
1.2 10 7.7 7.2 3.5 1.7 2.5 1.7 5.6 0.8 5.2 3.3 7.6 4.5 4.7 3.5 2.4 4.0 -
Fragment sizesd)(kb) NP 0.8 10 6.9 6.9 9 2.0 4.0 1.15
15 0.5 4.9 6.4 8.3 5.2
-
3.8 3.5 3 .o 2. I 7.6
4.9
Region -
Described in"
0
151 113. 141 [5. 141
0 0 0 0 0 0
0
[ S . 141
(5. 141
l jI IS, 141
A A A A A A A
1151 [I51 I41 I121 1.1. Ihj
L L L B J C
(17. 181 1191 [31 120,211 12 I , 221 123.241
I4
a) The probes are listed in 5 ' , 3' direction (Fig. 1). Cross-hybridizations were observed for p207-6 and the L region probes. p313-1 detects an Alu element insertion in the d copy with several restriction cleavages. p516-16 was tested only with five individuals. b) DDP(N) and DDP(G) refers to duplication-differentiatingpolymorphisms which can be used only for haplotype N or G, respectively (see text). c) In double digests the polymorphic sites are underlined. d) Data on the distal (d) and proximal (p) copies of haplotypes N and G are given. Equation marks (=) indicate that the sizes are identical for Nd and Gd and for Np and Gp, respectively. e) References are to the description of the probes and/or to initial polymorphism studies. f) The clone p313-1 is a 0.5-kb EcoRI fragment cloned in Bluescript SK- [15]. g) The clone 516-16 is a 0.96-kb BamHI/HpaI fragment cloned in pUC19; as probe a 360-bp AccI fragment from this clone was used t121. h) The clone m659-2 is a 1.2-kb Sau3A fragment cloned in M13mplO [16]. i) The clone HK134-6 is a 0.8-kb BamHIlEcoRI fragment cloned in M13mplO [18]. k) The clone m127-3 is a 1.1-kb Sau3A fragment in M13mplO [19].
The haplotype which is maximally different from haplotype N is called haplotype G after the cell line GM607. It is defined by yielding at various DDP sites just one band which corresponds either to the p or the d situation of haplotype N. Accordingly, there are DDP which are valid for both haplotypes studied and DDP which are useful only for haplotypes Nor G [DDP(N)or DDP(G) inTable 11.The latter two DDP types can be regarded also as RFLPThe cell line GM607 is homozygous for the allele G at seven out of eight RFLP sites (see below). The structures of the two cosmid clones cos 607/7 and cos 607/30 which were isolated from a library prepared from GM607 DNA [5] are consistent with this definition of haplotype G. In Fig. 4 blot hybridizations of DNA digests are shown which were prepared from individuals with different combinations of haplotypesN, G and 11in their genomes. Since the densitometry does not always allow an exact determination of the band intensity ratios, additional data on such ratios were provided by dilution experiments of the type shown in Fig. 5. Additional restriction cleavages were also helpful in certain cases: in BglIIKpnI-digested DNA of individual 5 the presence of both haplotypes N and G can be directly visualized by the appearance of three bands with
the DDP probe m144-2 (Fig. 4).The band intensity ratio of 1:2 : 1is as expected from the restriction map of the region (Fig. 2 of [ 5 ] , preceding report) with the intensities of the separated fragments of the proximal copies being half of the one of the two co-migrating distal copy fragments. The figure also illustrates the finding that the father (and also the brother, Table2) of ind.11 carries a rare haplotype which was found for the germ-line allele GM607 (Tables 2 and 4). Ind.17, on the other hand, carries haplotype N in combination with haplotype 11 (Tables 3 and 4). The 14 DDP probes used in the current work are shown in Fig. 1and Table 1; since m144-2 recognizes three differentiating restriction sites, 16 DDP sites are being discussed. Five of them are RFLP sites at the same time belonging t o the DDP(N) or DDP(G) categories. In addition, three RFLP probes from the non-duplicated B-J,-C, part of the x locus were studied which are also shown in Fig. 1 and Table 1.The experimental results obtained at the DDP and RFLP sites are compiled in n b l e 2; for the DDP sites the relative signal intensities of the p or d copy derived fragments are given, and for the three RFLP sites the genotypes are noted.
Eur. J. Immunol. 1991.21: 1829-1835
W.Pargent, K. F. Schtible and H. G. Zachau
1832
Table 2. Results of the blot hybridizations of DNA digestsa)
DNA"
p107-h Hind 111
rnl44-2 Eco RI'I'
in 144-2 d =p d=p d=p
s+0
d=p
d'
AF
d=p d p' d' > p' d' = p' d'
d p'
P P NI) d P
P
All') DDP
Allc1
d=p d=p d=p d=p d=p d
P
d'
L rcgions
m14.1-1 Rsa I
d=p 11 = p d=p d=p d=p d=p d=p d-p dp d=p
d'
A regions
rnlS1-4 Barn HI d=p d=p d=p d=p d=p d
