GENOMICS
13, 1289-1295 (19%)
Extension of the Physical Map in the Region of the Mouse X Chromosome Homologous to Human Xq28 and Identification of an Exception to Conserved Linkage CYNTHIA
J. FAUST,* BARBARA LEviNsoN,t JANE GITSCHIER,t AND GAIL E. HERMAN**’
*Institute for Molecular Genetics and the Department tHoward Hughes Medical Institute and Department Received
March
of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; and of Medicine, University of California, San Francisco, California 94143 25, 1992;
We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to human Xq28 to include the loci Gdx (DXS254Eh), P3 (DXS253Eh), GGpd, Cf-8, and FSa. Gdx, P3, and G6pd are demonstrated to be physically linked to the Xlinked visual pigment locus (Rsup) within a maximal distance of 340 kb, while G6pd and Cf-8 are approximately 900 kb apart. These studies favor a gene order of ten-Rsvp-Gdx-P3-GGpd-(Cf-8)-tel and extend the physical map of this region to 5 million bp. In conjunction with previous physical mapping studies in both mouse and human, the results suggest conserved linkage for loci in this region of the mouse X chromosome and human Xq28. However, employing pulsed-field gel electrophoresis and genetic pedigree analysis of interspecific backcross progeny, we have found close linkage of a clone encoding a mouse homolog for human factor VIII-associated gene A (FSA) to DXPas8, thus revealing the first exception to conserved gene order between murine and human loci in the region. cl 1992 Academic
Press,
Inc.
INTRODUCTION
While X-chromosomal linkage is conserved among mammals, gene order is not always preserved. In particular, at least five chromosomal rearrangements exist between the X chromosomes of human and mouse to account for the gene order in the two species (Davisson, 1987; Amar et al., 1988). Within each homologous block, the gene order appears to be conserved; however, in some autosomal regions of extensive synteny, small rearrangements have been observed (Meisler and Seldin, 1991; Siracusa and Abbott, 1991). On the mouse X chromosome, a large region spanning from Hprt to the X-linked visual pigment. gene (Rsup) 1 To whom correspondence lecular Genetics, One Baylor
should Plaza,
be addressed at Institute for MoS911, Houston, TX 77030. 1289
revised
May
21, 1992
demonstrates apparent conserved linkage with human Xq26-Xq28. Mapping of this region of the human X chromosome has been hindered by high recombination frequencies and conflicting genetic and physical maps from different laboratories (Davies et al., 1987; Arveiler et al., 1989; Kenwrick and Gitschier, 1989). Our laboratory has previously developed genetic and physical maps for several loci in the region of the mouse X chromosome homologous to proximal human Xq28 (Herman et al., 1991; Faust and Herman, 1991). These loci included Gabra3, encoding the a3 subunit of the GABA, receptor; DXPas8, defined by the anonymous Xq28 probe St14 (DXS52); LlCam, which encodes the neural cell adhesion molecule Ll; and Rsup. Mapping of these loci in our well-characterized interspecific backcross provided a sound basis for the accurate construction of a 3 millionbp physical map using pulsed-field gel electrophoresis. These maps established a gene order of ten-Gabra$ DXPas8-Ll Cam-Rsvp-tel and caused a reevaluation of the gene order for human Xq28. Recently, physical maps for human Xq28 that demonstrate conserved gene order with the mouse for these loci have been constructed (Poustka et al., 1991; Dietrich et al., 1992). In this study, we have extended our pulsed-field gel map to include the loci Gdx(DXS254Eh), P3(DXS 253Eh),’ GGpd, Cf-8, and F8a. The human homologs for the Gdx and P3 genes have been localized approximately 40 kb 3’ to glucose B-phosphate dehydrogenase (GGPD) (Alcalay and Toniolo, 1988). GDX is located approximately 1 kb 3’ to P3 in both species (Brockdorff et al., 1989; Filippi et al., 1990), yielding a gene order of GdX (Gdxj-P3(P3)-GGPD(G6pd). In human, coagulation factor VIII (F8) is located within 600 kb of GGPD (Patterson et al., 1987; Arveiler et al., 1989). Brockdorff et al. (1989) have linked the corresponding mouse locus, Cf-8, ’ The proper designation for the loci P3 and Gdx are DXS253Eh and DXS254Eh, respectively, refiecting anonymous human loci expressed in the mouse. Since the colloquial locus names are more commonly used, particularly in human, we have used them throughout the text here. 0888.7543/92
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1290
FAUST ET AL. TABLE
1
DNA Probes Used in This Study Probe
Locus
pKpn-sac (human 5’ partial cDNA) pF8.9 (human 3’ partial cDNA) pGEM4-M2 (mouse partial cDNA) G28B (mouse genomic) pGD-P-25A (human partial cDNA) GGpD/mMZ (mouse complete cDNA) G28A (mouse genomic) XgMM25 (mouse genomic)’
Cf.8
Cf-8
Hybridization
conditions” II II
F85
Gdx G6pd G6pd P3 Rsvp
I I II I I III
Wash
conditio&
Source Genetics B. Levinson J. Gitschier P. Avner M. Persico Massimo P. Avner J. Nathans
Institute (Levinson et al., 1990) (Avner et al., 1987) (Persico et al., 1986) Zollo (Avner et al.., 1987)
fl Hybridization conditions were as follows. I, hybridization at 65°C in 1 M NaCI, 10% dextran sulfate (Pharmacia), 1% SDS, and 200 pg/ml denatured herring sperm DNA. II, hybridization at 42°C in 40% formamide, 1 M NaCl, 10% dextran sulfate, 1% SDS, and 200 pg/ml denatured herring sperm DNA. III, protocol I plus 200 rg/ml total mouse DNA sonicated to an average size of 1 kb. * Wash conditions were as follows: A, three washes with 3~ SSC, 0.05% SDS. B, sequential washes of 2x SSC, 0.1% SDS; 1X SSC, 0.1% SDS; 0.5X SSC, 0.1% SDS. All washes were performed at 65°C for 30 min. 1X SSC is 0.15 M NaCl, 0.015 M Na citrate, pH 7.0. ’ Probe was preannealed for 2 h at 65°C with 1 mg/ml total mouse DNA sonicated to an average molecular weight of 1 kb.
and the Gdx, P3, G6pd cluster within 400 kh. In this report we present data that enable orientation of these loci with respect to the centromere. Factor VIII-associated gene A (F8A) is a transcribed gene originally identified in intron 22 of the human F8 gene (Levinson et al., 1990). Some of the unique characteristics of this gene are that it is small (t2 kb), contains no introns, is G-C rich, and is transcribed in the opposite direction of F8. Two additional copies of F8A are apparently located telomeric to F8 in Xq28 (Kenwrick and Gitschier, 1989). Mouse genomic and cDNA clones corresponding to F8A have been isolated. The human and mouse genes demonstrate 85% identity at the amino acid level. Preliminary pulsed-field mapping data indicate that the mouse gene (F8a) is not located within Cf-8 (Levinson et al., 1992). Utilizing both pulsed-field gel and genetic analysis, we now localize F8a in the mouse and demonstrate that it represents the first example in which gene order is not conserved between human and mouse in this region.
MATERIALS
AND METHODS
Pulsed-field gel electraphoresis. Murine DNA plugs were prepared from the thymus of 2- to 3-week-old normal male and female progeny of B6CBA-A”-J/A-Bpa females X (C57BL/6JAW-J X CBA) F, males. This mating scheme results in X chromosomes derived from the inbred C57BL/6JA”-J strain (Herman et al., 1991). Plug preparation, digestion, and gel electrophoresis were performed as previously described (Faust and Herman, 1991). Southern blotting and hybridization. Southern blotting was performed using Sure Blot hybridization membranes (Oncor). DNA probes were labeled using the random hexamer technique (Feinberg and Vogelstein, 1984) and were used at a concentration of l-2 X lo6 cpm/ml of hybridization solution. The DNA probes used in this study, as well as the hybridization and washing conditions employed for each probe, are listed in Table 1. For rehybridization, filters were boiled for 30 min in a solution of 0.1X SSC, 1% SDS and placed under film overnight to ensure removal of all probe. Genetic analysis. The F8a probe pGEM4-M2 was mapped in backcross progeny from an interspecific backcross of B6CBA-A”-J/A-Bpa and Mus spretus, as previously described (Herman et al., 1991). The
panel has been expanded to 467 backcross animals with single recombination events
progeny and includes between Cf-9 and Dmd.
42
RESULTS We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to Xq28 to include the loci Gdx, P3, GGpd, Cf-8, and F8a. A composite physical map is shown in Fig. 1. The sizes of pulsed-field gel fragments obtained with probes for the five loci are summarized in Table 2. Physical linkage of Rsvp with Gdx, P3, and G6pd. Primary evidence for linkage of Rsvp with Gdx, P3, and G6pd was observed with BssHII partial digests (Fig. 2A, Table 2). Gdx, P3, and G6pd were present on a series of partial digestion fragments ranging from 100 to 340 kb, while Rsvp was present on a 170-kb complete and 230and 340-kb partial digestion products. Other overlapping pulsed-field gel fragments indicating linkage of these four loci include common 780-kb CZaI, as well as 510- and 650-kb Sal1 partial digestion fragments (Table 2). Double digests with BssHII in combination with other enzymes could not further localize Rsvp and the Gdx, P3, G6pd cluster with respect to the ends of the 340-kb BssHII partial digestion fragment; thus, the maximum distance between Rsvp and the G6pd cluster is approximately 340 kb (Fig. 1). While multiple CZaI, SalI, and BssHII partial digestion products indicate the presence of several of these sites within 1 million bp of Rsvp and the G6pd cluster, their exact positions have not been determined; therefore, the sites have not been included on the physical map shown in Fig. 1. Physical linkage of Gdx, P3, GGpd, and Cf-8. The loci Gdx, P3, and G6pd were detected on lOO-kb BssHII, as well as on 180- and 800-kb Sac11 partial digestion fragments (Table 2). Gdx and P3 were linked on 70-kb NaeI and 60-kb Sac11 fragments, while P3 and G6pd were observed on common 60-kb BssHII and 800-kb EagI fragments. These data indicate a gene order of Gdx-P3-
MAPPING
Gabra.3
Fda DXPas8
LlCam
OF Gdx, GGpd, P3, Cf-8, AND
Rsvp
F8a
1291
Cf-8 tel
ten 3OOkb
GdxP3
FIG.
1.
G6,ni
Composite
physical map of the loci studied. Gene order and physical distances (in kilobases) in the upper map, with the exception of from Faust and Herman (1991) and Herman et al. (1991). In the lower map, physical distances on either side of the slash marks are drawn to scale. Overlapping fragments that could be exactly positioned are indicated by bars below the map; others are given in the text or in Table 2. Restriction sites located ~10 kb apart are positioned on the same vertical line. The placement of the clusters of restriction sites flanking Rsup is in part taken from Faust and Herman (1991). We have also assumed that there are single NotI, MU, and NruI sites between Rsup and Gdx. If additional ones do exist, they must be clustered within 100 kb of the sites as drawn. The placement of the NueI sites around the Gdr and P3 genes was inferred from double digests with BssHII/NaeI and BssHII/SacII. The 1350-kb Nru.1 and 1400-kb MluI fragments are drawn distally at a distance of 1165 and 1215 kb, respectively, based on addition of smaller fragments between Gdx and Cf-8. The discrepancy most likely reflects inaccurate sizing of the large pulsed-field gel fragments containing these loci. The arrow drawn below the Cf-8 locus indicates direction of transcription. ten, centromere; ml, telomere; B, BssHII; C, CluI; E, EngI M, M&I; Ne, Naef; N, NotI; Nu, NruI; S, SUCII.
F8a and Cf-8, are taken
GGpd, which was verified by BssHII/NueI and BssHII/ Sac11 double digests (Fig. 1). These analyses confirm previous data linking Gdx and P3 within 50 kb of G6pd (Brockdorff et al., 1989). Physical linkage of Gdx, P3, and G6pd to Cf-8 was observed on large NruI, MuI, and Not1 fragments (Fig. 2B). To determine the relative orientation of the Gdx, P3, G6pd cluster with respect to Cf-8, several other restriction enzymes were tested. With the enzyme NaeI, G6pd was localized on an 880-kb fragment that did not hybridize with probes for either Gdx or P3 (Fig. 3A). A human F8 cDNA probe specific for the 5’ end of the gene hybridized to a 470-kb NaeI fragment, but a human 3’FB cDNA probe recognized both this 470-kb fragment and the 880-kb G6pd fragment (Figs. 3B and 3C). These data suggest that the 3’ end of Cf-8 is closer to G6pd and is consistent with data regarding the transcriptional orientation of the human F8 locus (Kenwrick and Gitschier, 1989). It also implies that the 3’ probe crosses an NueI site in the murine Cf-8 gene, although the human probe itself lacks an NaeI site (data not shown). The presence of an NueI site within the Cf-8 gene was confirmed by double digestion with BssHII/NueI (Table 2). Single di-
gests with BssHII yielded a 240-kb fragment to which both the 5’ and 3’ factor VIII probes hybridized. After double digestion with BssHII/NaeI, the 5’ probe hybridized to a 155-kb fragment, while the 3’probe recognized both 155- and 85-kb fragments. BssHII/NueI digestion produced a 50-kb fragment recognized by probes for GGpd, placing this locus within 50 kb of the proximal end of the 880-kb NueI fragment. These data enable us to define a minimum physical distance between G6pd and Cf-8 of 800 kb, with a maximum of approximately 1040 kb (Fig. 1). While the human F8 locus spans 187 kb (Kenwrick and Gitschier, 1989), the size of the mouse Cf-8 locus is unknown; assuming similar gene lengths, the average distance between G6pd and Cf-8 would be approximately 900 kb. This distance is discrepant with previous data linking G6pd and Cf-8 within 240 kb (Brockdorff et al., 1989). The linkage of G6pd with Cf-8 also enables orientation of the G6pd cluster with respect to RSVP, favoring a gene order of Rsvp-Gdx-P3GGpd-(Cf-8). Genetic mapping of the F8a locus. F8a was originally included in these analyses to help complete the physical map distal to Rsup and possibly aid in crossing the evo-
1292
FAUST
ET
TABLE Summary
AL.
2
of Pulsed-Field
Gel Fragment Restriction
LOCUS
BssHII
ClaI
EagI
MlUI
170 230* 340*
450 780’
210 260’
370 550’
210 260*
390
Gdx
40 100* 120* 130% 180* 340*
110 130’ 210* 470’ 560* 660’ 780*
13, 1289-1295 (19%)
Extension of the Physical Map in the Region of the Mouse X Chromosome Homologous to Human Xq28 and Identification of an Exception to Conserved Linkage CYNTHIA
J. FAUST,* BARBARA LEviNsoN,t JANE GITSCHIER,t AND GAIL E. HERMAN**’
*Institute for Molecular Genetics and the Department tHoward Hughes Medical Institute and Department Received
March
of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; and of Medicine, University of California, San Francisco, California 94143 25, 1992;
We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to human Xq28 to include the loci Gdx (DXS254Eh), P3 (DXS253Eh), GGpd, Cf-8, and FSa. Gdx, P3, and G6pd are demonstrated to be physically linked to the Xlinked visual pigment locus (Rsup) within a maximal distance of 340 kb, while G6pd and Cf-8 are approximately 900 kb apart. These studies favor a gene order of ten-Rsvp-Gdx-P3-GGpd-(Cf-8)-tel and extend the physical map of this region to 5 million bp. In conjunction with previous physical mapping studies in both mouse and human, the results suggest conserved linkage for loci in this region of the mouse X chromosome and human Xq28. However, employing pulsed-field gel electrophoresis and genetic pedigree analysis of interspecific backcross progeny, we have found close linkage of a clone encoding a mouse homolog for human factor VIII-associated gene A (FSA) to DXPas8, thus revealing the first exception to conserved gene order between murine and human loci in the region. cl 1992 Academic
Press,
Inc.
INTRODUCTION
While X-chromosomal linkage is conserved among mammals, gene order is not always preserved. In particular, at least five chromosomal rearrangements exist between the X chromosomes of human and mouse to account for the gene order in the two species (Davisson, 1987; Amar et al., 1988). Within each homologous block, the gene order appears to be conserved; however, in some autosomal regions of extensive synteny, small rearrangements have been observed (Meisler and Seldin, 1991; Siracusa and Abbott, 1991). On the mouse X chromosome, a large region spanning from Hprt to the X-linked visual pigment. gene (Rsup) 1 To whom correspondence lecular Genetics, One Baylor
should Plaza,
be addressed at Institute for MoS911, Houston, TX 77030. 1289
revised
May
21, 1992
demonstrates apparent conserved linkage with human Xq26-Xq28. Mapping of this region of the human X chromosome has been hindered by high recombination frequencies and conflicting genetic and physical maps from different laboratories (Davies et al., 1987; Arveiler et al., 1989; Kenwrick and Gitschier, 1989). Our laboratory has previously developed genetic and physical maps for several loci in the region of the mouse X chromosome homologous to proximal human Xq28 (Herman et al., 1991; Faust and Herman, 1991). These loci included Gabra3, encoding the a3 subunit of the GABA, receptor; DXPas8, defined by the anonymous Xq28 probe St14 (DXS52); LlCam, which encodes the neural cell adhesion molecule Ll; and Rsup. Mapping of these loci in our well-characterized interspecific backcross provided a sound basis for the accurate construction of a 3 millionbp physical map using pulsed-field gel electrophoresis. These maps established a gene order of ten-Gabra$ DXPas8-Ll Cam-Rsvp-tel and caused a reevaluation of the gene order for human Xq28. Recently, physical maps for human Xq28 that demonstrate conserved gene order with the mouse for these loci have been constructed (Poustka et al., 1991; Dietrich et al., 1992). In this study, we have extended our pulsed-field gel map to include the loci Gdx(DXS254Eh), P3(DXS 253Eh),’ GGpd, Cf-8, and F8a. The human homologs for the Gdx and P3 genes have been localized approximately 40 kb 3’ to glucose B-phosphate dehydrogenase (GGPD) (Alcalay and Toniolo, 1988). GDX is located approximately 1 kb 3’ to P3 in both species (Brockdorff et al., 1989; Filippi et al., 1990), yielding a gene order of GdX (Gdxj-P3(P3)-GGPD(G6pd). In human, coagulation factor VIII (F8) is located within 600 kb of GGPD (Patterson et al., 1987; Arveiler et al., 1989). Brockdorff et al. (1989) have linked the corresponding mouse locus, Cf-8, ’ The proper designation for the loci P3 and Gdx are DXS253Eh and DXS254Eh, respectively, refiecting anonymous human loci expressed in the mouse. Since the colloquial locus names are more commonly used, particularly in human, we have used them throughout the text here. 0888.7543/92
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1290
FAUST ET AL. TABLE
1
DNA Probes Used in This Study Probe
Locus
pKpn-sac (human 5’ partial cDNA) pF8.9 (human 3’ partial cDNA) pGEM4-M2 (mouse partial cDNA) G28B (mouse genomic) pGD-P-25A (human partial cDNA) GGpD/mMZ (mouse complete cDNA) G28A (mouse genomic) XgMM25 (mouse genomic)’
Cf.8
Cf-8
Hybridization
conditions” II II
F85
Gdx G6pd G6pd P3 Rsvp
I I II I I III
Wash
conditio&
Source Genetics B. Levinson J. Gitschier P. Avner M. Persico Massimo P. Avner J. Nathans
Institute (Levinson et al., 1990) (Avner et al., 1987) (Persico et al., 1986) Zollo (Avner et al.., 1987)
fl Hybridization conditions were as follows. I, hybridization at 65°C in 1 M NaCI, 10% dextran sulfate (Pharmacia), 1% SDS, and 200 pg/ml denatured herring sperm DNA. II, hybridization at 42°C in 40% formamide, 1 M NaCl, 10% dextran sulfate, 1% SDS, and 200 pg/ml denatured herring sperm DNA. III, protocol I plus 200 rg/ml total mouse DNA sonicated to an average size of 1 kb. * Wash conditions were as follows: A, three washes with 3~ SSC, 0.05% SDS. B, sequential washes of 2x SSC, 0.1% SDS; 1X SSC, 0.1% SDS; 0.5X SSC, 0.1% SDS. All washes were performed at 65°C for 30 min. 1X SSC is 0.15 M NaCl, 0.015 M Na citrate, pH 7.0. ’ Probe was preannealed for 2 h at 65°C with 1 mg/ml total mouse DNA sonicated to an average molecular weight of 1 kb.
and the Gdx, P3, G6pd cluster within 400 kh. In this report we present data that enable orientation of these loci with respect to the centromere. Factor VIII-associated gene A (F8A) is a transcribed gene originally identified in intron 22 of the human F8 gene (Levinson et al., 1990). Some of the unique characteristics of this gene are that it is small (t2 kb), contains no introns, is G-C rich, and is transcribed in the opposite direction of F8. Two additional copies of F8A are apparently located telomeric to F8 in Xq28 (Kenwrick and Gitschier, 1989). Mouse genomic and cDNA clones corresponding to F8A have been isolated. The human and mouse genes demonstrate 85% identity at the amino acid level. Preliminary pulsed-field mapping data indicate that the mouse gene (F8a) is not located within Cf-8 (Levinson et al., 1992). Utilizing both pulsed-field gel and genetic analysis, we now localize F8a in the mouse and demonstrate that it represents the first example in which gene order is not conserved between human and mouse in this region.
MATERIALS
AND METHODS
Pulsed-field gel electraphoresis. Murine DNA plugs were prepared from the thymus of 2- to 3-week-old normal male and female progeny of B6CBA-A”-J/A-Bpa females X (C57BL/6JAW-J X CBA) F, males. This mating scheme results in X chromosomes derived from the inbred C57BL/6JA”-J strain (Herman et al., 1991). Plug preparation, digestion, and gel electrophoresis were performed as previously described (Faust and Herman, 1991). Southern blotting and hybridization. Southern blotting was performed using Sure Blot hybridization membranes (Oncor). DNA probes were labeled using the random hexamer technique (Feinberg and Vogelstein, 1984) and were used at a concentration of l-2 X lo6 cpm/ml of hybridization solution. The DNA probes used in this study, as well as the hybridization and washing conditions employed for each probe, are listed in Table 1. For rehybridization, filters were boiled for 30 min in a solution of 0.1X SSC, 1% SDS and placed under film overnight to ensure removal of all probe. Genetic analysis. The F8a probe pGEM4-M2 was mapped in backcross progeny from an interspecific backcross of B6CBA-A”-J/A-Bpa and Mus spretus, as previously described (Herman et al., 1991). The
panel has been expanded to 467 backcross animals with single recombination events
progeny and includes between Cf-9 and Dmd.
42
RESULTS We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to Xq28 to include the loci Gdx, P3, GGpd, Cf-8, and F8a. A composite physical map is shown in Fig. 1. The sizes of pulsed-field gel fragments obtained with probes for the five loci are summarized in Table 2. Physical linkage of Rsvp with Gdx, P3, and G6pd. Primary evidence for linkage of Rsvp with Gdx, P3, and G6pd was observed with BssHII partial digests (Fig. 2A, Table 2). Gdx, P3, and G6pd were present on a series of partial digestion fragments ranging from 100 to 340 kb, while Rsvp was present on a 170-kb complete and 230and 340-kb partial digestion products. Other overlapping pulsed-field gel fragments indicating linkage of these four loci include common 780-kb CZaI, as well as 510- and 650-kb Sal1 partial digestion fragments (Table 2). Double digests with BssHII in combination with other enzymes could not further localize Rsvp and the Gdx, P3, G6pd cluster with respect to the ends of the 340-kb BssHII partial digestion fragment; thus, the maximum distance between Rsvp and the G6pd cluster is approximately 340 kb (Fig. 1). While multiple CZaI, SalI, and BssHII partial digestion products indicate the presence of several of these sites within 1 million bp of Rsvp and the G6pd cluster, their exact positions have not been determined; therefore, the sites have not been included on the physical map shown in Fig. 1. Physical linkage of Gdx, P3, GGpd, and Cf-8. The loci Gdx, P3, and G6pd were detected on lOO-kb BssHII, as well as on 180- and 800-kb Sac11 partial digestion fragments (Table 2). Gdx and P3 were linked on 70-kb NaeI and 60-kb Sac11 fragments, while P3 and G6pd were observed on common 60-kb BssHII and 800-kb EagI fragments. These data indicate a gene order of Gdx-P3-
MAPPING
Gabra.3
Fda DXPas8
LlCam
OF Gdx, GGpd, P3, Cf-8, AND
Rsvp
F8a
1291
Cf-8 tel
ten 3OOkb
GdxP3
FIG.
1.
G6,ni
Composite
physical map of the loci studied. Gene order and physical distances (in kilobases) in the upper map, with the exception of from Faust and Herman (1991) and Herman et al. (1991). In the lower map, physical distances on either side of the slash marks are drawn to scale. Overlapping fragments that could be exactly positioned are indicated by bars below the map; others are given in the text or in Table 2. Restriction sites located ~10 kb apart are positioned on the same vertical line. The placement of the clusters of restriction sites flanking Rsup is in part taken from Faust and Herman (1991). We have also assumed that there are single NotI, MU, and NruI sites between Rsup and Gdx. If additional ones do exist, they must be clustered within 100 kb of the sites as drawn. The placement of the NueI sites around the Gdr and P3 genes was inferred from double digests with BssHII/NaeI and BssHII/SacII. The 1350-kb Nru.1 and 1400-kb MluI fragments are drawn distally at a distance of 1165 and 1215 kb, respectively, based on addition of smaller fragments between Gdx and Cf-8. The discrepancy most likely reflects inaccurate sizing of the large pulsed-field gel fragments containing these loci. The arrow drawn below the Cf-8 locus indicates direction of transcription. ten, centromere; ml, telomere; B, BssHII; C, CluI; E, EngI M, M&I; Ne, Naef; N, NotI; Nu, NruI; S, SUCII.
F8a and Cf-8, are taken
GGpd, which was verified by BssHII/NueI and BssHII/ Sac11 double digests (Fig. 1). These analyses confirm previous data linking Gdx and P3 within 50 kb of G6pd (Brockdorff et al., 1989). Physical linkage of Gdx, P3, and G6pd to Cf-8 was observed on large NruI, MuI, and Not1 fragments (Fig. 2B). To determine the relative orientation of the Gdx, P3, G6pd cluster with respect to Cf-8, several other restriction enzymes were tested. With the enzyme NaeI, G6pd was localized on an 880-kb fragment that did not hybridize with probes for either Gdx or P3 (Fig. 3A). A human F8 cDNA probe specific for the 5’ end of the gene hybridized to a 470-kb NaeI fragment, but a human 3’FB cDNA probe recognized both this 470-kb fragment and the 880-kb G6pd fragment (Figs. 3B and 3C). These data suggest that the 3’ end of Cf-8 is closer to G6pd and is consistent with data regarding the transcriptional orientation of the human F8 locus (Kenwrick and Gitschier, 1989). It also implies that the 3’ probe crosses an NueI site in the murine Cf-8 gene, although the human probe itself lacks an NaeI site (data not shown). The presence of an NueI site within the Cf-8 gene was confirmed by double digestion with BssHII/NueI (Table 2). Single di-
gests with BssHII yielded a 240-kb fragment to which both the 5’ and 3’ factor VIII probes hybridized. After double digestion with BssHII/NaeI, the 5’ probe hybridized to a 155-kb fragment, while the 3’probe recognized both 155- and 85-kb fragments. BssHII/NueI digestion produced a 50-kb fragment recognized by probes for GGpd, placing this locus within 50 kb of the proximal end of the 880-kb NueI fragment. These data enable us to define a minimum physical distance between G6pd and Cf-8 of 800 kb, with a maximum of approximately 1040 kb (Fig. 1). While the human F8 locus spans 187 kb (Kenwrick and Gitschier, 1989), the size of the mouse Cf-8 locus is unknown; assuming similar gene lengths, the average distance between G6pd and Cf-8 would be approximately 900 kb. This distance is discrepant with previous data linking G6pd and Cf-8 within 240 kb (Brockdorff et al., 1989). The linkage of G6pd with Cf-8 also enables orientation of the G6pd cluster with respect to RSVP, favoring a gene order of Rsvp-Gdx-P3GGpd-(Cf-8). Genetic mapping of the F8a locus. F8a was originally included in these analyses to help complete the physical map distal to Rsup and possibly aid in crossing the evo-
1292
FAUST
ET
TABLE Summary
AL.
2
of Pulsed-Field
Gel Fragment Restriction
LOCUS
BssHII
ClaI
EagI
MlUI
170 230* 340*
450 780’
210 260’
370 550’
210 260*
390
Gdx
40 100* 120* 130% 180* 340*
110 130’ 210* 470’ 560* 660’ 780*
